A Deep Dive into the Trophic Ecology of Engraulis ringens: Assessing Diet Through Stomach Content and Stable Isotope Analysis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper employs various methods to study the feeding habits of the Peruvian anchovy along the Chilean coast, and discusses the changes in anchovy feeding with latitude, individual size, and its relationship with the physical condition of the fish. It provides a scientific basis for the role of anchovies in the local ecosystem, as well as for the conservation and development of resources, demonstrating a certain level of scientific validity and innovation.Moreover, the paper has some problems. I would suggest minor modification before acceptance.

 

Line 223: The weight of fish is affected by their feeding conditions. It is suggested that the authors supplement the explanation that the weight of the anchovies in this study is net weight or total weight.

Line 306, Part 4.1: The feeding habits of fish are influenced by the external environment, and they generally prioritize prey with high biomass for feeding. In this study, the types of food consumed by anchovies change with latitude. It is recommended that the authors supplement the explanation of the changes in the types of zooplankton and other bait organisms in three different sea areas.

Line 360: Fish feeding is affected by factors such as reproduction. In this study, the authors categorized anchovies into juveniles and adults. Are there any individuals in the reproductive phase? If so, it is suggested that the authors provide additional clarification.

Author Response

We thank reviewers for their constructive comments. In this text we have included the suggested modifications, as well the justification for those comments that we believe are correct and should not be changed. The reviewers' comments are marked "C" and the response to those comments is marked "R" to make it easier to follow in the subsequent round of evaluation.

 

C: The weight of fish is affected by their feeding conditions. It is suggested that the authors supplement the explanation that the weight of the anchovies in this study is net weight or total weight.

R: Line 243. We added clarification about the measurements. In both cases, we used total weight and length.

 

C: The feeding habits of fish are influenced by the external environment, and they generally prioritize prey with high biomass for feeding. In this study, the types of food consumed by anchovies change with latitude. It is recommended that the authors supplement the explanation of the changes in the types of zooplankton and other bait organisms in three different sea areas.

R: Line 339-346. We added an explanation detailing the variations in zooplankton and other prey items across the three fishing areas according to relevant references. Although we do not have environmental data accompanying our samples of anchoveta, we rely on relevant literature to confirm that the Humboldt Current System is dominated by copepods and euphausiids as the main components of the zooplankton community. Also, anchovetas, and small pelagic fish in general, are not caught with bait. Purse seine nets are used to catch them.

 

C: Fish feeding is affected by factors such as reproduction. In this study, the authors categorized anchovies into juveniles and adults. Are there any individuals in the reproductive phase? If so, it is suggested that the authors provide additional clarification.

R: Line 438, 440-442. We clarified that the reference to gonadal maturation applies specifically to adult individuals. Additionally, while our study did not specifically target individuals in the reproductive phase, we indicated that the elevated SMI values in adults suggest they were likely preparing for spawning during the austral summer months. This preparation is associated with increased energy investment in gonadal development and gamete production, as evidenced by the favorable body condition observed in adult individuals in Zone A.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, 

I found your study very interesting, however I suggest to provide more information about the species and have various other comments and suggestion which should be incorporated in the text of the ms - added as a reviewed file below. I suggest to accept the ms after this revisions. 

PH

Here is copy of my comments to editors, as this will not be shown to authors, but I guess can be known:

Dear editors,

Overall, I found this  study very interesting, well prepared and important  from the view of fish ecologist. I believe, the manuscript can gain larger audience, however it should be highly improved in the quality of the presentation of the results and overal of the study itself. 

I understand the motives that lead to presenting the importance of the study, however I think that the study would benefit more from presenting the biology of the species itself, factors that can influence it, and dive deeply into the evaluation the reasons that lead to the use of just such food sources. For example, how large can be fish as adults, how they migrate, why the copepods are a dietary source and what drives the availability of food sources for these fishes. Also, inevitably, Humboldt currents and ElNiňo it must not be forgotten when mentioning the ecological requirements of the species and conditions for the fish. 

