Diversity and Regional Variation of Endosymbionts in the Green Peach Aphid, Myzus persicae (Sulzer)

Round 1

Reviewer 1 Report

Dear Authors,

Thank you for the opportunity to review your manuscript “Diversity and regional variation of endosymbionts in the green 2 peach aphid, Myzus persicae (Sulzer).” It is an excellent study, which combines both fundamental and applied aspects of very popular and important nowadays endosymbiotic research. However, I have found some places in your manuscript, which remain unclear to me, or that look kind of unpolished. From the reader's perspective, it would be beneficial to address them.

1.      Please italicize all species and genera mentioned in the text.

2.      Aphids from some Australian samples and Chinese samples were genotyped to de- 99

I believe, it is necessary to mention the number of genotyped samples here.

3.      DNA was extracted by homogenising individual aphids in a 200 μl solu- 102

Is this that one individual per population was used to assess its clonality?

4.      Was the reason for the absence of microsatellite-defined clones in Table 1 the insufficient number of collected field samples?

5.      How did you identify some of the bacteria you detected, as non-endosymbionts? What were your criteria for such decisions (line 200, Figure 2)?

6.      In the Results & Discussion section you separated two paragraphs (line 336), not connected to the placement of the figures. Does this division have any specific meaning?

7.      I believe, it would be beneficial to separate the Conclusion or Summary from the Results.

8.      I am not sure if the term “difference of endosymbionts diversity” is applicable to your conclusions. I feel it somehow is a quantitative difference like you might compare some numbers in two sample sets. In your study, in one case you observed some endosymbionts diversity (China samples). In Australian samples, you haven’t detected any. It is confusing and misleading because, in reality, you discuss the presence and absence of the endosymbionts, a sort of qualitative difference. However, I might be wrong.

Despite all my concerns and criticisms your study definitely has scientific merits and it is definitely worth to be published in the Diversity journal.

Respectfully,

 

Reviewer.

Author Response

We appreciate the constructive feedback from the reviewers. We’ve addressed these points and improved the manuscript and look forward to further evaluation. The following is a point-by-point response to the reviewer’s feedback.

Reviewer 1

 

1. Please italicize all species and genera mentioned in the text.

All the species and genera have been italicized.

2. Aphids from some Australian samples and Chinese samples were genotyped to de- 99

I believe, it is necessary to mention the number of genotyped samples here.

This has been amended.

3. DNA was extracted by homogenising individual aphids in a 200 μl solu- 102

Is this that one individual per population was used to assess its clonality?

Two to 12 individuals from each population were genotyped. We’ve revised this in the text at line 115.

4. Was the reason for the absence of microsatellite-defined clones in Table 1 the insufficient number of collected field samples?

No, it was not the reason. We genotyped the clones involved in a pesticide bioassay for another project. We considered genotype information from 27 Australian populations and 13 Chinese populations to be sufficient to present clonal diversity for the M. persicae populations, so we did not genotype all field samples in this study.

5. How did you identify some of the bacteria you detected, as non-endosymbionts? What were your criteria for such decisions (line 200, Figure 2)?

We focused on the secondary endosymbionts which have been reported in aphids. These are Serratia symbiotica, Hamiltonella defensa, Regiella insecticola, Rickettsia, Rickettsiella, Spiroplasma, Wolbachia and Arsenophonus, as well as the primary endosymbiont Buchnera. All the other bacteria identified were placed in the group ‘non-endosymbionts’, please see lines 262-264.

 

6. In the Results & Discussion section you separated two paragraphs (line 336), not connected to the placement of the figures. Does this division have any specific meaning?

The two paragraphs have been combined.

7. I believe, it would be beneficial to separate the Conclusion or Summary from the Results.

The conclusion has been separated from the results section.

8. I am not sure if the term “difference of endosymbionts diversity” is applicable to your conclusions. I feel it somehow is a quantitative difference like you might compare some numbers in two sample sets. In your study, in one case you observed some endosymbionts diversity (China samples). In Australian samples, you haven’t detected any. It is confusing and misleading because, in reality, you discuss the presence and absence of the endosymbionts, a sort of qualitative difference. However, I might be wrong.

As we mentioned in the draft, a previous study detected the Regiella infection in M. persicae populations collected from Bacchus Marsh, Australia on wild mustard, in 2003. A few other studies reported secondary endosymbiont infections (but very rarely) in GPA from South America (Gallo-Franco et al. 2019), and the UK (Henry et al. 2015). We therefore consider it reasonable to use the term ‘endosymbiont diversity’ but we have clarified our usage now at line 408.

