Genetic Diversity of Tamarixia radiata Populations and Their Associated Endosymbiont Wolbachia Species from China

Round 1

Reviewer 1 Report

This manuscript is written on the experiment in the last manuscript, and no improvement can be seen basically. In particular, no scientific or rational explanations are given in that this study is confined in some regions of China with one region of Ecuador. The date of collections and experiments are not provided either. The species name of Wolbachia must be referred to, unless it was a new species and a report was being prepared.

The authors report in this manuscript the genetic variation in both a parasitoid, Tamarixia radiata, and the symbiotic microorganism, Wolbachia. The point to consider the manuscript for the publication in the journal is what results are novel in the study and what value the results have for readers of the journal. The results are new in that some genetic variations in these organisms were revealed, although the sequence of the genomes had been already known before this study. The data collection was confined almost in China, and what cannot be understood in their study is why only Ecuador was considered for this study. No strong reasoning can be found in the selection of geographical locations. If the authors found any genetic variations that can be developed for the establishment of IPM as biocontrol agents in these species (for example in L36-37), the manuscript may be considered for the publication in the journal. Similar descriptions are repeated in the manuscript, but it is not clearly indicated how such genetic variations can be included in the managements of this citrus disease. Due to these points, it seems to be rather reluctant to recommend the manuscript for the publication in the journal.

Here are mentioned major points that should be considered for revising the manuscript, whether a new one will be resubmitted to this journal or else.

1. English and wrong spelling
There are lots of typographical errors, not pointed individually. Spelling can be checked by a standard word processing software such as MS Word. English should be checked by a native English speaker before the resubmission.
2. Date of collection and treatment
The date of collections and treatments must be mentioned. The preparation of samples is explained too simply. It should be described such as how the samples were prepared (how many replicates were set in each treatment) and when it was done.
3. Scientific name
All organisms used for the study must be referred to with the author of the species. Also all scientific names must be italicised. The species epithet of Wolbachia must be referred to. Any new species of the genus were found or at least suggested?
4. It is unclear why the authors included other congeneric species or conspecific specimens of other regions that were not directly treated in this study. The conclusion that the authors obtained may vary which species or regions had been considered.

Author Response

Response to Reviewer 1 Comments

Comment: This manuscript is written on the experiment in the last manuscript, and no improvement can be seen basically. In particular, no scientific or rational explanations are given in that this study is confined in some regions of China with one region of Ecuador. The date of collections and experiments are not provided either. The species name of Wolbachia must be referred to, unless it was a new species and a report was being prepared.

The authors report in this manuscript the genetic variation in both a parasitoid, Tamarixia radiata, and the symbiotic microorganism, Wolbachia. The point to consider the manuscript for the publication in the journal is what results are novel in the study and what value the results have for readers of the journal. The results are new in that some genetic variations in these organisms were revealed, although the sequence of the genomes had been already known before this study. The data collection was confined almost in China, and what cannot be understood in their study is why only Ecuador was considered for this study. No strong reasoning can be found in the selection of geographical locations. If the authors found any genetic variations that can be developed for the establishment of IPM as biocontrol agents in these species (for example in L36-37), the manuscript may be considered for the publication in the journal. Similar descriptions are repeated in the manuscript, but it is not clearly indicated how such genetic variations can be included in the managements of this citrus disease. Due to these points, it seems to be rather reluctant to recommend the manuscript for the publication in the journal

Response: Thank you very much for your valuable comments and suggestions that certainly help in improving this manuscript. In this MS, we have presented the genetic variation in both a parasitoid, Tamarixia radiata, and its symbiotic bacteria, Wolbachia. Due to limited regional distribution of T. radiata, we selected some already reported provinces located in the south of China [Jiangxi (Ganzhou), Guangdong (Zhaoqing), Fujian (Fuzhou)] for the T. radiata collection, and the selection of these geographical zones was again mainly dependent upon the availability of T. radiata. In a previous study (Ramos-Aguila et al., 2021) life table of Ecuador population of T. radiata has already been reported without any comparative molecular data of species diversity. Therefore, we decided to include Ecuador papulation of T. radiata (which is already maintained in our laboratory) for a preliminary comparative molecular analysis with other T. radiata papulations collected from China. However, we mainly focused on the T. radiata populations in different geographical locations in China for further diversity analysis.There are some published literature references where the Ecuador population of T. radiata has been selected based on the source and previously reported study from the laboratory.

