Aquatic Insects in Habitat-Forming Sponges: The Case of the Lower Mekong and Conservation Perspectives in a Global Context

Round 1

Reviewer 1 Report

Specific Comments:

Abstract

-       The abstract is not well written and difficult to follow.

 

Introduction

-       This section is not written well and lacks citations for many sentences. The prose is unclear in many cases, missing citations and the grammar is problematic in many cases and requires more attention syntax.

-       What is the aquatic insect biodiversity like in Thai rivers not so impacted by pollution? Knowing this might be good to compare with their findings. What is the aquatic insect diversity on sponges elsewhere? Knowing this would be good to compare to the Mekong region.  

-       Referring to the numbers of insect orders on sponges, the authors note that 10 orders have been observed, but where, is that globally? Do they mean to specific region?

 

Materials and Methods

Study Site: This section rambles on and includes results information (lines 122-125).

-       What is the range of depths for this river?

-       Were sponges found at deeper depths? Were they even looked for a deeper depths?

Sampling Methods:

-       Again, results (lines 128-130) were inserted but also the grammar is an issue. Fourteen specimens were collected…specimens of what, sponges?

-       When were they collected? This makes a huge difference as to when biodiversity might be the highest.

-       How were sponges physically collected, by hand, cut away with a knife – authors say carefully detached but how? How was sponge size controlled? In other words, did the authors collect sponges of different sizes or within a certain size range to not skew diversity/abundance counts? Some of their taxonomic keys are outdated and geographically wrong, they needed to use more recent identification sources (e.g., Merritt et al 2019 would have been most updated but those taxa are for North America;  Epler 2001 is for Chironomidae of North and South Carolina USA not for eastern Thailand; Morse’s 1994 key was for China). The other keys seem geographically appropriate.  

-       What does medium water level mean? (line 131)

-       What magnification was used in their microscopy?

-       Are there identification keys for Thai tricoptera and Chironomid cases?

-       Why was light intensity not measured, sponges are known to harbor photosynthesizing algae which some of these insects may feed upon, that parameter would have been interesting to know.

-       For a study interested in documenting biodiversity, using light microscopy only on the fractionated remains found in larval insect midguts is a bit superficial, why did they not utilize any molecular techniques to establish maybe a cDNA library of the dietary components found within these insects? Also, did the authors separate the insects into functional feeding groups?

-       Diversity indices used not mentioned, could have had a Statistical Analysis subheading for this information.

 

Results

Water Quality Data:

-       First sentence information should have been in the methods where they mentioned water quality parameters (lines 157-166). In a system such as this, not sure why the authors chose two shallow depths, it is no surprise there was no statistical differences here. The authors state on line 172 shallow and deeper depths but what constitutes a deeper depth, 1m? At what time of day was DO collected, this would make a difference.

-       If data for suspended solids, turbidity, nitrate, and phosphate at different water depths

was not obtained, why include this in the methods?


Aquatic Insect collections:

-       This section is not clearly written at all, needs revised.

-       Why were only 14 sponges sampled?

-       Because the time of sponge collection was not mentioned, it seems like 14 sponges were collected at one time. This one sample date could significantly affect the number of taxa observed on the sponges if some or many were in the adult state. Therefore, a once and done snapshot provides a limited perspective and is a significant flaw in this study.

-       Always spell out generic names initially and especially at the start of sentences, it is impossible to know what genus if not done so.

-       Results are confusingly stated, e.g., lines 183-186 unclearly written.

-       Lines 187-192 probably refer to the order Trichoptera but this is not clear and should be stated after the genera are mentioned here.

-       The prose concerning the Jaccard index findings is very unclear, the authors are not clear on what they were comparing in terms of similarity, was it 0.3m to 1.0m depths? I feel this is what they should have done, calculated a mean Jaccard for each depth and state how similar they were, but as written, it is not comprehendible in terms of what these results may mean.

-       A major issue is for a study documenting biodiversity of aquatic insects inhabiting sponges for this region, to only identify your taxa for the most part to generic level defeats the biodiversity purpose. Authors should have identified all taxa to species level.

 

Gut Content Analysis:

-       If the idea on this analysis was to determine if the insects ate sponges, why do this?