Reviewer 2 Report

The authors investigated endosymbionts of the green peach aphid, Myzus persicae, using short-read 16S rRNA gene amplicon sequencing, especially between China and Australian populations, which were putative native, and invasive populations, respectively. Experimental setup and analysis were basically no-problem, while the resultant patterns of infection were similar to previous studies, as the authors mentioned. This is descriptive research, but I believe the accumulation of case studies would be one of essential parts of science. The loss of secondary or facultative symbionts in newly established or introduced population is itself interesting phenomenon; in this sense, the present study can contribute to the future studies.

 

-General comments:

1. In this manuscript, it was unclear why the authors should have to “understand the diversity and regional variation of endosymbionts” in Australia (Line 70-71). In other words, the necessity of understanding the diversity and infection status of secondary symbionts in the pest aphids was not explicitly represented in Introduction and Abstract. Clarifying question of this research will make it easier for a wider reader to understand the content of this paper.

 

2. I could not well understand why Chinese populations were concluded as native ones. More specifically, which regions in China are most likely? The authors omitted the details in the manuscript, but they may add specific explanations in the Introduction section, if possible.

 

3. The authors discuss the differences in their results from previous studies in Line 250-273. I agree with that secondary symbionts such as Spiroplasma and Rickettsia can be detected more often in China as authors indicated. However, a previous study successfully detected additional endosymbionts including Wolbachia; then, the authors tried to explain the difference from previous reports by the sampling vias and technical sensitivity.

I wonder why the authors did not discuss about the regional and seasonal difference; how different are the sampling points from the previous study? Also, there are some reports wherein the relative abundance of secondary endosymbiont can seasonally change:

Smith, A. H., O’Connor, M. P., Deal, B., Kotzer, C., Lee, A., Wagner, B., ... & Russell, J. A. (2021). Does getting defensive get you anywhere?—Seasonal balancing selection, temperature, and parasitoids shape real‐world, protective endosymbiont dynamics in the pea aphid. Molecular Ecology, 30(10), 2449-2472.

Zhao, D., Zhang, Z., Niu, H., & Guo, H. (2021). Win by quantity: a striking Rickettsia-bias symbiont community revealed by seasonal tracking in the whitefly Bemisia tabaci. Microbial ecology, 81(2), 523-534.

 

4. In the present study, the authors successfully detected Spiroplasma and Rickettsia in some Chinese populations. It may be worth to analyze their phylogenetic status. Are they specific to Myzus aphids? Or the same as bacteria from pea-aphids? These would be important for the authors’ argument, “loss of secondary symbiont in invasive regions”. Although the length of the sequence is short (V3-V4 region, ~500 bp), it will be possible to use sequences from 16S amplicon seq to phylogenetic analysis. Here is one of examples:

Nakabachi, A., Inoue, H., & Hirose, Y. (2022). Microbiome analyses of 12 psyllid species of the family Psyllidae identified various bacteria including Fukatsuia and Serratia symbiotica, known as secondary symbionts of aphids. BMC microbiology, 22(1), 1-21.

 

-Specific comments:

 

Line 181-183, Table 1

I could not figure out “Microsatellite defined clones”. Is this the number of repeats of certain microsatellite loci? Or, just following some previous studies?

 

Line 238, Figure 2

The authors should explain color label; red and blue seems to be corresponded to Chinese and Australian populations, respectively.

I could not find the definition of “other not endosymbiotic bacteria.” (Graph legend is blurry in my PDF and difficult to read; is this okay?) This should be improved.

I recognized some samples exhibit high rate of “other non-symbiotic bacteria” (for examples, China_Sichuan_Dayand, AUS_Osborne_2, 3). Are they pathogenic bacteria? Or unknown endosymbionts? This is not mentioned and explained in the manuscript.

 

Line 255-256, 317-318

Statistical methods and used software should be included in the Materials and methods section.

 

Line 337-345

There are some other papers in which investigate endosymbionts both in native and introduced populations. It may be worth to include them. For example, in the little fire ant and RIFA, Wolbachia was rarely detected in the introduced ranges (Rey et al., 2013, Yang et al., 2010). These are similar to introduced populations of thrips (Nguyen et al., 2016). In the aphid Cinara cedri, Wolbachia was detected in the recently introduced population in Japan (Nozaki et al., 2022).