What are the most important characteristics of the fishing zones used in this study (A, B, C?). What this may  mean for food sources and the fish themselves? What would happen with various scenarious of climatic changes? This would much more increase the reader's sense that it is important to explore this. This could be presented right away in introduction, and further discussed later. I suggest authors shoul incorporate this into the ms. 

I wonder why authors did not evaluated trophic position of juveniles/adults in the study. This would bring an important message about the structure of trophic interactions in ecosystem.  This will also help to interpret the data, especially the high c:n ration in zone can be more discussed in the light of suggested results. It would help the manuscript to add this analysis, and I hope the authors will do so. 

Discussion:

The results have the potential to provide a part of implications for conservation of marine ecosystems, however in discussion and previously in introduction, the authors do not offer any description or explanation of what their results might mean for the entire ecosystem, and hence, they did not bring any implications for it. This, I guess is their role as an authors.

I like the rest of the discussion. 

Comments for author File: Comments.pdf

Author Response

We thank reviewers for their constructive comments. In this text we have included the suggested modifications, as well the justification for those comments that we believe are correct and should not be changed. The reviewers' comments are marked "C" and the response to those comments is marked "R" to make it easier to follow in the subsequent round of evaluation.

C: l. 21 I wonder why there is not evaluated trophic position of juveniles/adults in the study. It is very easy and can bring important message about the structure of trophic interactions in ecosystem. Moreover, if data on the position of copepods and euphausiids in dN are known according to the presentation of the data. This will also help to interpret the data, especially the high c:n ration in zone can be more discussed in the light of suggested results.

It would help the manuscript to add this analysis. It is inevitable part of evaluation of trophic (isotopic) niches. Please add.

R: We incorporated trophic position data for juveniles and adults across all three zones, with a detailed discussion of the results. The methods can be found between lines 231-237, results in lines 308-310, and the new Table 4 starting at line 311. The discussion of these findings is located in lines 408-428.

Regarding trophic niche evaluations, we decided not to include metrics such as Layman's isotopic niche analysis. The concept of isotopic niche is inherently complex, and its integration could detract from the primary focus of our study. Instead, we opted for more direct approaches that align with the study's objectives, providing clearer insights into species interactions within the ecosystem.

 

C: zones of what - Humboldt Ecosystem?

R:  Line 23 fixed. We specified: Fishing zones of Humboldt Ecosystem in Chilean waters. 

 

C: what are significant implications - What specific recommendations do you have?

R: Line 27-29. We have already mentioned the implications in the text: Implications for fisheries management and conservation efforts, such as the development of targeted management strategies that address variations in the trophic structure of anchoveta across different life stages and fishing zones.

 

C: I would add: Humboldt currents, food webs, peruvian anchoveta, upwelling

R: Line 30. We added anchoveta and food webs.

 

C: ...diet analysis to better predict responses to shifting marine environments.

R: Line 44-45 text fixed.

 

C: latin name in first mention missing

R: Line 49-50 fixed with latin names.

 

C: This is the same as above.

R: Line 53 we deleted the repeated text

 

C: This is already well know, this part of text can be significantly shortened

R: Line 54-65. It is an important part of our work, is stated concisely, and feel it should be retained.

 

C: not only the predator

R: Line 62. We changed predator to individual

 

C: reproductive abilities

R: Line 76 text fixed

 

C: resulting in

R: Line 76 fixed. We changed word resulted by resulting

 

C: This was already mentioned in the previous text and the reader is already convinced of the importance of this approach. I would suggest deleting this from the text

R: Line 81 we deleted the repeated text

 

C: latin names

R: Line 80 fixed with latin name

 

C: please specify

R: Line 91. We specify what energy density means and its unit of measurement

 

C: And what was the best predictor - the traditional or newer indices - which was meant as a goal of the study?

R: Line 93-94 text fixed.

