First Eastern Mediterranean Record of Xenoligophoroides cobitis, the Only Dactylogyrid Monogenean Infecting Mediterranean Gobies: Just Arrived or Missed the Boat?

Round 1

Reviewer 1 Report

The title (“Just arrived or missed the boat? First Eastern .....”) Generates expectations that are not developed in the article.  Just a few lines in the discussion regarding the ideas underlying by Johnson et al. 2003 and is not clear if Xenoligophoroides cobitis just arrived or missed the boat.

Line 32 Lack of sequence data of closely related dactylogyrid monogeneans prevents either proposing a sister-group for Xenoligophoroides

In Genbank 1834 sequences are available for the related Ancyrocephalidae and more than 2400 for Dactylogyridae.

 Moreover in lines 191 -202 author discuss molecular similarity with related monogenean, then a sister-group can be defined.

According to  Dmitrieva et al. (2018)  Xenoligophoroides,  is a member of  Ancyrocephalidae, but a member of Dactylogyridae  (Kmentová et al., 2022). WoRMS also indicated Ancyrocephalidae. Please clarify the status of your material. Such definition will facilitate the inclusion of a sister-group.

Lines 88-90 et al.  measurements followed Sasal et al. [16], whose study was the most recent publication on dactylogyrid parasites of European gobies at the onset of this work...

What about Dmitrieva et al (2018)? refer to the same species.

158-160 Anecdotic and non-related with the scope of this article. Delete.

Table 2. Most importat than the number of sequenced fishes, is the number of parasites per fish. I understand that the article refers to monogenean and not their gobid’s host.

If accession code for Genbank is not available, do not put “xxxxx-xx” in the table.

Nothing about Table 2 in the discussion. If sequences (for fish species) are not analyzed or discussed, delete table 2. Absolutely unneccessary in the scope of this contribution.

Line 257:  this does suggest an influence of host size on the size of the hard parts of X. cobitis

Please explain how the host body size explain size of hard parts of the parasite or is merely a speculation?

Table 3. What about the significance of the differences in measurements?

Line 293: In contrast to the species-rich assemblage of Gyrodactylus on European (sand) gobies, only a single species of Xenoligophoroides is known. This could be due to, for example, a recent colonization of the “Gobius-lineage” from another host lineage, or to “missing the boat” of diversification of these gobies, i.e. absence on the founder populations of diverging goby lineages.

Note that the potential effect of sample effort is not considered. Small sample size does not ensure absence of a parasite. For parasites with prevalence of 5% a sample size of near 60 host specimens is required to ensure one infected fish specimens.

Are all known gobid species sampled for monogenea? Note your comment line 57: Goby parasites also hold  a lot of potential for biodiversity discovery.

 

Author Response

The title (“Just arrived or missed the boat? First Eastern .....”) Generates expectations that are not developed in the article.  Just a few lines in the discussion regarding the ideas underlying by Johnson et al. 2003 and is not clear if Xenoligophoroides cobitis just arrived or missed the boat.

RESPONSE: we do not pretend to solve this question, but merely intend to open up this discussion by offering two explicit hypotheses on the potential mechanism of (lack of) diversification of Xenoligophoroides, as it is a feature that, in our opinion, renders this an enigmatic parasite taxon. We regret of course to have generated unmet expectations. Therefore, we have now moved the “Just arrived…” part to the end of the title, hoping to give the correct impression of an open question rather than of something we promise to solve in this short communication.

Line 32 Lack of sequence data of closely related dactylogyrid monogeneans prevents either proposing a sister-group for Xenoligophoroides

In Genbank 1834 sequences are available for the related Ancyrocephalidae and more than 2400 for Dactylogyridae.

 Moreover in lines 191 -202 author discuss molecular similarity with related monogenean, then a sister-group can be defined.

RESPONSE: we respectfully disagree: describing sequence similarity with the closest matches on GenBank is absolutely not akin to establishing sister-group relations. Only careful phylogenetic analyses can provide information on this.

According to  Dmitrieva et al. (2018)  Xenoligophoroides,  is a member of  Ancyrocephalidae, but a member of Dactylogyridae  (Kmentová et al., 2022). WoRMS also indicated Ancyrocephalidae. Please clarify the status of your material. Such definition will facilitate the inclusion of a sister-group.

