Cuticular Swabs and eDNA as Non-Invasive Sampling Techniques to Monitor Aphanomyces astaci in Endangered White-Clawed Crayfish (Austropotamobius pallipes Complex)

Round 1

Reviewer 1 Report

The manuscript described a non-invasive sampling techniques to monitor Aphanomyces astaci in crayfish. The experiments design is interesting and the results provide a meaningful technique for disease surveillance on similar species the paper can be published in the journal after minor revision.

1.    Only figure S1 listed in supplementary materials, all the tables can’t be found either in manuscript or supplementary files.

2.    Line 232, the “Crayfish specimens stored frozen at -20 °C since the plague episode were assumed as equally infested”. Why the crayfish haven’t been detected by qPCR to comfirm their infection condition? Furthermore, “Twenty specimens were thawed at room temperature and were virtually longitudinally divided by swabbing each half separately with a sterile cotton swab”,  if the pathogen distribution was not uniformly,  the initial pathogen load will afect the later result?

3.    Line 86-106, the authors mentioned different genotypes A. astaci discovered in different crayfish species, so in present study, which genotype was detected?

4.    Line 521-524, “the efficacy of absolute ethanol was confirmed to store both swabs and glass microfiber filters for up to a week at room temperature, beyond which we recommend storing samples at a refrigerated temperature “,  so why the samples are recommended in absolute ethanol not too long time ? not more than one week?

5.    Line 559, the basis for sufficient number of samples “at least 30 swabs and 4 sampling points for eDNA analysis” ?

Author Response

Thank you for your useful suggestions. We appreciate the time and effort that you have dedicated to providing valuable feedback on our manuscript. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewer, highlighting the changes within the manuscript.
Please see below (and in the attachment) the answers point by point to your specific comments. The revised manuscript has been uploaded with the changes in yellow.

Yours faithfully,
Andrea Basso

(On behalf of authors)

Reviewer 1 comment

1. Only figure S1 listed in supplementary materials, all the tables can’t be found either in manuscript or supplementary files.

Authors answer

We are sorry for the inconvenience. We submitted all the supplementary files together in a ".zip" archive, to be downloaded concurrently with the manuscript. We find that this kind of problem can often be due to incorrect file encoding during compression or uploading, so we would like to encourage all reviewers to contact directly the journal to require the missing files, to get a complete view of the manuscript.

In this case, supporting tables were provided in “.docx” format and reviewer 2 did not declare any problem to revise them. However, now we have included the updated version of all supplementary materials also in “.pdf” format including them among the uploaded files.

Reviewer 1 comment

2. Line 232, the “Crayfish specimens stored frozen at -20 °C since the plague episode were assumed as equally infested”. Why the crayfish haven’t been detected by qPCR to comfirm their infection condition? Furthermore, “Twenty specimens were thawed at room temperature and were virtually longitudinally divided by swabbing each half separately with a sterile cotton swab”, if the pathogen distribution was not uniformly,  the initial pathogen load will afect the later result?

Authors answer

The crayfish were tested when the mortality episode occurred (2014) and the outbreak of crayfish plague caused by Aphanomyces astaci (Genotype A) was confirmed through molecular biology analyses (end-point PCR followed by sequencing and RAPD-PCR). We did not report the results of these analyses in this manuscript, because they were performed before the experiments described in the present study (during LIFE RARITY project), but we indicated in the paper (Line 229) the references of the relative publications [63,67].  We have added some details about the techniques used to clarify the point.

Regarding the second part of the comment, probably yes, with a low level of pathogen, this approach could affect the results, but the 40 swabs were randomly mixed before dividing them into the four groups subjected to different storage conditions to avoid this bias. For clarity, we added this detail to the manuscript, referring to the Simple Random Sampling method (SRS).

In any case, it is not a problem detecting the pathogen in moribund/dead animals, given the large amount of Aphanomyces present and released into the environment during the crayfish plague (WOAH). Instead, the distribution of pathogen in carrier crayfish cuticles is most likely heterogeneous: soft/hard cuticles, melanisation areas, wound edges, etc. In this case, for monitoring purposes, swabbing is useful because it allows testing all surfaces of the specimens instead of small sections of cuticles.

Reviewer 1 comment

3. Line 86-106, the authors mentioned different genotypes A. astaci discovered in different crayfish species, so in present study, which genotype was detected?