 

C: nutritional health - what does it mean specifically, you should specify this in the beginning of the text - are there some definitions of this health status? If so, it should be provided and cited within the first mention in the text, as this seems to be important factor in this study

R: This definition is mentioned earlier in the text (Lines 69-71).

 

C: This was already mentioned in l. 102

R: Line 102. We deleted the repeated text and relocated the corresponding references.

 

C: l. 113 Are there any differences between juvenile and adult stages in Peruvian coast

R: Line 120-128 we mentioned: there are differences in prey consumption between these life stages.

 

C: l. 122-124 - This was not addressed in this manuscript - so I would suggest to be more careful about this statement and change that accordingly.  Specifically, you evaluated neither physiology, nor you made a comparison between physiology and nutritional health.

R: Line 223-227. We employed a mixing model to assess digestive physiology and have now added a detailed explanation in the methods section to clarify how this analysis was used to trace the assimilation of specific nutrients and energy sources. Our prediction (b) is directly related to digestive physiology, as it examines how variations in the efficiency of the digestive system may influence the isotopic signatures of these populations, particularly in relation to changes in diet and environmental conditions.

 

C: l.127-130 This was already meant - I would focus on the biology of species rather than constantly pointing out that your study is important.

Please provide here more information about the anchoveta - about the area which cover this species, if it is migrant, how long is their lifespan, how many offstpring it produces, how long it is in various stages of their life etc. This is much more important for the reader to understand the biology of species. Also, it would be interesting to meant something about the Humboldt current system and how this may be influenced by El Niňo and climatic changes and how this may influence the species and its prey - this in inevitably the important part of your study, although the diet and health is the aim of your study.

R: Line 107-119. We added biological information about the anchoveta and the influence of El Niño on trophic ecology.

 

C: l. 160 - permanent and seasonal upwelling can be also mentioned, as the zonation of your study is according to it

R: Line 164-170. We already indicated that both Fishing Zone A (n=334) and Fishing Zone B (n=137) are characterized by the presence of highly productive, permanent upwelling throughout the year, while Fishing Zone C (n=464) is characterized by seasonal upwelling with variations in productivity. Additionally, these three fishing zones represent independent populations of anchovetas, each with distinct breeding and feeding areas.

 

C:  Again, this is only the sentence about the importance of the study. This is possible to mean only once for the attentive reader. l. 138-140 - please delete

R: We deleted repetitive text

 

C: l. 132-137 - shift this part of the text to be to line no. 77 next the part about the larval survival

R: Based on the flow of the text, this paragraph is placed here as it discusses an example related to the anchoveta species.

 

C: which one? be more precise, this is important to meant even if this is introduction - this would provide reader better insight and enhance clearness of the text

R: Line 151 fixed. Three different fishing zones in Chilean waters of the GHE

 

C: ? Previously, you meant that C and N isotopes are for the evaluating the position in the food chain etc. - this does not make sense

R: In Line 62-65 we explain SIA and SCA serve as complementary biotracers to assess the diet, more than just evaluating TP.  

 

C: l. 158 - three "zones" (I guess)
R: Line 165 fixed

 

C: l. 164 - Be much more specific within definition of the categories - just to cite a study is not appropriate

R: Line 172-173 fixed. We added an explanation

 

C: l. 174 - grams?
R: Line 182 fixed

C: l. 201 - how many individuals in each category? What was the SL, TL and weight of fish in the study?

R: Information is in table 5.

 

C: l. 205 Pee Dee Belemnite is not commonly used in stable isotope analysis

R: Line 213-216. We clarify the use of international standards. In response to your inquiry, we would like to emphasize that our laboratory adheres to certified standards rather than internal protocols. Specifically, we employ the USGS40 and USGS41a reference materials for stable isotope measurements, rather than the PDB standard.