RESPONSE: the article by Kmentová et al. (2022, Int. J. Parasitol.) is the most recent and most comprehensive phylogeny of dactylogyrid monogeneans available and we therefore consider it the most authoritative reference at the moment. In it, Ancyrocephalinae is considered a subfamily of Dactylogyridae. As we stated in our manuscript, these authors could not propose a sister-group for Xenoligophoroides, though they considered all dactylogyrid genera of which representatives have been sequenced.

Lines 88-90 et al.  measurements followed Sasal et al. [16], whose study was the most recent publication on dactylogyrid parasites of European gobies at the onset of this work...

What about Dmitrieva et al (2018)? refer to the same species.

RESPONSE: as we intended to explain with the sentence the reviewer cites above, the article by Dmitrieva et al. (2018) was not yet published at the time the measurements for our current study were carried out.

158-160 Anecdotic and non-related with the scope of this article. Delete.

RESPONSE: we respectfully disagree with the referee on the importance of host data in a study like ours. We consider the goby specimens that were deemed free from monogeneans after screening as important “negative records”. Sampling limitations in general lead to many host-symbiont interactions remaining undetected, which hampers understanding of e.g. host-parasite interactions, host-specificity and species richness of parasites (Dallas et al. 2017, PLoS Computational Biology). Therefore it is regrettable that negative results in parasitology are underreported (Cruz-Laufer et al. 2021, Hydrobiologia). We explicitly insist on reporting host specimens that were screened and on which no monogenean ectoparasites were detected: they are part of our sampling effort, and also provide information on infection parameters on various host specimens, host populations and host species.

There are numerous calls from the parasitological and pathobiological community to voucher host specimens to allow further scrutiny (e.g. verification of taxonomic identification) (Thompson et al. 2021, mBio) and this practice can also provide important information to better understand host taxonomy and distribution (Brooks 1993, Journal of Parasitology). Therefore, we consider it crucial to report new observations on the fish species studied in the respective parasitological study, certainly as in the case of our current manuscript, the host specimens were vouchered in a curated ichthyological collection. 

Table 2. Most importat than the number of sequenced fishes, is the number of parasites per fish. I understand that the article refers to monogenean and not their gobid’s host.

RESPONSE: we entirely agree on the importance of infection parameters, which is why they were already reported in the first version of our manuscript, on lines 171-176 (183-186 in the revised version).

If accession code for Genbank is not available, do not put “xxxxx-xx” in the table.

RESPONSE: we typically await the reviewing procedure before depositing sequences to GenBank. Obviously we do not intend to publish without accession numbers. They have now been added on lines 116-118 and Table 2 (hosts) and lines 157-158 (parasites).

Nothing about Table 2 in the discussion. If sequences (for fish species) are not analyzed or discussed, delete table 2. Absolutely unneccessary in the scope of this contribution.

RESPONSE: depositing host voucher specimens and host sequences ensures traceability of the host-parasite relationships discussed in a parasitological study; to allow the verification of the identification of hosts and parasites, and to ensure correspondence between host and parasite data, accession numbers should be mentioned in the respective publications. We consider this good practice and follow in the footsteps of numerous recent studies taking the same approach (e.g. Justine et al. 2013, PLoS ONE; Bouguerche et al. 2019, Parasitology Research; Bouguerche et al. 2020, Parasite; Bouguerche et al. 2021, Parasite; Ayadi et al. 2022, PeerJ). We have now explicitly mentioned the goal of including this data under “3.1 Host records” and have cited one of these references with it.

Line 257:  this does suggest an influence of host size on the size of the hard parts of X. cobitis

Please explain how the host body size explain size of hard parts of the parasite or is merely a speculation?

RESPONSE: thanks for this question. What the exact mechanisms is in this particular case is beyond the scope of this study, and we were inspired by Dmitrieva et al. (2018) in considering this possibility. A mechanism potentially explaining this phenomenon in general, together with some examples from the literature, is now cited on line 273-279.

Table 3. What about the significance of the differences in measurements?