 

• Ramos Aguila, L.C.; Atlihan, R.; Ashraf, H.J.; Keppanan, R.; Lei, L.; Bamisile, B.S.; Cerda, H.; Wang, L. Temperature-Dependent Biological Control Effectiveness of Tamarixia radiata (Hymenoptera: Eulophidea) Under Laboratory Conditions. J. Econ. Entomol. 2021, doi:10.1093/jee/toab130.

 

The date of collection has been mentioned in the methodology and highlighted in red.

In the present study, the genus Wolbachia is being reported for the first time from T. radiata. Based on blast search and phylogenetic analysis of wsp gene, we could identify Wolbachia pipientis associated with T. radiata. We have incorporated species names in revised MS at required positions for clarity.  

Tamarixia radiata is an important biological control of D. citri; until now, its distribution and genetic differentiation are unclear in China. Population genetics tools have already been successfully applied to insect pest management studies characterizing gene flow among populations (Groot et al., 2011, Medina et al., 2012; Silva-Brandão et al., 2015). Therefore, exploring the genetic diversity and differentiation of T. radiata is the basis for improving biocontrol technology for pest management. The present study is the first attempt to compare the population distribution and genetic variations of T. radiata, using COI gene as a molecular marker in different geographical areas that can be helpful to control the citriculture economic loss caused by D. citri.

 

• Silva-Brandão, K.L.; Santos, T. V; Cônsoli, F.L.; Omoto, C. Genetic Diversity and Structure of Brazilian Populations of Diatraea saccharalis (Lepidoptera: Crambidae): Implications for Pest Management. Econ. Entomol. 2015, 108, 307–316, doi:10.1093/jee/tou040.
• Groot, A. T., A. Classen, O. Inglis, C. A. Blanco, J. Lo´pez, Jr., A. T. Vargas, C. Schal, G. Heckel, and G. Scho¨fl.2011. Genetic differentiation across North America in the generalist moth Heliothis virescens and the specialist subflexa. Mol. Ecol. 13: 2676–2692.
• Medina, R. F., S.M. Reyna, and J. S. Bernal. 2012. Population genetic structure of a specialist leafhopper on Zea: likely anthropogenic and ecological determinants of gene flow. Entomol. Exp. Appl. 142: 223–235.

 

#1. English and wrong spelling

There are lots of typographical errors, not pointed individually. Spelling can be checked by a standard word processing software such as MS Word. English should be checked by a native English speaker before the resubmission

Response: The manuscript has been checked thoroughly and edited the spelling mistakes. As suggested, the English has been revised in the new version of the manuscript by a native English speaker.

 

#2. Date of collection and treatment

The date of collections and treatments must be mentioned. The preparation of samples is explained too simply. It should be described such as how the samples were prepared (how many replicates were set in each treatment) and when it was done.

Response: The date of collections is mentioned in the methodology and highlighted in red. Furthermore, the methodology and treatment of samples were also elaborated in detail, and a supplementary figure was also given. Five replicates were used and added in the text, also highlighted in red.

 

 

 

Figure S2: Rearing bottle for Tamarixia radiata

 

3#. Scientific name

All organisms used for the study must be referred to with the author of the species. Also all scientific names must be italicised. The species Wolbachia must be referred to. Any new species of the genus were found or at least suggested?

