Thermal Limits of the Estuarine Amphipod Melita palmata Under Different Salinities and Its Relevance for Aquaculture Production

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review for the paper “Thermal limits of the estuarine amphipod Melita palmata under different salinities and its relevance for aquaculture production” by Luísa Marques, Daniela P. Rodrigues, Rafael C. Duarte, Ricardo Calado submitted to “Animals”.

 

This research paper investigates the physiological interplay between salinity and thermal tolerance in the estuarine amphipod, Melita palmata. The authors conducted an analysis of the critical thermal maximum of individuals collected from distinct sites within a coastal lagoon system, characterised by varying salinity regimes. They found that amphipods originating from and acclimated to the lowest salinity conditions exhibited a significantly reduced upper thermal limit compared to conspecifics from more marine-influenced sites. A key finding was the absence of significant sex-based differences in thermal tolerance, contrasting with patterns observed in other amphipod taxa. The results indicate that the prevailing environmental salinity, rather than individual sex or size, is a primary determinant of thermal sensitivity in this species. The results of this study may have important implications for predicting species vulnerability within rapidly changing estuarine ecosystems.

 

Introduction

 

L 101-102. I think the authors should clarify how Melita palmata manages osmoregulation.

 

Materials and Methods

 

Section 2.2. I would suggest that the authors include details about the annual temperature patterns in the study area. It would also be helpful to explain why they chose January to investigate upper thermal limits. Could they share the salinity and temperature readings during collection?

 

L 168-169. It is unclear why the ionic makeup of this synthetic seawater is suitable compared to natural seawater. The authors should definitely outline that.

 

L 174. The authors need to explain why they thought this acclimation period was enough. Did the authors check stress response indicators in the amphipods during that time?

 

L 188. I would suggest the authors provide a clear, operational definition for what they mean by "loss of motor coordination".

 

Results

 

L 235. It would be great to get some morphometric information on the amphipods used in the experiments. Were these adults? What about their physiological state and reproductive status?

 

In my opinion, the authors' presentation of results does not clearly address the covariate’s effects. I think they should update the text.

 

Discussion

 

I think it would be insightful for the authors to talk about whether the observed trend of osmotic stress decreasing thermal resilience is more about acclimation (plasticity) or local adaptation.

 

L 316-318. It is unclear why the lack of any significant sex effect in this study might differ from what’s found in other amphipod species. The authors should explore specific life-history or ecological traits of M. palmata that might shed light on this.

 

L 349-353. The authors should provide evidence from their sites or the literature to support these ecological explanations for the higher BCI (absence of direct competitors" and "increased food availability).

Author Response

Reviewer #1

This research paper investigates the physiological interplay between salinity and thermal tolerance in the estuarine amphipod, Melita palmata. The authors conducted an analysis of the critical thermal maximum of individuals collected from distinct sites within a coastal lagoon system, characterised by varying salinity regimes. They found that amphipods originating from and acclimated to the lowest salinity conditions exhibited a significantly reduced upper thermal limit compared to conspecifics from more marine-influenced sites. A key finding was the absence of significant sex-based differences in thermal tolerance, contrasting with patterns observed in other amphipod taxa. The results indicate that the prevailing environmental salinity, rather than individual sex or size, is a primary determinant of thermal sensitivity in this species. The results of this study may have important implications for predicting species vulnerability within rapidly changing estuarine ecosystems.

Reply: The authors acknowledge the positive feedback from Reviewer #1.

 

Introduction

L 101-102. I think the authors should clarify how Melita palmata manages osmoregulation.

Reply: The reviewer’s suggestion was carefully considered, and the following paragraph has been incorporated into the manuscript, and now reads as follows: “As other amphipods, tolerance to salinity changes is linked to active ionic regulation, including increased gill Na⁺/K⁺-ATPase activity [50], adjustments in ion and water fluxes [51], and the involvement of gill ionocytes in Na⁺ and Cl⁻ uptake [52].” (Lines 119 - 122). The following references were included in this section:

50. Brooks, S.J.; Lloyd Mills, C. Gill Na+, K+-ATPase in a Series of Hyper-Regulating Gammarid Amphipods. Enzyme Characterisation and the Effects of Salinity Acclimation. Comp Biochem Physiol A Mol Integr Physiol 2006, 144, 24–32, doi:10.1016/J.CBPA.2006.01.023.
51. Brooks, S.J. The Osmoregulation of Selected Gammarid Amphipods; 2003.
52. Henry, R.P.; Lucu, Č.; Onken, H.; Weihrauch, D. Multiple Functions of the Crustacean Gill: Osmotic/Ionic Regulation, Acid-Base Balance, Ammonia Excretion, and Bioaccumulation of Toxic Metals. Front Physiol 2012, 3 NOV, 36402, doi:10.3389/FPHYS.2012.00431.