Authors answer

With reference to the in-field tests, unfortunately, it was not possible to identify the genotype of A. astaci in the positive samples, because the molecular protocols for genotype identification (i.e. Minardi et al., 2018; Di Domenico et al., 2021) require a higher amount, good integrity and quality of A. astaci DNA (> 10^4 copies/µl) compared to the qPCR targeting ITS region. (Vralstad et al., 2009). Following the suggestion of another reviewer, we chose to summarize that paragraph about the different genotypes of A. astaci, since genotype identification was not performed in the present study.

Reviewer 1 comment

4. Line 521-524, “the efficacy of absolute ethanol was confirmed to store both swabs and glass microfiber filters for up to a week at room temperature, beyond which we recommend storing samples at a refrigerated temperature “, so why the samples are recommended in absolute ethanol not too long time ? not more than one week?

Authors answer

Because, in the present study, we tested the different storage solutions for no longer than a week at room temperature (an appropriate period of time for our sampling purposes). Considering that absolute ethanol gets diluted by the water released from the samples and that, as mentioned in the present manuscript, several studies report DNA degradation when samples are stored in a solution of 70% ethanol [123], we advise a refrigerating temperature for long-term storage.

Reviewer 1 comment

5. Line 559, the basis for sufficient number of samples “at least 30 swabs and 4 sampling points for eDNA analysis” ?

Authors answer

Generally, and especially during monitoring and/or surveillance of a pathogen, sampling should be carried out to provide the best representativeness of the population within the practical constraints imposed by different environments and investigated species. The Central Limit Theorem (CLT) provides the basal guideline to define the number of specimens sampled to achieve adequate coverage.

Swabs sampling

Furthermore, the guideline of the WOAH aquatic manual (CHAPTER 1.4.) provides helpful information to perform sampling and analyses with surveillance purposes, correlating the specificity and sensibility of the assay with a population of infinite specimens. Thus, accordingly to that, assuming a prevalence of the pathogen in a carrier population of 10% (Filipová et al. 2013; Schrimpfet al., 2013) and considering a sensibility of 95% and a specificity of 100% for the qPCR assay to investigate the presence of A. astaci, 30 should be the minimum number to take to avoid false positive in carrier population.

Last but not least, our choice considers (i) the status of A. pallipes populations, which are often limited to a few individuals and/or fragmented along the waterway and (ii) the overnight time needed to perform the monitoring.

• Filipová L, Petrusek A, Matasová K, Delaunay C, Grandjean F (2013) Prevalence of the Crayfish Plague Pathogen Aphanomyces astaci in Populations of the Signal Crayfish Pacifastacus leniusculus in France: Evaluating the Threat to Native Crayfish. PLOS ONE 8(7): e70157. https://doi.org/10.1371/journal.pone.0070157
• Schrimpf, A., Maiwald, T., Vrålstad, T., Schulz, H.K., Śmietana, P. and Schulz, R. (2013), Absence of the crayfish plague pathogen (Aphanomyces astaci) facilitates coexistence of European and American crayfish in central Europe. Freshwater Biology, 58: 1116-1125. https://doi.org/10.1111/fwb.12112
• Chapter 1.4 – Aquatic animal health surveillance. In Aquatic Animal Health Code 2009; Office international des épizooties: Paris, 2009; pp. 1–34. https://www.woah.org/fileadmin/Home/eng/Health_standards/aahc/2009/en_chapitre_1.1.4.htm
• Sheldon M. Ross. "Introductory Statistics," Section 7.4. Academic Press, 2017.
• Chang, H. J., K. Huang, and C. Wu. "Determination of sample size in using central limit theorem for weibull distribution." International Journal of Information and Management Sciences, 17, No. 3. 2006, pp. 153-174.

eDNA sampling

Regarding eDNA sampling, multiple samples for each creek help to overcome inconveniences, i.e. carryover of chloroform or inhibitors, that can occur during the extraction protocols and affect downstream applications. Four sampling points were chosen to have a minimum of independent replicates for the watercourse, increasing both the variability between samples and the chance of detecting the presence of A. astaci.

The use of independent replicates to avoid false negative results and to increase sample diversity was suggested also by other authors (Strand et al., 2019: uses 3 replicates/site; Rusch et al., 2020: uses 2 replicates/site; West et al., 2020: uses 6 replicates/site).