-       The insect diet analyses presented are for only 4 taxa yet 19 were found, why were only four taxa examined? Unclear.

-       These insects feed on a variety of items and clearly not just sponge bits, if the authors looked deeper into their diet, that information may have shed even more light in terms of conservation.

-       The paragraph (lines 219-223) on pupal cases and midge tubes is irrelevant and does not belong here or in the study unless the authors used these to identify any of the taxa in the study.

 

Discussion:

-       This section lacked on overarching connection that explained why this study was critical to understanding the biodiversity of this region, e.g., in the opening paragraph of this section. Instead the authors launched into

-       Section 4.3 discusses results that were not stated in the results or methods. This entire section discusses aspects not mentioned at all before, such as biomasses, correlations of sponge weight and insects, protozoa found e.g., cysts. This information needs to appear earlier, how were they collected, weighed etc.  

-       It seemed like the authors were cherry-picking certain aspects to discuss in depth in this section (entire subheading 4.4) that were not mentioned or superficially mentioned in the results e.g., Sisyridae (2.3% of all taxa). There is a high possibility that the authors sampled when the sisyrids had pupated or emerged as adults, the same for other taxa as well.

-       How do the environmental parameters affect sponges? Since the sponges provide the habitats, how does water quality, DO, flow etc affect them? What about parameters like suspended sediment, this is a silty river right? Have sponge populations declined? 

-       Section 4.5: here there are bits discussed not mentioned in the results, e.g., diatoms in caddisfly guts, why was this not mentioned? How did they identify diatoms?

-       Much of the discussion is arm-waving without support or at least direct support from this study.

Author Response

Response - Reviewer 1

Introduction:

Lines 41-52: We deleted the first paragraph of the introduction.

Line 69: from several regions of the world with the references in lines 72-73.

Material and methods:

Study site:

Line 112: The range of depths for this river varies and the maximum depth in the study site is 8 m.

Line 124-125: We deleted texts and sentences.

• For this study, we did not collect sponge samples from the river bottom.

Sampling methods:

Line 131: Fourteen specimens of sponges Corvospongilla siamensis were collected in February 2008.

Line 138: We deleted the sentence.

Line 139-140: Sponge samples were collected by hand-net. Sponges size/biomass not controlled for this study.

Line 150: This work was conducted in February 2008. We deleted Merritt & Cummins (1996). We used taxonomic keys for identification to genus level. There are no identification keys for Thai Trichoptera and chironomid cages, we used insect samples with their cages for identification.

• We did not focus on light intensity for this study. We did not use molecular techniques for this study.

Results:

Water quality data now as Supplementary material:

• We agree and move the first sentence to Line 165.
• DO data were collected at the same time.
• The deep zone is 1.0 m.

We would like to present the information on suspension solids, turbidity, nitrate, and phosphate.

Aquatic insect collections:

• Sponges samples in the study site at two different water depths (0.3 m and 1 m) were limited, so we collected only fourteen samples for this study.
• We can identify at the genus level.

Gut content analysis:

We would like to to compare with the previous reports from other locations

        - We selected only four dominant taxa to perform gut content analysis.

• We agree on this point: insect diet investigation will be conducted in the further study.
• We mentioned about pupa case and midge tubes in lines 156-159.

Discussion:

• Section 4.3: we deleted the point of sponge weight correlation from this study (line 344).
• Section 4.4: we would like to present this point.
• Lines 348-355: We agree and deleted this part.
• Only water temperature can affect sponges in the study site, but we did not mention it in this study

Section 4.5: diatom was mentioned in the result (line 253). It is a common diatom.

 

Reviewer 2 Report

This manuscript presents the results of a study of the invertebrate fauna on and in sponges colonizing a raft in the Pong River, a tributary of the Mekong in Thailand. The authors have made some interesting observations and extend knowledge of the sponge-associated fauna, and how some insects associate with sponges. However, the manuscript is greatly overwritten and needs to be much better focused on the specific field and laboratory work undertaken in this specific study. The study is based on the sampling of 14 sponges on a raft on a single occasion, so it is not possible to generalize greatly from such a limited base. This applies even more to the single set of water quality measurements. They really provide only background information on the physico-chemistry of the river. My impression is that the present paper represents part of a larger study encompassing more fieldwork given comments about the luxurious and extensive sponge fauna of the river. It may also have its origin in a thesis by the first-named author, in which the context of the present study has been expanded upon extensively. Thus, the present manuscript includes over 130 references which need to be cut back to those particularly pertinent to the current study.