Materials and Methods

Section 2.2.

I would suggest that the authors include details about the annual temperature patterns in the study area.

Reply: We appreciate this suggestion. To address this, we have added a table summarising the annual temperature patterns in the study area, including the mean and standard deviation for each site and tidal condition across all seasons. These values derive from our own year-round measurements, not from previously published sources. This table has been included in the supplementary materials as Table S1, providing a clear overview of the thermal regime experienced throughout the year. In the main manuscript, the corresponding reference to this table now reads: “Additional details on annual water temperatures (mean ± SD) across seasons and tidal stages for the three sampling sites are presented in Table S1.” (Lines 188 - 189).

 

It would also be helpful to explain why they chose January to investigate upper thermal limits.

Reply: We thank the reviewer for their suggestion, and to best accommodate this request, we added to the manuscript the following information that now reads: “January was chosen because it represents one of the coldest periods of the year, when organisms are likely acclimatised to the lowest ambient thermal conditions. Under these conditions, organisms are particularly suitable for upper-thermal-limit trials, as using winter-collected individuals will maximize the contrast between their acclimation baseline and the elevated temperatures applied during the experiment, thereby enhancing the ecological and physiological relevance of the results. This approach aligns with evidence showing that, in aquatic ectotherms, thermal tolerance is strongly shaped by recent thermal history and acclimation temperature [60]. Seasonal plasticity has also been widely reported, where organisms collected during colder periods tend to exhibit lower baseline temperatures and broader thermal safety margins, which makes their responses to heat-stress trials more pronounced and enhances the realism of assessments under extreme warming scenarios [60,61]. By performing experiments on winter-collected organisms, we minimize the effects of prior warm acclimation and better simulate extreme warming events, providing a more accurate estimate of species’ upper thermal limits and vulnerability to climate-induced thermal stress.” (Lines 159 - 174). The following references were included in this section: 

60. Waterbury, C.R.; Sutton, T.M.; Kelley, A.L.; López, J.A. Effects of Temperature Acclimation on the Upper Thermal Tolerance of Two Arctic Fishes. Conserv Physiol 2024, 12, doi:10.1093/CONPHYS/COAE001.
61. Hopkin, R.S.; Qari, S.; Bowler, K.; Hyde, D.; Cuculescu, M. Seasonal Thermal Tolerance in Marine Crustacea. J Exp Mar Biol Ecol 2006, 331, 74–81, doi:10.1016/J.JEMBE.2005.10.007.

Could they share the salinity and temperature readings during collection?

Reply: We thank the insightful suggestion. We have added a table summarising the mean and standard deviation of the environmental conditions recorded at each sampling site on the sampling day. This table is now provided as supplementary materials as Table S2, offering a clear overview of the salinity and temperature conditions at the time of collection of the specimens. In the main manuscript, the corresponding reference to this table now reads: “Salinity and temperature were measured in situ, with three replicate measures being recorded for each parameter at each sampling site. (Table S2)” (Lines 192 - 194).

L 168-169. It is unclear why the ionic makeup of this synthetic seawater is suitable compared to natural seawater. The authors should definitely outline that.

Reply: We thank the reviewer for their suggestion. Most CTmax studies that use local water involve freshwater amphipods, where ionic variability is less influential. In contrast, thermal-tolerance studies on marine and brackish crustaceans routinely employ salinity-controlled or synthetic media to minimize ionic drift during experiments. Therefore, and to best accommodate this request, we added to the manuscript the following information to clarify this point that now reads: “Synthetic water was used to maintain a constant and reproducible salinity throughout the trial [67–69], ensuring that individuals from each sampling site were kept at the salinity corresponding to their site of origin” (Lines 223 - 225). The following references were included in this section:

67. Morritt, D.; Ingólfsson, A. Upper Thermal Tolerances of the Beachflea Orchestia Gammarellus (Pallas) (Crustacea: Amphipoda: Talitridae) Associated with Hot Springs in Iceland. J Exp Mar Biol Ecol 2000, 255, 215–227, doi:10.1016/S0022-0981(00)00299-9.
68. Missionário, M.; Bertalan, R.; Calado, R.; Dupont, S.; Costa, P.M.; Vinagre, C.; Madeira, D. Living on the Edge: Temperature and Salinity Performance Curves across Levels of Biological Organization in a Shallow Water Shrimp. Mar Environ Res 2026, 214, 107749, doi:10.1016/J.MARENVRES.2025.107749.
69. Collins, M.; Truebano, M.; Spicer, J.I. Consequences of Thermal Plasticity for Hypoxic Performance in Coastal Amphipods. Mar Environ Res 2022, 177, doi:10.1016/j.marenvres.2022.105624.