Response: As per comments, all the organisms used for the study have been provided with species' author names highlighted in red as well as all scientific names have been italicized. In our study, Wolbachia has been reported first time in T. radiata, based on the wsp gene analysis, and our results are parallel previously published data. Based on blast search and phylogenetic analysis of wsp gene, we could identify Wolbachia pipientis associated with T. radiata. We have incorporated species names in revised MS at required positions for clarity.

 

4#Comment: It is unclear why the authors included other congeneric species or conspecific specimens of other regions that were not directly treated in this study. The conclusion that the authors obtained may vary which species or regions had been considered.

Response: In this study, we used different species of Tamarixia in the phylogenetic tree (ITS1 and ITS2) to demonstrate the identification of T. radiata populations from different geographical zones in China and make our results more evident to investigate the genetic diversity and phylogenetic relationships among the parasitoids. However, phylogenetic analysis showed that Tamarixia radiata populations from China and Ecuador formed a distinct clade.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors

Thank you for the opportunity to read and review your important research. I found it well written, thought through and reported and I congratulate you on your research. I support the publication of you research subject to the minor corrections I’ve identified that need to please be addressed. I have made some corrections and comments on the pdf you submitted and I ask you please see the attached manuscript for my comments. I have also included some comments below. The manuscript would also benefit from academic/language editing, this is something I recommend to all. I think that the English was of a very high standard, but there are small typos and grammatical mistakes that can be addressed by a fresh pair of eyes that an editor will bring when they read the manuscript. I wish you luck with your corrections and publication of your manuscript.

 

Additional comments to address

• Italicise all scientific names
• Spelling mistake L 37, diverse
• Please include a map of sample sites/locations. This is especially important for the Chinese locations samples because it is unclear to reader how geographically distant populations are from each other. The inter-country comparisons imply bigger geographic distance, but not sure of geographic distance within the Chinese locations sampled. A map would make it clearer.
• A comparison was made between China and Ecuador, but there are other countries added to analyses and figures. Can you please make it clearer in your methods why Ecuador was chosen and also mention the other countries you included in analyses too.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 2 Comments

 

Comments and Suggestions for Authors

Comment#1: Thank you for the opportunity to read and review your important research. I found it well written, thought through and reported and I congratulate you on your research. I support the publication of you research subject to the minor corrections I’ve identified that need to please be addressed. I have made some corrections and comments on the pdf you submitted and I ask you please see the attached manuscript for my comments. I have also included some comments below. The manuscript would also benefit from academic/language editing, this is something I recommend to all. I think that the English was of a very high standard, but there are small typos and grammatical mistakes that can be addressed by a fresh pair of eyes that an editor will bring when they read the manuscript. I wish you luck with your corrections and publication of your manuscript.

Response: The authors would like to thank Reviewer 2 very much for his/her relevant/pertinent comments and suggestions. We have addressed all your comments and included your suggestions as suggested in the revised agronomy-1305799-R1. In addition, the English has been edited and revised in the new version of the manuscript by a native English speaker, as suggested.

 

Comment#2: Italicise all scientific names

Response: All the scientific names have been Italicise in the text and also highlighted in red.

 

Comment#3: Spelling mistake L 37, diverse

Response: Changed as suggested: divers to “diverse”. Line 37.

 

Comment#4: Please include a map of sample sites/locations. This is especially important for the Chinese locations samples because it is unclear to reader how geographically distant populations are from each other. The inter-country comparisons imply bigger geographic distance, but not sure of geographic distance within the Chinese locations sampled. A map would make it clearer

Response: A map is provided as by the reviewer's suggestion between China populations that clear the geographical distance in between populations of T. radiata.(Figure S2)

 

 

Figure S1: A map showing different geographical zones selected for Tamarixia radiata collection (source: https://simplemaps.com).

 

Comment#5: A comparison was made between China and Ecuador, but there are other countries added to analyses and figures. Can you please make it clearer in your methods why Ecuador was chosen and also mention the other countries you included in the analyses too.