RESPONSE: at the very modest sample size we are dealing with here, we deem formal statistical testing biologically irrelevant and uninterpretable.

Line 293: In contrast to the species-rich assemblage of Gyrodactylus on European (sand) gobies, only a single species of Xenoligophoroides is known. This could be due to, for example, a recent colonization of the “Gobius-lineage” from another host lineage, or to “missing the boat” of diversification of these gobies, i.e. absence on the founder populations of diverging goby lineages.

Note that the potential effect of sample effort is not considered. Small sample size does not ensure absence of a parasite. For parasites with prevalence of 5% a sample size of near 60 host specimens is required to ensure one infected fish specimens.

Are all known gobid species sampled for monogenea? Note your comment line 57: Goby parasites also hold  a lot of potential for biodiversity discovery.

RESPONSE: gobies are in fact a relatively well-studied fish taxon when it comes to their monogenean fauna, lending the suggested differences in species richness on gobies between Gyrodactylus and Xenoligophoroides on gobies some credibility, in our opinion. However we whole-heartedly agree with the referee that gobies do remain undersampled and have underlined this on lines 316-318.

Reviewer 2 Report

General comments

The manuscript entitled "Just arrived or missed the boat? First Eastern Mediterranean record of Xenoligophoroides cobitis, the only dactylogyrid monogenean infecting Mediterranean gobies" (manuscript ID: diversity-1775232) reports the description of a species of ectoparasite in gobies in the Greek territory. 

Although the title suggests the novelty of the work, reading the manuscript shows how the data dates back to 2008, making them unique (according to the available knowledge), but not current. This aspect, which represents a great limiting factor of the manuscript, must absolutely be highlighted in order to contextualize the data, which probably do not represent a picture of the current situation. The authors may consider resubmitting the work following new samplings. 

The data are presented in a truly unclear way: Table 2 shown that almost all the species of gobies are infected by parasites, while the results say the exact opposite, indicating as positive the only species that is negative in the table. It is therefore required to check the results and standardize them. 

There are also doubts about the data obtained following sequencing. The low ID percentages do not allow to arrive at a species determination, so this point must also be reviewed by the authors. 

Specific comments

Table 1: add the expected base pairs (bp) size of the three amplicons.

Line 149: the GenBank accession numbers of your sequences have not been entered. Please provide the numbers. 

Line 156: change "scrutinized" with "checked".

Line 158: write in italics "Gobius couchi".

Table 2: the GenBank accession numbers of your sequences have not been entered. Please provide the numbers. 

Line 170-171: "Among the five goby species studied, only representatives of Gobius cobitis were in- 170 fected by monogenean ectoparasites". In Table 2 you say that only one specimen was sampled, which was even negative. 

Line 248: change "versus" with "compared to".

Line 295: "recent colonization of the “Gobius-lineage”": what is meant by recent?

Author Response

The manuscript entitled "Just arrived or missed the boat? First Eastern Mediterranean record of Xenoligophoroides cobitis, the only dactylogyrid monogenean infecting Mediterranean gobies" (manuscript ID: diversity-1775232) reports the description of a species of ectoparasite in gobies in the Greek territory. 

Although the title suggests the novelty of the work, reading the manuscript shows how the data dates back to 2008, making them unique (according to the available knowledge), but not current. This aspect, which represents a great limiting factor of the manuscript, must absolutely be highlighted in order to contextualize the data, which probably do not represent a picture of the current situation. The authors may consider resubmitting the work following new samplings. 

RESPONSE: we fail to see the relevance of the sampling date when reporting a first record from a certain geographical area: our study deals with phylogeographical and biogeographical patterns, hence considers evolutionary rather than ecological timescales. A difference of 14 years with the present has no implication on the fact that the species and genotypes reported occurred where we report them.

The data are presented in a truly unclear way: Table 2 shown that almost all the species of gobies are infected by parasites, while the results say the exact opposite, indicating as positive the only species that is negative in the table. It is therefore required to check the results and standardize them. 

RESPONSE: Table 2 makes no claims about parasite infections at all, it purely presents data on the number of host specimens that were studied, and the number of host specimens sequenced. We have tried to make the heading and caption of Table 2 clearer now.