 

C: l. 233 - There is neither mention, nor methodics about evaluation of plankton in this study - should be added into the text

R: Methodology of plankton identification for SCA is described in lines 180-186

 

C: This is not trophic variation, but dietary variation (analysis)

R: Line 257 we changed trophic to dietary

 

C: Trophic patterns can not be "ontogenetic" or "fishing zones”, I would suggest something like: "Anchoveta trophic niche use in fishing zones and life stages"

R: Line 268 fixed.  We used “Trophic patterns of anchoveta across life stages and fishing zones”

 

C: l. 241-243 Whereas I believe in this trend, do you have any statistical results for this?

R: The trend is presented in Figure 2, and the Bayesian analysis is presented in lines 270-279.

 

C: l.251 Why did you use Bayesian analysis to evaluate these data - according to methodics, you had almost 1 thousand fish stomachs. Hence, it would be enough to evaluate them as they were

R: Irrespective of the number of stomachs, Bayesian analyses offer superior precision compared to frequentist inferences. Furthermore, the Bayesian approach prioritizes assessing the credibility or plausibility of the hypothesis rather than making a binary decision of rejection or non-rejection regarding the examined problem. This stems from the decision rule inherent in Bayesian analysis, which relies on calculating the posterior probabilities of the contrasted hypotheses, with the evaluation contingent upon the obtained results. The conceptual advantage of this approach lies in the fact that these posterior probabilities represent the true (subjective) probabilities of the hypotheses, reflecting the observed data and the prior distribution.

 

C: In Zone A, the weight importance of phytoplankton is high in comparison to B and C zones. This should be presented (and discussed).

R:  We presented the results in line 260-261 and discussed in lines 334-335 and 411-413.

 

C: l. 276 - F1,6 - I wonder about the evaluation of zones? Degrees of freedom are "1", however zones were 3.

R: Line 296 fixed

 

C: Body condition in various fishing zones and life stages

R:  Line 313 fixed

 

C: Anyway, how large were juveniles and adults in different fishing zones + S.D. should be provided in results. This my inevitably play an important role.

R: We provided this information in Table 5. 

 

C: l. 293-294 - This seems to me not to be true. When looking to the table, it more looks like SMI in zone A (11.8) differ from the zone C and D, which are quite similar (10.8 and 10.7) in juveniles. Similarly, in evaluation of SMI in adults (l. 295-296), SMI in zone A (11.16) differ from B and C (9.63 and 9.87).  Or the results in table are misleading.

R: Line 318, 320 corrected

 

C: l. 304 and everywhere across the manuscript - I would suggest to use the name of indices (SMI), rather than "nutritional health". This seems to me misleading.  I would use "trophic status", "scaled mass index" or "body condition index.

R: The body condition index is used to assess the overall physical state of an organism, reflecting its energy reserves and general health. For this reason, we use the term "nutritional health" in our manuscript.

 

C: l. 310 - plants assimilate, animals feed!

R: Line 333 fixed.

 

C: l. 311-312 This is little bit a grey literature. I understand why it is used, however if they are used, the results and outcomes of the studies should be precisely provided and interpreted.

Hence, what I do not understand is the evaluation and interpretation of author of this ms - in Introduction you stated: "Studies show that the diet of anchoveta varies between juvenile and adult stages, 110 with both predominantly consuming zooplanktonic organisms such as copepods and 111 euphausiids [20-23]. Notably, research along the Peruvian coast has shown a low representation of 113 phytoplankton in their diet [20-21].

So, it does not make me sense why you provide that "phytoplankton played a more prominent role...".  Please present cited works better, more precisely in introduction and compare better in discussion OR do not use them.

R:  We revised the text to clarify how our results differ from some previous studies.

 

C: l. 315-320 These are just empty word...

There is anything about anchoveta distribution in this manuscript, nor you did not address species composition in the community...  Concerning "Important implications for the understanding the trophic dynamics" - yes, your results have the potential to provide a part of implications, however here in discussion and previously in introduction, you do not offer no description or explanation of what your results might mean for the entire ecosystem, and hence, you did not bring any implications. This, I guess is your role as an authors, if you will not do this work, you do not provide nothing than the raw data, which is not enough.