• Strand, D.A., Johnsen, S.I., Rusch, J.C., Agersnap, S.; Larsen, W.B.; Knudsen, S.W.; Møller, P.R.; Vrålstad, T.; Tulloch, A. Monitoring a Norwegian freshwater crayfish tragedy: eDNA snapshots of invasion, infection and extinction. J Appl Ecol. 2019; 56: 1661– 1673. https://doi.org/10.1111/1365-2664.13404.
• Rusch J.C., Mojžišová M., Strand D.A., Svobodová J., Vrålstad T., Petrusek A. (2020) Simultaneous detection of native and invasive crayfish and Aphanomyces astaci from environmental DNA samples in a wide range of habitats in Central Europe. NeoBiota 58: 1-32. https://doi.org/10.3897/neobiota.58.49358.
• West, K.M., Stat, M., Harvey, E.S., Skepper, C.L., DiBattista, J.D., Richards, Z.T., Travers, M.J., Newman, S.J., Bunce, M. (2020). eDNA metabarcoding survey reveals fine-scale coral reef community variation across a remote, tropical island ecosystem. Molecular Ecology, 29, 1069–1086. https://doi.org/10.1111/mec.15382.

We summarized the part above and edited the text highlighting changes in the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

I have read this manuscript with pleasure. The subject is important for the conservation of ICS and the monitoring of A. astaci. I believe that both the results and the thorough methodological approach will be of help to researchers in this field. The methodology and study design are appropriate, clearly presented and discussed, and technically sound.

I have only minor comments.

line 22: I would change to "collect A. astaci DNA" as not only zoosporangia were the source of A. astaci DNA in the samples (as the authors also mention in the manuscript).

line 43: Add "sense stricto" to A. pallipes in this sentence.

line 46: I assume that "they" refers to all Austropotamobius spp. or to all A. pallipes? Please clarify.

line 63: The word "other" should be deleted.

lines 85-100: I would shorten this part about the genotypes of A. astaci, as it is not directly relevant to the results presented.

lines 95-97: Virulence should be analysed by infection experiments. I think this sentence can be deleted.

line 102: NICS usually (but not always) carry A. astaci on the cuticle without showing clinical signs of the disease.

lines 232-234. After this "longitudinal division" of 20 animals and the swabbing, I assume that these 40 halves were somehow randomly mixed to divide them into 4 groups that were subjected to different storage? This could be explained in the text.

lines 253-254: I do not understand why the water samples were taken a month later than the swab samples and not at the same time. There was no need to wait for the results of the swab analyses. Regardless of the reason, I think this fact does not affect the quality of the results obtained and presented.

line 298: For better readability, the authors could refer here to the supplementary material where the (higher) annealing temperature is mentioned.

line 429: 2 swabs

lines 480-507: In Pavić et al. (2020), where cuticular biofilm samples were collected by brushing, the A. astaci load was also similar after invasive and non-invasive sampling. The sample comprised mainly native crayfish species. So this is consistent with the results of this study on A. pallipes (compared to carrier species) and can be mentioned in the discussion. I like the hypothesis that this similarity could be due to the deeper penetration of A. astaci into native species.

Supplementary materials:

I think qPCR and RT-PCR mean the same thing here and there is no reason to distinguish between them.

Table S4: Add "swabs" in the column Samples (next to EtOH, Lysis Buffer, Dry). There is a spelling mistake in the word "quantities".

Author Response

Thank you for your constructive criticisms. We appreciate the time and effort that you have dedicated to providing valuable feedback on our manuscript. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewer, highlighting the changes within the manuscript.
Please see below (and in the attachment) the answers point by point to your specific comments. The revised manuscript has been uploaded with the changes in yellow.

Yours faithfully,
Andrea Basso
(On behalf of authors)

Reviewer 2 comment

line 22: I would change to "collect A. astaci DNA" as not only zoosporangia were the source of A. astaci DNA in the samples (as the authors also mention in the manuscript).

Authors answer

Done; we edited the text highlighting changes in the manuscript.

Reviewer 2 comment

line 43: Add "sense stricto" to A. pallipes in this sentence.

Authors answer

Done; we edited the text highlighting changes in the manuscript.

Reviewer 2 comment

line 46: I assume that "they" refers to all Austropotamobius spp. or to all A. pallipes? Please clarify.

Authors answer

Done; we edited the text highlighting changes in the manuscript.

Reviewer 2 comment

line 63: The word "other" should be deleted.

Authors answer

Done; we edited the text highlighting changes in the manuscript.

Reviewer 2 comment

lines 85-100: I would shorten this part about the genotypes of A. astaci, as it is not directly relevant to the results presented.

Authors answer

Done; we agree with the comment and shorten the paragraph highlighting changes in the manuscript, and reporting below the modified section. However, we think that this part can not be shortened yet to not lose useful details to understand the need for specific procedures for monitoring.