There are also doubts about the data obtained following sequencing. The low ID percentages do not allow to arrive at a species determination, so this point must also be reviewed by the authors. 

RESPONSE: As not all extant monogenean species have been sequenced, we have to respectfully disagree with this comment at a general level: a failure to find near-identical sequences on NCBI GenBank has absolutely no bearing on species-level identification of a given parasite. In addition, at the level of this particular study, we do not understand why the referee suggests our species identification is doubtful, as (1) we clearly state on which basis we morphologically identified the worms, citing literature references on lines 190-191, offering photographic evidence (Figure 1) and rendering voucher specimens available for follow-up research by depositing them in a curated collection; and (2) we actually did find highly similar published sequences, and compare these with our new sequences on lines 216-219 of the manuscript, thus providing genetic support for the proposed species identification.

Specific comments

Table 1: add the expected base pairs (bp) size of the three amplicons.

RESPONSE: done.

Line 149: the GenBank accession numbers of your sequences have not been entered. Please provide the numbers. 

RESPONSE: these have now been provided.

Line 156: change "scrutinized" with "checked".

RESPONSE: done.

Line 158: write in italics "Gobius couchi".

RESPONSE: done.

Table 2: the GenBank accession numbers of your sequences have not been entered. Please provide the numbers. 

RESPONSE: these have now been provided.

Line 170-171: "Among the five goby species studied, only representatives of Gobius cobitis were in- 170 fected by monogenean ectoparasites". In Table 2 you say that only one specimen was sampled, which was even negative. 

RESPONSE: Table 2 does not suggest only a single individual of G. cobitis was sampled, nor does this table mention anything about parasite infection. The table shows that 2 specimens of G. cobitis were inspected for infection, and one of them was sequenced for host identification.

Line 248: change "versus" with "compared to".

RESPONSE: done.

Line 295: "recent colonization of the “Gobius-lineage”": what is meant by recent?

RESPONSE: long after the Gobius-lineage diversified. We have now clarified this in the text (line 319).

 

 

Reviewer 3 Report

This is an interesting article which, in my opinion, uses an important approach applying the morphological description and identification followed by sequencing. Some scientists have limited their research to molecular studies only, leaving aside the morphological studies.

The Ms is well written and well documented. The Introduction is clear and focuses on the topic.

The Discussion section is well conducted, well documented and leads to the final hypotheses of the origin of this monogenean species, Xenoligophoroides cobitis, in the giant goby, Gobius cobitis, in the Mediterranean.

 

A minor revision is needed:

-          all scientific names should be in italics;

-          the names of the authors corresponding to the original description of fish or parasite species should be written using “et” and not “and” because the complete taxa of a species is written in Latin;

-          there are some GenBank accession numbers missing;

-          abbreviations of scientific names should be used in a standardised manner.

Author Response

This is an interesting article which, in my opinion, uses an important approach applying the morphological description and identification followed by sequencing. Some scientists have limited their research to molecular studies only, leaving aside the morphological studies.

The Ms is well written and well documented. The Introduction is clear and focuses on the topic.

The Discussion section is well conducted, well documented and leads to the final hypotheses of the origin of this monogenean species, Xenoligophoroides cobitis, in the giant goby, Gobius cobitis, in the Mediterranean.

RESPONSE: we thank the reviewer for the positive appraisal of our study.

A minor revision is needed:

-          all scientific names should be in italics;

RESPONSE: this has now been corrected.

-          the names of the authors corresponding to the original description of fish or parasite species should be written using “et” and not “and” because the complete taxa of a species is written in Latin;

RESPONSE: this has now been changed.

-          there are some GenBank accession numbers missing;

RESPONSE: these have now been added.

-          abbreviations of scientific names should be used in a standardised manner.

RESPONSE: this has been done.

Round 2

Reviewer 1 Report

Dear Authors

I revised the amended MS and I agree with the changes, but allow me to insist, I accept that the information given in table 2 is important, but it should not be in the body of the work, but as supplementary material, this work refers to monogeneans and not to fishes

 

Reviewer 2 Report

The authors responded comprehensively to the comments.