The paper needs a reasonably large amount of attention given to the language and would benefit from the authors using the MDPI English editing service. The Methods and Results sections are the best written, but the Introduction is overly ponderous and needs considerable revision. Some excellent images are included in the paper and most enhance understanding of the work. Although Figures 9 and 10 relate to conservation issues they are the least useful in my opinion. Table 3, the world-wide list of sponge-associated insects is really outside the scope of the study and breaks up its continuity by being included in the body of the text. Nevertheless, it is a valuable compilation, and I suggest it be included as an appendix or supplementary information.

Specific issues

Line 56. I doubt that sponges play a KEY functional role in most rivers around the world, although they may do at your particular study site. I suggest you delete the word ‘key’.

End of Introduction: the rationale for the study and its primary objectives are clearly stated.

Study area: the description emphasizes the variety of things and activities that impinge upon the river and suggest they have negative effects. However, your measurements indicate that water quality is good. I suggest you insert “Nevertheless’ before ‘generally’ in line 99.

Sampling: sponges were photographed and weighed in the field. I gather some at least were photographed in vivo, presumably while still attached to the raft. Clarify. I assume a large and perhaps variable amount of water was present in the sponges when they were weighed. Was this taken into account? I note that no results are presented for sponge weights. Why was that?

Identification of insects: In what way were the North American guides by Eppler (2001) and Merritt & Cummins (1996) useful for the identification of Thai aquatic insects? What levels of identification were accepted using them?

Cluster analysis: The method description needs to state what linkage method was employed and that presence: absence data were used.

Line 153. Indicate here why SEM of caddis cases and chironomid tubes was done.

Lines 156-159. I suggest this short paragraphed be moved forward to line 133 where the name of the sponge is given for the first time.

Line 168. By the two water depths I assume you mean the 7 sponges on the raft and the 7 on the ropes below. Make this clear.

Results: Table 1. I assume the SD is based on the 7 sponge samples. State n = 7 after 7 in the parentheses. If max and min values are included in the table mention them in the caption. The significant difference for DO is less than 0.2 mg/l, i.e., very small and not worth discussing in the depth you have. I suggest you greatly limit discussion of the water chemistry in 4.2.

Cluster analysis. I am not sure how useful the cluster analysis has been. Little is learnt from it and you do not draw out any additional understanding. It is unsurprising that Sponge 1 separates out first as only 2 species were found on it. An interesting question would be why? On Figure 5 what is the upper cluster diagram. If it is to be included it needs to be explained in the caption and discussed in the text.

Table 2. What is meant by Trichoptera fam. gen.?

Gut content analysis: What are the percentage values given? How were they derived? In the Methods section it is stated that gut analyses were qualitative so I assume no counts or other measurements were made.

Discussion: You say that many insects are adapted to inhabit sponges in the Mekong River. A lot of the species you found look as though they would be found on a wide variety of substrata that happen to include sponges. I don’t think inferences of adaptation are needed here. Just say they can inhabit sponges.

Lines 306-318. Greatly downplay or delete this section of discussion.

Line 319. Not results from this study though.

Line 337. Mention of sponge weight correlation. See earlier comment, and include the results if you want to discuss them.

Lines 341-348. Not relevant to this paper. Delete.

Section 4.5. This is one of the most relevant sections of the discussion.

Line 420. 3007 new species of what? Sponges? Unlikely – clarify.

Conclusions: the opening sentence is difficult to follow. Again, I note that sponges can be locally important habitat forming species.

Author Response

Response - Reviewer 2

Specific issues

Line 56: We agree and deleted the word ‘key’

Line 100: We agree and insert ‘Nevertheless’

Line 131: insert “of sponges”

Sampling:

Line 144: Each sponge was photographed in the field. We deleted the point of biomass from this study.