Rey, O., Estoup, A., Facon, B., Loiseau, A., Aebi, A., Duron, O., ... & Foucaud, J. (2013). Distribution of endosymbiotic reproductive manipulators reflects invasion process and not reproductive system polymorphism in the little fire ant Wasmannia auropunctata. PLoS One, 8(3), e58467.

Yang, C. C., Yu, Y. C., Valles, S. M., Oi, D. H., Chen, Y. C., Shoemaker, D., ... & Shih, C. J. (2010). Loss of microbial (pathogen) infections associated with recent invasions of the red imported fire ant Solenopsis invicta. Biological Invasions, 12(9), 3307-3318.

Nguyen, D. T., Spooner-Hart, R. N., & Riegler, M. (2016). Loss of Wolbachia but not Cardinium in the invasive range of the Australian thrips species, Pezothrips kellyanus. Biological invasions, 18(1), 197-214.

Nozaki, T., Y. Kobayashi, and S. Shigenobu. "First record of the cedar bark aphid, Cinara cedri cedri Mimeur, 1936 (Hemiptera: Aphidoidea) in Japan, and identification of infecting Wolbachia strains." BioInvasions Records 11.4 (2022): 900-911.

 

Line 347-355

This paragraph seems to be ambiguous. Please clarify.

I could not understand why the authors concluded that “any secondary endosymbionts artificially introduced into M. persicae outside of China are unlikely to compete with secondary endosymbionts already present in field populations”. Moreover, what the unpublished data (from authors’ group?) means is unclear. The third sentence looks contradict to the fourth one.

 

Line 352-355

What is “favorable characteristics?” Favorable for pest control?

 

Line 356-362

I felt that the content of this paragraph had little relevance to the results of the present study. What the unpublished data (from authors’ group?) means is unclear. The authors may add more data, analysis and explanation, or omit this paragraph.

 

Line 375

The authors indicated that there are no Supplemental materials, but I could download a word file. If this is a supplemental data, authors must add some explanation.

Author Response

We appreciate the constructive feedback from the reviewers. We’ve addressed these points and improved the manuscript and look forward to further evaluation. The following is a point-by-point response to the reviewer’s feedback.

Reviewer 2

 

1. In this manuscript, it was unclear why the authors should have to “understand the diversity and regional variation of endosymbionts” in Australia (Line 70-71). In other words, the necessity of understanding the diversity and infection status of secondary symbionts in the pest aphids was not explicitly represented in Introduction and Abstract. Clarifying question of this research will make it easier for a wider reader to understand the content of this paper.

 

The introduction has been amended particularly at lines 73-83.

 

2. I could not well understand why Chinese populations were concluded as native ones. More specifically, which regions in China are most likely? The authors omitted the details in the manuscript, but they may add specific explanations in the Introduction section, if possible.

 

More details and references have been added to clarify this point (lines 63-71).

 

3. The authors discuss the differences in their results from previous studies in Line 250-273. I agree with that secondary symbionts such as Spiroplasma and Rickettsia can be detected more often in China as authors indicated. However, a previous study successfully detected additional endosymbionts including Wolbachia; then, the authors tried to explain the difference from previous reports by the sampling vias and technical sensitivity.

I wonder why the authors did not discuss about the regional and seasonal difference; how different are the sampling points from the previous study? Also, there are some reports wherein the relative abundance of secondary endosymbiont can seasonally change:

Smith, A. H., O’Connor, M. P., Deal, B., Kotzer, C., Lee, A., Wagner, B., ... & Russell, J. A. (2021). Does getting defensive get you anywhere?—Seasonal balancing selection, temperature, and parasitoids shape real‐world, protective endosymbiont dynamics in the pea aphid. Molecular Ecology, 30(10), 2449-2472.

Zhao, D., Zhang, Z., Niu, H., & Guo, H. (2021). Win by quantity: a striking Rickettsia-bias symbiont community revealed by seasonal tracking in the whitefly Bemisia tabaci. Microbial ecology, 81(2), 523-534.

 

 

Contamination is a very common issue in DNA metabarcoding process. The Wolbachia abundance/reads detected in each single sample in Xu et al. were very low. And also, neither further filtering nor PCR validation was conducted. Therefore we do feel that the Wolbachia infection was likely contamination. However, we added some details discussing the difference in sampling points as well as the possibility of seasonal differences (e.g. lines 280-284).