L 174. The authors need to explain why they thought this acclimation period was enough. Did the authors check stress response indicators in the amphipods during that time?

Reply: We thank the reviewer for this remark, and to best accommodate this request, we added to the manuscript the following information that now reads: “The 24-hour acclimation period was considered appropriate to allow individuals to recover from handling and transport and to verify that only healthy specimens were used in the CTmax trial. This procedure ensures that any observed responses during the CTmax trial reflect natural thermal limits rather than transient effects from collection or transport.” (Lines 219 - 223). With this addition, the previous sentence: “Given these near-natural conditions, a 24-hour acclimation period was considered appropriate, as the amphipods were exposed to minimal osmotic stress relative to the location where they were collected.” was deleted from the revised version to avoid redundancy.

 

L 188. I would suggest the authors provide a clear, operational definition for what they mean by "loss of motor coordination".

Reply: We thank the reviewer for this observation and to accommodate it best we added to the revised manuscript and now reads as follows: “The endpoint (Tend-point) was defined as the temperature at which an individual no longer maintained equilibrium, exhibiting disorganized or ineffective movements and failing to right itself when gently disturbed, marking its critical thermal threshold, with water temperature at this specific moment being recorded.” (Lines 240 - 243)

 

Results

L 235. It would be great to get some morphometric information on the amphipods used in the experiments. Were these adults? What about their physiological state and reproductive status?

Reply: We appreciate the reviewer’s observation. To address this point, we have added the following clarification to the main text: “All amphipods used in the CTmax experiment were healthy and active adults, as confirmed after the 24-h acclimation period. Ovigerous females were intentionally excluded to avoid behavioural and physiological biases associated with brooding. The final dataset consisted of males and non-ovigerous females from all three sampling locations, with the mean TL and DW of each group being summarized in Table S3.” (Lines 291 – 295)

In my opinion, the authors' presentation of results does not clearly address the covariate’s effects. I think they should update the text.

Reply: We thank the reviewer for this suggestion. We agree that the influence of the covariate (dry weight, DW) on CTmax required a clearer presentation. The revised text clarifies the role of the covariate in the ANCOVA framework. We added further explanation to the manuscript, and it now reads as follows: “The covariate DW did not significantly affect CTmax (F_1,53 = 3.80, p = 0.057), indicating that thermal limits were independent of individual body mass.” (Lines 323 - 325).

 

Discussion

I think it would be insightful for the authors to talk about whether the observed trend of osmotic stress decreasing thermal resilience is more about acclimation (plasticity) or local adaptation.

Reply: Thank you for this valuable suggestion. We agree that clarifying this duality strengthens the interpretation of our results. To best accommodate this request, we added to the manuscript new information that reads as follows: “Although our design cannot disentangle short-term plasticity from long-term adaptation, both remain plausible explanations for our findings. Future studies on this topic can be specifically designed to answer whether the observed trend of osmotic stress decreasing thermal resilience is a result of acclimation (plasticity) or local adaptation. Salinity and temperature interact to affect metabolism, ion regulation, and thermal tolerance in estuarine invertebrates. For instance, amphipods from low‑salinity habitats have shown lower heat resistance, likely reflecting the energetic costs of osmoregulation under hypoosmotic conditions [37]. At the same time, some crustacean populations exhibit distinct tolerance phenotypes linked to their environmental conditions or thermal regimes, suggesting that population‑level divergence may arise under spatially heterogeneous habitats [81,82].” (Lines 359 – 369). The following references were included in this section:

37. Vereshchagina, K.P.; Lubyaga, Y.A.; Shatilina, Z.; Bedulina, D.; Gurkov, A.; Axenov-Gribanov, D. V.; Baduev, B.; Kondrateva, E.S.; Gubanov, M.; Zadereev, E.; et al. Salinity Modulates Thermotolerance, Energy Metabolism and Stress Response in Amphipods Gammarus Lacustris. PeerJ 2016, 4, doi:10.7717/PEERJ.2657.
38. Leong, W.; Sun, P.Y.; Edmands, S. Latitudinal Clines in Temperature and Salinity Tolerance in Tidepool Copepods. J Hered 2017, 109, 71–77, doi:10.1093.
39. Lou, F.; Han, Z.; Gao, T. Transcriptomic Responses of Two Ecologically Divergent Populations of Japanese Man-tis Shrimp (Oratosquilla Oratoria) under Thermal Stress. Animals 2019, Vol. 9, Page 399 2019, 9, 399, doi:10.3390/ANI9070399.