Identification of insects:

Line 150: We deleted Merritt & Cummins (1996), references can use to identify the genus level.

Cluster analysis:

Line 148: average linkage method, Jaccard’s similarity index.

Line 159: Cases of trichopterans and tubes of chironomids were examined and photographed to determine whether these insects use the sponge spicules to construct their cases or tubes as reported for other biogeographic areas.

Lines 161-164: We agree and move the short paragraph forward to line 134

Line 180: to make it clear, we moved the first sentence of 3.1 to line 165.

Line 313-329: We agree.

Results:

Line 189: n = 3 (measurements of water quality parameters)

Table 2: We cannot identify this sample of Trichoptera to genus level because the samples were damaged. Then we use “Trichoptera fam. gen.”

Gut content analysis: the percentage value is the presence of spicules in the guts of the four dominant taxa.

Table 3: We would like to give more information about this point in the text.

Discussion:

Line 275: We agree and modified as  “can inhabit sponges”

Line 319: We agree and deleted the sentence.

Line 343: We deleted the point of sponge weight correlation from this study.

Lines 347-354: We agree and deleted this part.

Line 431: 3,007 new species of vascular plants, fishes, amphibians, reptiles, birds, and mammals.

Conclusions:

Line 470, 476: deleted “key” 

References:

Deleted: Merritt & Cummins, 1996

Reviewer 3 Report

The aquatic insect fauna found on or in freshwater sponges is poorly known and the current paper from the Lower Mekong River should interest river ecologists. However, I think the paper is too long and contains too many details that are not relevant or not justified by the extent of the data. In addition, although the text is generally understandable, there are instances where the English is not idiomatic or is incorrect. This can best be corrected by having the manuscript edited by an English speaker.

The first paragraph of the Introduction (lines 41-52) is hardly needed because these facts are well known. I think this section of the paper should start at the second paragraph. The Methods are generally satisfactory, but the second paragraph (lines 106-109) seems redundant and I do not believe the cluster analysis (lines 147-149) is necessary to elucidate faunal patterns. The water quality data (Table 1) do not need to be given in detail. These are more or less summarised in the text; the table could be placed in an appendix. The heart of the paper is Table 2 and the photos (Figs. 3-9); these give a good impression of the extent of the fauna and how this differs between shallow and deep regions. Thus Fig.5 (cluster analysis) is not needed and does not clarify the distribution of taxa any better than Table 2. The section on gut contents is fascinating but Figs.6 (a,b) showing the gut divisions of a caddis larva are not needed to appreciate Figs 6 (c,d).

The length of the Discussion is not justified by the extent of the results and should be considerably shortened. Delete Table 3; the information it contains is barely mentioned in the text. Section 4.2 (lines 280-327) tries to associate environmental factors with the depth distribution of the fauna. However, too few environmental data are available to be able to do so confidently. Although oxygen levels appear different between 0.3m and 1.0m, the actual difference is so slight that I doubt it has any ecological significance. This section should be reduced to a single paragraph. Section 4.3 contains little that is relevant to this study and should be deleted. Section 4.4 deals with the spongillaflies. These are worth mentioning but again too much detail and too many references are referred to. Shorten this section. Section 4.5 can also be shortened: delete lines 408-417 which are simply speculation. Also delete lines 431-438 (section 4.6) as these have no bearing on the results of this study. Finally, the long section of conclusions is not needed. In fact, I don’t think the Discussion needs to be split into sections and it would be easier to read without them.

The reference list is far too long. Many of these references should be cut. The abstract should also be shortened.

Author Response

Response - Reviewer 3

Lines 41-52: We agree and deleted the first paragraph of the introduction.

Lines 106-109: We agree and deleted the paragraph.

Table 1: We would like to show a detail of the table in the text.

Fig 6 (a,b) We would like to highlight the position of gut divisions of a caddis larva.

Table 3: We removed table 3 to supplementary material.

Section 4.2: We agree, this section was reduced by deleting the lines 289-291, 312-328.

Section 4.3: We agree, this section was reduced by deleting the lines 343-344, 347-354.