The studies carried out with different marker genes (RAPD, mitochondrial DNA, chitinase) [59-61] on A. astaci isolates reveal five genetic clusters (genotypes A-E) linked to specific host species [58]. Genotype A is related to its first introduction in Europe [39] and has been isolated from native crayfish Astacus sp. [40] and Austropotamobius sp. [41,42]. Furthermore, in some cases, this genotype exhibits a low pathogenicity, persisting with a low prevalence in ICS populations acting as carriers [43]. Further genotypes (B and C) have been isolated in Europe from imported signal crayfish (Pacifastacus leniusculus (Dana, 1852) [44]), while genotype D has been detected in red swamp crayfish (Procambarus clarkii (Girard, 1852) [51]) [39,45, 52,53] and has been described as highly pathogenic in indigenous crayfish [54]. Lastly, genotype E has been found in an introduced population of spiny-cheek crayfish (Faxonius limosus (Rafinesque, 1817) [55]) [56].

Reviewer 2 comment

lines 95-97: Virulence should be analysed by infection experiments. I think this sentence can be deleted.

Authors answer

Done; we edited the text to shorten the paragraph on A. astaci genotypes’ highlighting changes in the manuscript

Reviewer 2 comment

line 102: NICS usually (but not always) carry A. astaci on the cuticle without showing clinical signs of the disease.

Authors answer

Done; we edited the text highlighting changes in the manuscript

Reviewer 2 comment

lines 232-234. After this "longitudinal division" of 20 animals and the swabbing, I assume that these 40 halves were somehow randomly mixed to divide them into 4 groups that were subjected to different storage? This could be explained in the text.

Authors answer

Done; we edited the text highlighting changes in the manuscript, including the reference to the Simple Random Sampling method (SRS).

Reviewer 2 comment

lines 253-254: I do not understand why the water samples were taken a month later than the swab samples and not at the same time. There was no need to wait for the results of the swab analyses. Regardless of the reason, I think this fact does not affect the quality of the results obtained and presented.

Authors answer

The time discrepancy between swabs and eDNA sampling is partly due to the Covid-19 restrictions in force in Italy during the summer of 2020 and partly to logistic reasons (samplings were performed by 2 teams located in different regions). We know that the results of swabs and eDNA are not directly comparable, but we believe that the concordance between the results may still be of great interest.

Reviewer 2 comment

line 298: For better readability, the authors could refer here to the supplementary material where the (higher) annealing temperature is mentioned.

Authors answer

Done; we edited the text highlighting changes in the manuscript

Reviewer 2 comment

line 429: 2 swabs

Authors answer

Done; we edited the text highlighting changes in the manuscript

Reviewer 2 comment

lines 480-507: In Pavić et al. (2020), where cuticular biofilm samples were collected by brushing, the A. astaci load was also similar after invasive and non-invasive sampling. The sample comprised mainly native crayfish species. So this is consistent with the results of this study on A. pallipes (compared to carrier species) and can be mentioned in the discussion. I like the hypothesis that this similarity could be due to the deeper penetration of A. astaci into native species.

Authors answer

Done; we edited the text highlighting changes in the manuscript

Reviewer 2 comment

Supplementary materials:

I think qPCR and RT-PCR mean the same thing here and there is no reason to distinguish between them.

Authors answer

Although qPCR and Real-Time PCR are often used as synonyms by several authors, the terminology indicates a different rank of detail. “Quantitative PCR” (qPCR) should be used to indicate assays where an estimate of the initial quantity, either absolute or relative, is made by comparing the “Ct” between standards (with known quantity) against unknown samples or by normalizing the results based on the signal given by a known gene (reference or housekeeping). On the other hand, “Real-Time PCR” has a broader meaning, referring essentially to a technique in which the level of the amplicons can be evaluated during the run (and not after the end of thermal protocol), but with a higher sensitivity than end-point PCR. Therefore, in this case the results can be interpreted also qualitatively (as presence/absence).

Useful reference:

• Miriam Abbadi, Michele Gastaldelli, Francesco Pascoli, Gianpiero Zamperin, Alessandra Buratin, Giulia Bedendo, Anna Toffan, Valentina Panzarin, Increased virulence of Italian infectious hematopoietic necrosis virus (IHNV) associated with the emergence of new strains, Virus Evolution, Volume 7, Issue 2, December 2021, veab056, https://doi.org/10.1093/ve/veab056

We edited the text highlighting changes in the manuscript in order to clarify this point.