L 316-318. It is unclear why the lack of any significant sex effect in this study might differ from what’s found in other amphipod species. The authors should explore specific life-history or ecological traits of M. palmata that might shed light on this.

Reply: We thank the reviewer for this observation. Unfortunately, no studies have directly examined sex differences in Melita palmata, so these explanations are inferred from related species. Nonetheless, we added to the manuscript information that will further clarify the potential reasons for the lack of a sex effect, and it now reads: “In the present study, ovigerous females were not included, and sexual size dimorphism in M. palmata is relatively small, in comparison to other amphipod species already investigated, which may explain the absence of a detectable sex effect. Additionally, both males and females inhabit the same highly variable estuarine environment, likely experiencing similar selective pressures on thermal tolerance [86].” (Lines The following reference was included in this section:

86. Premate, E.; Fišer, Ž.; Biró, A.; Copilaş-Ciocianu, D.; Fromhage, L.; Jennions, M.; Borko, Š.; Herczeg, G.; Balázs, G.; Kralj-Fišer, S.; et al. Sexual Dimorphism in Subterranean Amphipod Crustaceans Covaries with Subterranean Habitat Type. J Evol Biol 2024, 37, 487–500, doi:10.1093.

 

L 349-353. The authors should provide evidence from their sites or the literature to support these ecological explanations for the higher BCI (absence of direct competitors" and "increased food availability).

Reply: We thank the reviewer for this comment. These ecological factors were not measured at our study sites, and no studies from Ria de Aveiro specifically address this topic, so we cannot confirm their influence without being too speculative. The suggested explanations for the higher BCI (fewer competitors or greater food availability) remain plausible hypotheses that certainly warrant further investigation to be either accepted or refuted.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

This study examines the Critical Temperature Maximum (CTmax), a common experimental method for assessing the upper temperature limit, normal organism functioning, and comparative resistance between species under given conditions. In this study, the method was applied to one amphipod species, a potential aquaculture target, under varying water salinity.

The authors write that they tested whether energy requirements related to osmoregulation affect their thermal stability and whether this relationship differs between the sexes, potentially reflecting sex-specific physiological needs related to reproduction or differences in body size. In fact, all the work is based on studying the heat tolerance (Critical thermal maxima) of males and females.  Energy needs involve the study of amount of energy substrates (like glucose) or the activity of enzymes that metabolize them, oxidative metabolic markers. This is not in the works, so I recommend rearranging the phrase (lines 113-116).

Lines 88-89: Please reconsider as: Recently, amphipods have gained attention as a potential sustainable feed source for aquaculture due to their unique biochemical composition. They contain high levels of …

Line 91. …in water salinity…

Line 93. Here it is necessary to write about individual species sensitivity, as the tolerant salinity ranges of amphipods differ. Mаny species of amphipods live in deep-water Lake Baikal and are purely freshwater. It is also important to emphasize the need for a marine species that has a high tolerance for salinity and other environmental factors for use in aquaculture.

Line 99. Could you put an image of this species of amphipods? For example, provide a link to a photo in the supplemental material  or figure 1 with view of amphipods by pointing to the link in this place.

Line 101. … changes in salinity

Line 102. …for environmental stress.

Line 106. … to achieve more sustainable…

Line 112. .. from three locations that differ in overall salinity (15, 20 and 30),…

Lines 113-116. Delete.

Line 165. Critical thermal maximum reference and body size of Melita palmata

Line 219. Provide reference for calculation of a body condition index or provide more detailed description.

Figures. Enlarge the fonts in the captions on the drawings, very small, difficult to make out. It may be better to transfer the station names to the captions.

Line 372. …. conspecific populations 

The research has been done at a good level, and after the corrections mentioned above, it can be accepted for publication.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Reviewer #2

This study examines the Critical Temperature Maximum (CTmax), a common experimental method for assessing the upper temperature limit, normal organism functioning, and comparative resistance between species under given conditions. In this study, the method was applied to one amphipod species, a potential aquaculture target, under varying water salinity.