Response: In this MS, we have presented the genetic variation in both a parasitoid, Tamarixia radiata, and its symbiotic bacteria, Wolbachia. Due to limited regional distribution of T. radiata, we selected some already reported provinces located in the south of China [Jiangxi (Ganzhou), Guangdong (Zhaoqing), Fujian (Fuzhou)] for the T. radiata collection, and the selection of these geographical zones was again mainly dependent upon the availability of T. radiata. In a previous study  (Ramos-Aguila et al., 2021) from our laboratory,  the life table of the Ecuador population of T. radiata has already been reported without any comparative molecular data of species diversity. Therefore, we decided to include Ecuador papulation of T. radiata (which is already maintained in our lab) for a preliminary comparative molecular analysis with other T. radiata populations collected from China. However, we mainly focused on the T. radiata populations in different geographical locations in China for further diversity analysis. 

There are some published literature references where the Ecuador population of T. radiata has been selected based on the source and previously reported study from the laboratory.

• Ramos Aguila, L.C.; Atlihan, R.; Ashraf, H.J.; Keppanan, R.; Lei, L.; Bamisile, B.S.; Cerda, H.; Wang, L. Temperature-Dependent Biological Control Effectiveness of Tamarixia radiata (Hymenoptera: Eulophidea) Under Laboratory Conditions. Econ. Entomol. 2021, doi:10.1093/jee/toab130.

 

As per suggestion, we have incorporated details of additional data used from different counties to support ITS1 and ITS2 analysis. All modifications have been highlighted in red font.

 

Line# 103 Comment: Ecuador.  (add: to act as an outgroup)

Response: We have included an Ecuador papulation for preliminary molecular study. We had already published the life table of the Ecuador population of T. radiata, and molecular work is not done on this population so far. The morphological analysis also showed that the Ecuador population and Chinese population have no difference (data not showed); however, molecular analysis showed a slight variation between China populations and Ecuador population. Therefore we cannot use it as an outgroup.  However, we have used different species of Tamarixia for ITS1, ITS2, and the COI analysis using Fragaria vesca as an outer group.

 

Line# 133 Comment: state concentration rather than volume of primers

Response: As per the comment, information has been added. 

 

Line# 254 (Figure#4) Comment: I don't recall seeing in the methods any mention of the inclusion of sequence data from countries other than China and Ecuador. Can you please include all the countries/samples that you used in analyses in your methods. The use of map indicating where you have samples from would also be very helpful.

Response: A map is being provided as per suggestion to show T. radiata populations collection regions and geographical distance (Figure S1). Information of other populations data used in ITS1 and ITS2 is incorporated in the methodology and highlighted red.

 

Line# 286 (Table#4) Comment: This is a small number of samples, can you please include a statement in your methods about the sample size and literature supporting the undertaking of statistics on small samples sizes being ok (for Tajimma's D)

Response:  According to the literature, the number of samples we used for  Tajimma's D test is acceptable. As genetic variability of a population of Aedes aegypti from Nova Londrina, using the mitochondrial ND4 gene, showed the highest genetic diversity values; however, the relatively smaller sample size from Nova Londrina

(n = 4). This area is a densely populated commercial center with people constantly traveling in and out, which may also contribute to the increase in gene flow between populations.

 

• Twerdochlib, A.L.; Bonna, A.C.D.; Leite, S.S.; Chitolina, R.F.; Westphal, B.; Navarro-Silva, M.A. Genetic variability of a population of Aedes aegypti from Paraná, Brazil, using the mitochondrial ND4 gene. Bras. Entomol. 2012, 56, 249–256.

 

Line# 320 Comment: use of a map would be helpful here

Response: As per suggestion, a map is being provided as a supplementary file (Figure S1) to show a clear geographical distance between populations of T. radiata.

 

Line# 324 Comment: please remove these values from brackets, also state these as a range in values.

Response: As per suggestion,  modifications have been incorporated, and the minimum and maximum range of values were mentioned and highlighted in red.

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.