 

 

4. In the present study, the authors successfully detected Spiroplasma and Rickettsia in some Chinese populations. It may be worth to analyze their phylogenetic status. Are they specific to Myzus aphids? Or the same as bacteria from pea-aphids? These would be important for the authors’ argument, “loss of secondary symbiont in invasive regions”. Although the length of the sequence is short (V3-V4 region, ~500 bp), it will be possible to use sequences from 16S amplicon seq to phylogenetic analysis. Here is one of examples:

Nakabachi, A., Inoue, H., & Hirose, Y. (2022). Microbiome analyses of 12 psyllid species of the family Psyllidae identified various bacteria including Fukatsuia and Serratia symbiotica, known as secondary symbionts of aphids. BMC microbiology, 22(1), 1-21.

 

This is a good suggestion and we analysed the phylogenetic status of Rickettsia from both ASV sequences and Sanger sequences (Fig S1). We also analysed the phylogeny of Spiroplasma using the Sanger sequencing data (Fig S2). We then discuss these new results briefly.

 

-Specific comments:

 

Line 181-183, Table 1

I could not figure out “Microsatellite defined clones”. Is this the number of repeats of certain microsatellite loci? Or, just following some previous studies?

 

The clonal numbers (ex: 209, 37, 188) are numbers we use to differentiate clones but these have no direct links to microsatellite loci. The 209 clone is one of the M. persicae “super clones” which carries all the resistance SNPs from kdr, MACE, E4FE4 (Umina et al., 2019).

There is no published information yet on the other M. persicae clones.

Umina, P.A., et al., Escalating insecticide resistance in Australian grain pests: contributing factors, industry trends and management opportunities. Pest Manag Sci, 2019. 75: p. 1494–1506

 

 

Line 238, Figure 2

The authors should explain color label; red and blue seems to be corresponded to Chinese and Australian populations, respectively.

I could not find the definition of “other not endosymbiotic bacteria.” (Graph legend is blurry in my PDF and difficult to read; is this okay?) This should be improved.

I recognized some samples exhibit high rate of “other non-symbiotic bacteria” (for examples, China_Sichuan_Dayand, AUS_Osborne_2, 3). Are they pathogenic bacteria? Or unknown endosymbionts? This is not mentioned and explained in the manuscript.

 

The color label has been explained in the figure legend.  The term of ‘other non-endosymbionts’ has been explained at lines 262-265. ‘Other non-endosymbionts’ include unknown endosymbionts as well as other bacteria. Their taxonomic identities were assigned to ASVs with the SILVA 16S rRNA database.

 

 

Line 255-256, 317-318

Statistical methods and used software should be included in the Materials and methods section.

 

The information on software used has been included in various places (e. g. line 273).

 

Line 337-345

There are some other papers in which investigate endosymbionts both in native and introduced populations. It may be worth to include them. For example, in the little fire ant and RIFA, Wolbachia was rarely detected in the introduced ranges (Rey et al., 2013, Yang et al., 2010). These are similar to introduced populations of thrips (Nguyen et al., 2016). In the aphid Cinara cedri, Wolbachia was detected in the recently introduced population in Japan (Nozaki et al., 2022).

Rey, O., Estoup, A., Facon, B., Loiseau, A., Aebi, A., Duron, O., ... & Foucaud, J. (2013). Distribution of endosymbiotic reproductive manipulators reflects invasion process and not reproductive system polymorphism in the little fire ant Wasmannia auropunctata. PLoS One, 8(3), e58467.

Yang, C. C., Yu, Y. C., Valles, S. M., Oi, D. H., Chen, Y. C., Shoemaker, D., ... & Shih, C. J. (2010). Loss of microbial (pathogen) infections associated with recent invasions of the red imported fire ant Solenopsis invicta. Biological Invasions, 12(9), 3307-3318.

Nguyen, D. T., Spooner-Hart, R. N., & Riegler, M. (2016). Loss of Wolbachia but not Cardinium in the invasive range of the Australian thrips species, Pezothrips kellyanus. Biological invasions, 18(1), 197-214.

Nozaki, T., Y. Kobayashi, and S. Shigenobu. "First record of the cedar bark aphid, Cinara cedri cedri Mimeur, 1936 (Hemiptera: Aphidoidea) in Japan, and identification of infecting Wolbachia strains." BioInvasions Records 11.4 (2022): 900-911.