Authors answer

Done; we edited the text highlighting changes in the manuscript

Reviewer 2 comment

Table S4: Add "swabs" in the column Samples (next to EtOH, Lysis Buffer, Dry). There is a spelling mistake in the word "quantities".

Authors answer

Done; we edited the text highlighting changes in the manuscript

Author Response File: Author Response.pdf

Reviewer 3 Report

General comments:

In the paper by Basso et al: “Cuticular swabs and eDNA as non-invasive sampling techniques to monitor Aphanomyces astaci in endangered white-clawed crayfish (Austropotamobius pallipes complex) “, the authors compared the performance of invasive vs. non-invasive sampling in detection of the pathogen A. astaci DNA, propose an optimisation of the sampling protocol and sample storage prior to analysis and finally present the results of a pilot-study designed to test the performance of non-invasive sampling for monitoring the presence of A. astaci in the field.

The introduction is comprehensive and covers in details all important aspects of the work (taxonomy and distribution of the host species, the current state of knowledge about pathogen and disease, as well as pathogen detection methodology) providing therefore very good overview of the subject.

Experimental design and procedures are good presented, and applied methodology is in the line with current standards. The results are clearly presented in Figures 2-5, however, authors also often refer to supplementary tables (Tables S1-S5), which were not included in the provided supplementary material. The manuscript provides valuable guidelines for monitoring A. astaci for conservation purposes and should be published.

Specific comments:

Specific comments are provided in the attached pdf document

Comments for author File: Comments.pdf

Author Response

Thank you for your useful suggestions. We appreciate the time and effort that you have dedicated to providing valuable feedback on our manuscript. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewer, highlighting the changes within the manuscript.
Please see below (and in the attachment) the answers point by point to your specific comments. The revised manuscript has been uploaded with the changes in yellow.

Yours faithfully,
Andrea Basso
(On behalf of authors)

Reviewer 3 comment

line 26: please insert the full name of teh organization

Authors answer

Done; we edited the text highlighting changes in the manuscript

Reviewer 3 comment

line 253: Please explain why eDNA sampling was not performed at the same time as swabbing but one month later? Are the results sufficiently comparable?

Authors answer

Done; we edited the text to clarify the point.

Before to apply the procedure to the creeks we need preliminary results from in-house experiments to choose the right stocking solution and eDNA sampling procedures (i.e. filter porosity, water volume and replicates).

The time discrepancy between swabs and eDNA sampling is partly due to the Covid-19 restrictions in force in Italy during the summer of 2020 and partly to logistic reasons (samplings were performed by 2 teams located in different regions). We know that the results of swabs and eDNA are not directly comparable, but we believe that this does not affect the quality of the presented outcomes.

Reviewer 3 comment

line 269: It is not clear. Three in-field tests?

Authors answer

Done; we edited the text to clarify the point, highlighting changes in the manuscript

Reviewer 3 comment

line 295-296: please reformulate, (like....DNA isolates from cuticles, swabs and water samples were evaluated for the presence of A. astaci DNA)

Authors answer

Done; we edited the text highlighting changes in the manuscript

Reviewer 3 comment

line 238: please give the full name of the genes and indicate that they are mitochondrial.

Authors answer

Done; we edited the text highlighting changes in the manuscript

Reviewer 3 comment

line 347: Maybe you should refere here to Fig 1a?

Authors answer

Done; we edited the text highlighting changes in the manuscript

Reviewer 3 comment

line 390: PCR amplification of the 16S rRNA fragment (16S)

Authors answer

Done; we edited the text highlighting changes in the manuscript

Reviewer 3 comment

line 398: Please refer to Fig 1b.

Authors answer

Done; we edited the text highlighting changes in the manuscript

Reviewer 3 comment

several lines:

• Table S1 is not included in provided supplementary material
• Table S2 is not included in provided supplementary material
• Table S3 is not included in provided supplementary material
• Table S4 is not included in provided supplementary material

Authors answer

We are sorry for the inconvenience. We submitted all the supplementary files together in a ".zip" archive, to be downloaded concurrently with the manuscript. We find that this kind of problem can often be due to incorrect file encoding during compression or uploading, so we would like to encourage all reviewers to contact directly the journal to require the missing files, to get a complete view of the manuscript.

In this case, supporting tables were provided in “.docx” format and reviewer 2 did not declare any problem to revise them. However, now we have included the updated version of all supplementary materials also in “.pdf” format including them among the uploaded files.

Author Response File: Author Response.pdf