Section 4.4: We agree, this section was reduced by deleting lines 370-372, 375-387.

Line 366: insert “Larvae of Sisyra and Xenochironomus had never been found in the macroinvertebrate community along the Pong River (Sangpradub et al., 1998; Hanjavanit & Tangpirotewong, 2007; Sriariyanuwath et al., 2015). These reinforce the opinion that larvae of Sisyra and Xenochironomus in the Pong River are strictly associated with freshwater sponges”

Line 393: insert “and Xenochironomus”

Section 4.5: We agree and deleted lines 419-428.

Lines 431-438: We would like to give more information about this point.

Round 2

Reviewer 2 Report

My main concerns now are:

Title

The title of the paper could be more informative. What is the hotspot? I suggest it be reworded, perhaps more like “Aquatic insects in habitat-forming sponges of the lower Mekong River: diversity and conservation perspectives”

Abstract

The abstract is poorly written and too long, especially its first half. The changes made at lines 23-25 are very poorly expressed.

Table 2

 I still recommend that the current Table 2 be in Supplementary Materials as it is secondary to the actual study. On the other hand, the physico-chemical table (S1) is now in Supplementary materials could be embedded in the main text.

Use of measured physico-chemical data

The data are so limited that it is not possible to draw conclusions about effects on fauna. This passage needs to acknowledge that and delete explanations.

Cluster analysis

In my original report I suggested that the cluster analysis was not particularly useful. What it shows is that most deep samples clustered together as did most shallow ones. So, what can one deduce from that?

References

 Despite changes to the text there are still the same number of references cited. I guess you can live with that.

Other points

Figure 2.  The greenish C. siamensis appears to be yellow.

Line 156. State whether counts or measurements were made. In line 256 it is suggested that relative abundance was determined in some unstated manner.

Line 214. An additional brief comment is needed to explain the cluster. For example, “One cluster comprised the shallow-water samples and the other all but one of those from deer water”.

Table 2 (Line 289) should be in Supplementary Material.

Line 294. You need to state that your environmental data are VERY LIMITED. Because of this you cannot draw conclusions on its effects on the fauna.

Line 351. What is a vesicle rock?

Line 485. The heading should be “Synthesis”

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

This revision has much improved the original submission. As I said in my first review, I think the topic of the paper will interest river ecologists and as it is based on data from a tropical region, where little ecological data is forthcoming, it has added appeal. I still believe it is longer than it needs to be and in my comments below I have indicated where I think the MS could be shortened.

Lines 13-16: the first sentence of the abstract is not very clear. Rather than re-write it I suggest that these lines be deleted and the abstract could start at ‘Shallow water sponges …’ at the end of line 16.

Line 70: insert ‘insect’ between ‘several’ and ‘taxa’.

Line 71: re-write as ‘aquatic insects have been recorded as associated with sponges i.e. …’.

Line 80: delete this line. It is not clearly expressed and adds little.

Line 184: delete ‘the’.

Line 185: re-write as ‘except flow velocity and DO which were significantly higher at 1.0 m’.

Line 213: add after (Figure 7) ‘those from 0.3 m and those from 1.0 m’.

Figure 7: this figure could be simplified by removing the top cluster plot. The matrix is sufficient to indicate which samples the taxa occurred in. The taxon codes must be explained, otherwise the figure is not understandable.

Line 307: delete ‘can’ and replace with ‘could’.

Line 348: delete ‘of’ and replace with ‘in’.

Lines 406- 407: re-write as ‘ their life cycles in tropical sponges of Asia would be worthwhile’.

Line 410: re-write as ‘…spicules. These larvae…’.

Line 458: re-write as ‘neglected fauna is urgently needed.’ The rest of the sentence can be deleted.

Lines 460-461: delete ‘cage’ in both these lines.

Line 473: delete ‘are considered’.

Lines 475-476: re-write as ‘conservation and monitoring of freshwater sponges in tropical Asia are needed to understand their status’.

Lines 485-523: I think most of this last section could be deleted as it adds very little to your conclusions. Keep the last paragraph because this makes some useful suggestions.    

Author Response

Please see the attachment.

Author Response File: Author Response.docx