R: Line 360-368. We added a paragraph to clarify the implications of our ms in addition to the paragraph about anchoveta biology lines 107-119.

 

C: l. 321 - The results of your mixing models did not bring any information about the correlation between latitudinal variation and the composition of the diet - this is highly misleading, as 1) the mixing models are not incorporated in your study, and 2) MM are moreover not about the correlation.  In case of the chapter "trophic variation in zone" was meant, there is even no statistic evaluation for this statement.

R: Line 223-227. We added a paragraph to explain the methodology.
1) The mixing models in Table 3 were implemented through the R package SIMMR to estimate the assimilation proportions in the tissues based on stomach content observations, using data from SCA (stomach content analysis) and SIA (stable isotope analysis).

2) Using these mixing models, we were able to compare the main prey items through a PERMANOVA analysis. The results are presented in lines 292-294.

 

C: l. 323 - This is only a speculation. You did not evaluated the abundance of phyto/zooplankton - or provide citation.

R:  Line 339-346 we added a paragraph and references to explain changes in the trophic structure.

 

C: increased?

R: Line 449-442 text corrected.

 

C: l. 390 - some citation on resource partitioning would be valuable

Also, effect of learning to capture a prey may be important.

It would be interesting to provide some information about how schools of the anchoveta are formed - are there shoals of juveniles/adults or mixed shoals of both age category?

R:  Line 460 we provided a reference

Line 462-470 we added a paragraph and citation regarding the schools formed by anchovetas.

 

C: l. 395-396

How specifically this information will help for sustainable use of anchovetas fishing?

R: Line 475-482 We added a paragraph clarified the significance of our results for sustainable fisheries

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

 This paper analyzes the feeding habits and nutritional status spatial distribution characteristics of anchoveta using both stomach content analysis (SCA) and stable isotope analysis (SIA). It is considered to provide reliable results by performing SCA and SIA analyses on a very large number of specimens. The paper is very well-structured, and it summarizes and organizes the quantitative comparison results based on statistical analysis very well, so it seems that there are no major revisions necessary.

Even in its current state, this paper is deemed to have sufficient value for publication, but if possible, supplementing the following aspects might provide more detailed information:

1. Trend of Anchoveta's Condition Factor: In this paper, the health status of fish individuals is compared using the scaled mass index of the length-weight relationship. Currently, comparisons are made simply between juveniles and adults, but given that a large amount of data on the specimens measured in this study has been accumulated, it might reveal more detailed trends if the weight distribution trend according to the length of anchoveta is shown and compared by region.

2. Prey Availability: As emphasized in this paper, selective feeding (copepods vs. euphausiids) is presented as a very important factor in maintaining a healthy population. The proportion of major prey in the stomach contents is well organized and presented, but was information on the abundnace and species composition of prey (zooplankton) collected in the same area also investigated? Information on the prey in the environment (at least the proportion of major prey) can be very crucial in explaining the feeding characteristics by region claimed in this paper, and it is expected that it would be possible to calculate the prey selectivity index for direct comparison. (Quantitative collection of zooplankton is generally conducted during research cruises, so if there is data, it might be suggested for use as supplementary material.)

3. Interpretation of SIA Values: SIA is essential for quantitatively interpreting the contribution rate of food sources and the overall food web structure. This paper also compares the contribution rates of copepods and euphausiids based on SIA analysis.

   However, in the SIA values presented in Figure 3 and Table 2, there is a difference of more than 5‰ between the prey items and anchovetas. Generally, in the same food web, the carbon isotope signal shows a difference of 1-2‰ between prey and predator, but in this study, there is a very large difference.