 

Thank you, that is useful. We’ve now cited some of these papers at lines 383-5. It will be interesting in the future to undertake a comprehensive evaluation of this issue across different insect groups!

 

Line 347-355

This paragraph seems to be ambiguous. Please clarify.

I could not understand why the authors concluded that “any secondary endosymbionts artificially introduced into M. persicae outside of China are unlikely to compete with secondary endosymbionts already present in field populations”. Moreover, what the unpublished data (from authors’ group?) means is unclear. The third sentence looks contradict to the fourth one.

 

This has been clarified. The unpublished data was indeed from our group.

 

Line 352-355

What is “favorable characteristics?” Favorable for pest control?

 

This has been clarified.

 

Line 356-362

I felt that the content of this paragraph had little relevance to the results of the present study. What the unpublished data (from authors’ group?) means is unclear. The authors may add more data, analysis and explanation, or omit this paragraph.

 

We’ve observed quite a few colour morphs in M. persicae nature populations, and our group has recently found that Rickettsiella infection modified M. persicae body colour from light to dark green, hence our interest in this area. The absence of secondary endosymbionts in different colour morphs collected in field is therefore of direct interest to us, and we have mentioned endosymbiont names and provided some more information around our interest in this area (lines 405-406).

 

Line 375

The authors indicated that there are no Supplemental materials, but I could download a word file. If this is a supplemental data, authors must add some explanation.

 

This has been amended.

Reviewer 3 Report

This paper focuses on the diversity of endosymbionts of populations of the green pea aphid, and is an interesting contribution to the field. The study increases our understanding of the distribution of endosymbionts in native and invasive populations of a species.

 

Specific comments

Lines 43-45 clarify that primary symbionts are obligate, while secondary are facultative.

Line 62: What are the approximate years of introduction for other populations relevant to the study?

Line 65: Natural as opposed to clonal laboratory-grown? Please clarify this. Since your study included clones, it would be useful to introduce that idea in this section and state your hypotheses for clonal diversity of symbionts.

Figure 1: Longitude is cut off in the axis label.

Line 194: M. persicae in italics

Figure 3: Please enlarge the text of the figure labels and axis labels. In addition to showing the qPCR results, it would be helpful to show the prevalence data of Rickettsia and Spiroplasma symbionts across geographic regions on a map, similar to Figure 1. Were the four samples in which Rickettsia was detected located close together geographically?

Author Response

We appreciate the constructive feedback from the reviewers. We’ve addressed these points and improved the manuscript and look forward to further evaluation. The following is a point-by-point response to the reviewer’s feedback.

Lines 43-45 clarify that primary symbionts are obligate, while secondary are facultative.

 

This has been clarified.

 

Line 62: What are the approximate years of introduction for other populations relevant to the study?

 

We mention the introduction year for Australia at line 62, we have not been able to locate records of first detections in various states of Australia and other continents but this is also complicated by ongoing gene flow in the species which we now mention at lines 63-71.

 

Line 65: Natural as opposed to clonal laboratory-grown? Please clarify this. Since your study included clones, it would be useful to introduce that idea in this section and state your hypotheses for clonal diversity of symbionts.

 

Laboratory colonies were established from the field collections, we have now clarified this. Our recent studies showed that many of the endosymbionts could be stably maintained in laboratory cultures while others may eventually be lost so we feel it is important to emphasize that we are exploring natural populations as much as possible. However, we also now mention clones at line 88.

 

 

Figure 1: Longitude is cut off in the axis label.

 

This has been corrected.

 

Line 194: M. persicae in italics

 

This has been italicized.

 

Figure 3: Please enlarge the text of the figure labels and axis labels. In addition to showing the qPCR results, it would be helpful to show the prevalence data of Rickettsia and Spiroplasma symbionts across geographic regions on a map, similar to Figure 1. Were the four samples in which Rickettsia was detected located close together geographically?

 

Done. The sampling points of Rickettsia incidence were all in Yunnan province, but these were nevertheless separated by several hundred kilometers.  A map showing the prevalence locations of Rickettsia and Spiroplasma has been added in figure 3.

Round 2

Reviewer 2 Report

The authors responded to my comments and substantially improved the quality of this paper.

One issue: Figure S2 (phylogenetic analysis?) does not appear to be presented.

Author Response

Thanks for this comment. The Supplementary file did have the supplementary figure referred to in the manuscript. We have reattached this file. We suspect that the issue may be line 422 where only Fig S1 is mentioned but the manuscript is correct in this respect.