   Moreover, in the case of nitrogen isotope, in Zone B, anchovetas show a trophic level more than two levels higher than copepods and euphausiids (10.10, 7.3‰, respectively), with values up to 19‰. Assuming the nitrogen isotope signals of copepods and euphausiids are herbivore levels, the nitrogen isotope values for anchovetas essentially appear to be carnivore levels. From my personal experience, anchoveta species always show high nitrogen isotope values (sometimes higher than sharks), making interpretation difficult, and similar trends are observed in this study. If the authors can explain this phenomenon, it would greatly help in interpreting the marine food web including anchovetas. (This comment is not questioning the SIA values of this study but is a request for additional explanation if possible, however, it is not mandatory to include it.)

Author Response

We thank reviewers for their constructive comments. In this text we have included the suggested modifications, as well the justification for those comments that we believe are correct and should not be changed. The reviewers' comments are marked "C" and the response to those comments is marked "R" to make it easier to follow in the subsequent round of evaluation.

 

C: Trend of Anchoveta's Condition Factor: In this paper, the health status of fish individuals is compared using the scaled mass index of the length-weight relationship. Currently, comparisons are made simply between juveniles and adults but given that a large amount of data on the specimens measured in this study has been accumulated, it might reveal more detailed trends if the weight distribution trend according to the length of anchoveta is shown and compared by region.

R: Although analyzing the weight distribution trend relative to length in each region could provide detailed insights into the condition of anchovetas, separating individuals into juveniles and adults is more pertinent for studies on recruitment versus reproduction, which is critical for effective fisheries management and analysis.

 

C: Prey Availability: As emphasized in this paper, selective feeding (copepods vs. euphausiids) is presented as a very important factor in maintaining a healthy population. The proportion of major prey in the stomach contents is well organized and presented, but was information on the abundance and species composition of prey (zooplankton) collected in the same area also investigated? Information on the prey in the environment (at least the proportion of major prey) can be very crucial in explaining the feeding characteristics by region claimed in this paper, and it is expected that it would be possible to calculate the prey selectivity index for direct comparison. (Quantitative collection of zooplankton is generally conducted during research cruises, so if there is data, it might be suggested for use as supplementary material.)

R: Unfortunately, we don't have data on the zooplankton from the cruises on which we collected these samples.

 

C: Interpretation of SIA Values: SIA is essential for quantitatively interpreting the contribution rate of food sources and the overall food web structure. This paper also compares the contribution rates of copepods and euphausiids based on SIA analysis.

However, in the SIA values presented in Figure 3 and Table 2, there is a difference of more than 5‰ between the prey items and anchovetas. Generally, in the same food web, the carbon isotope signal shows a difference of 1-2‰ between prey and predator, but in this study, there is a very large difference.

R: The large δ13C difference may result from factors like regional variations in isotopic baselines or differences in prey isotopic signatures, but these do not affect the study's conclusions. The distinction between copepods, euphausiids, and anchovetas across regions remains clear despite this variability.

 

C: Moreover, in the case of nitrogen isotope, in Zone B, anchovetas show a trophic level more than two levels higher than copepods and euphausiids (10.10, 7.3‰, respectively), with values up to 19‰. Assuming the nitrogen isotope signals of copepods and euphausiids are herbivore levels, the nitrogen isotope values for anchovetas essentially appear to be carnivore levels. From my personal experience, anchoveta species always show high nitrogen isotope values (sometimes higher than sharks), making interpretation difficult, and similar trends are observed in this study. If the authors can explain this phenomenon, it would greatly help in interpreting the marine food web including anchovetas. (This comment is not questioning the SIA values of this study but is a request for additional explanation if possible, however, it is not mandatory to include it.)

R: Lines 382-393: We provided an explanation for the highly enriched δ15N values observed in anchovetas from Zone B, attributing this enrichment to the region's increased upwelling intensity. It is important to note that both δ13C and δ15N exhibited large standard deviations, reflecting considerable variability in the data.

Lines 408-428: Additionally, we incorporated the trophic position (TP) of anchovetas to further clarify this pattern.

Author Response File: Author Response.pdf