The authors write that they tested whether energy requirements related to osmoregulation affect their thermal stability and whether this relationship differs between the sexes, potentially reflecting sex-specific physiological needs related to reproduction or differences in body size. In fact, all the work is based on studying the heat tolerance (Critical thermal maxima) of males and females.  Energy needs involve the study of amount of energy substrates (like glucose) or the activity of enzymes that metabolize them, oxidative metabolic markers.

This is not in the works, so I recommend rearranging the phrase (lines 113-116).

Reply: The authors acknowledge the positive feedback from Reviewer #2. Concerning the suggestion to rearrange the phrase in lines 113-116 of our original manuscript, the sentence has been deleted as suggested by Reviewer #2 (see below).

Lines 88-89: Please reconsider as: Recently, amphipods have gained attention as a potential sustainable feed source for aquaculture due to their unique biochemical composition. They contain high levels of …

Reply: The sentence has been corrected according to the reviewer’s suggestion and now reads: “Recently, amphipods have gained attention as a potential sustainable feed for aquaculture due to their unique biochemical composition [31]. They contain high levels of protein, essential amino acids, and omega-3 polyunsaturated fatty acids [32–34].” (Lines 88 - 90). The following reference was included in this section:

31. Calado, R.; Carvalho, M.; Marques, L.; Rodrigues, D.P.; Sousa, J.P.; Rey, F.; Domingues, M.R.; Fernandes, J.F.; Silva, R.X.G.; Madeira, D.; et al. Why Marine Gammarids Belong to the Future Portfolio of Aquafeed Ingredients. Front Mar Sci 2025, 12, 1697384, doi:10.3389/FMARS.2025.1697384.

Line 91. …in water salinity…

Reply: We have revised the text to address the suggestion, and the revised sentence now reads: “Their tolerance to a wide range of environmental stressors, particularly fluctuations in water salinity, along with traits such as foraging plasticity, migratory behaviour, and drift capacity, has facilitated their successful colonization of diverse habitats worldwide”. (Line 92).

Line 93. Here it is necessary to write about individual species sensitivity, as the tolerant salinity ranges of amphipods differ. Mаny species of amphipods live in deep-water Lake Baikal and are purely freshwater. It is also important to emphasize the need for a marine species that has a high tolerance for salinity and other environmental factors for use in aquaculture.

Reply: The reviewer’s suggestion was carefully considered, and the following paragraph has been incorporated into the revised manuscript and now reads as follows: “Amphipods exhibit a broad spectrum of ecological affinities, while a considerable number of species are strictly freshwater, many others inhabit brackish, estuarine, or fully marine environments, and some are adapted even to deep-sea conditions [27,28]. Euryhaline species often display greater physiological robustness, as they are exposed to abiotic conditions varying over short and seasonal timescales. For instance, Gammarus lacustris populations from saline-lake habitats tolerate thermal and osmotic stress better than con-specifics from freshwater [37]. Similar patterns occur within the genus Gammarus, which includes several marine and estuarine species capable of maintaining internal osmotic balance across broad salinity ranges, reflecting strong physiological plasticity in fluctuating brackish and coastal environments [38–40]. Moreover, broad salinity tolerance appears associated with ecological plasticity and invasive potential in amphipods, underlining their evolutionary and ecological significance [41]. Given these interspecific differences in physiological and ecological plasticity, using an estuarine or marine amphipod species with broad salinity tolerance should be prioritized for aquaculture settings to achieve a stable biomass production, high survival, and maintenance of nutritional quality under variable conditions [32–34,41].” (Lines 95 – 110). The following references were included in this section:

27. Momtazi, F.; Saeedi, H. Exploring Latitudinal Gradients and Environmental Drivers of Amphipod Biodiversity Patterns Regarding Depth and Habitat Variations. Scientific Reports 2024 14:1 2024, 14, 1–12, doi:10.1038/s41598-024-83314-6.
28. Väinölä, R.; Witt, A.J.D.S.; Grabowski, A.M.; Bradbury, A.J.H.; Jazdzewski, A.K.; Sket, A.B.; Balian, E. V; Lévêque, C.; Segers, H.; Martens, K.; et al. Global Diversity of Amphipods (Amphipoda; Crustacea) in Freshwater. Hydrobiologia 2007 595:1 2007, 595, 241–255, doi:10.1007/S10750-007-9020-6.
29. Alberts-Hubatsch, H.; Slater, M.J.; Beermann, J. Effect of Diet on Growth, Survival and Fatty Acid Profile of Marine Amphipods: Implications for Utilisation as a Feed Ingredient for Sustainable Aquaculture. Aquac Environ Interact 2019, 11, 481–491, doi:10.3354/AEI00329.
30. Jiménez-Prada, P.; Hachero-Cruzado, I.; Guerra-García, J.M. Aquaculture Waste as Food for Amphipods: The Case of Gammarus Insensibilis in Marsh Ponds from Southern Spain. Aquaculture International 2021, 29, 139–153, doi:10.1007/S10499-020-00615-Z.
31. Suhaimi, H.; Rahman, M.I.A.; Ashaari, A.; Ikhwanuddin, M.; Rasdi, N.W. Adaptation and Potential Culture of Wild Amphipods and Mysids as Potential Live Feed in Aquaculture: A Review. PeerJ 2024, 12, e17092, doi:10.7717/PEERJ.17092/SUPP-2.
32. Vereshchagina, K.P.; Lubyaga, Y.A.; Shatilina, Z.; Bedulina, D.; Gurkov, A.; Axenov-Gribanov, D. V.; Baduev, B.; Kondrateva, E.S.; Gubanov, M.; Zadereev, E.; et al. Salinity Modulates Thermotolerance, Energy Metabolism and Stress Response in Amphipods Gammarus Lacustris. PeerJ 2016, 4, doi:10.7717/PEERJ.2657.
33. Normant, M.; Kubicka, M.; Lapucki, T.; Czarnowski, W.; Michalowska, M. Osmotic and Ionic Haemolymph Concentrations in the Baltic Sea Amphipod Gammarus Oceanicus in Relation to Water Salinity. Comp Biochem Physiol A Mol Integr Physiol 2005, 141, 94–99, doi:10.1016/J.CBPB.2005.04.007.
34. Delgado, L.; Guerao, G.; Ribera, C. Effects of Different Salinities on Juvenile Growth of Gammarus Aequicauda (Malacostraca: Amphipoda). Int J Zool 2011, doi:10.1155/2011/248790.
35. Bolt, S.R.L. Ecophysiological Responses to Salinity Changes in Selected Euryhaline Amphipods with Special Reference to Gammarus Duebeni. 1982.
36. Cuthbert, R.N.; Kotronaki, S.G.; Dick, J.T.A.; Briski, E. Salinity Tolerance and Geographical Origin Predict Global Alien Amphipod Invasions. Biol Lett 2020, 16, doi:10.1098/RSBL.2020.0354.

Line 99. Could you put an image of this species of amphipods? For example, provide a link to a photo in the supplemental material or figure 1 with view of amphipods by pointing to the link in this place.

Reply: We appreciate this suggestion and consider that adding a high-resolution image is certainly a plus to our study, as it helps illustrate the species’ size and key biological traits. Accordingly, the image has been added to the supplementary material, and its respective reference has been included in the main manuscript, that now reads as follows: “The estuarine amphipod Melita palmata (Montagu, 1804) (Figure S1) is a common species in temperate coastal lagoons, estuaries, and brackish waters throughout the North Atlantic, Mediterranean, and Black Sea regions [42–44].” (Lines 111 - 113).

 

Line 101. … changes in salinity and Line 102. …for environmental stress.

Reply: We have revised the text to address the suggestion, and the sentence now reads: “Due to its sensitivity to changes in salinity, pollution, and temperature, M. palmata is considered a valuable bioindicator for environmental stress.” (Lines 118 - 119).

 

Line 106. … to achieve more sustainable…

Reply: We have revised the text to address the suggestion, and the sentence now reads: “Such resilience is particularly advantageous in systems under daily or seasonal fluctuations in salinity and temperature, as well as in those promoting the reuse of brackish or marine water to achieve more sustainable production.” (Line 126).

 

Line 112. .. from three locations that differ in overall salinity (15, 20 and 30),…

Reply: We have revised the text to address the suggestion, and the sentence now reads: “In this study, we determined the CTmax of male and female M. palmata amphipods collected from three locations that differ in overall salinity conditions (15, 20, and 30) within a temperate coastal lagoon (Ria de Aveiro, Portugal).” (Line 132).

 

Lines 113-116. Delete.

Reply: The sentence has been deleted as suggested by Reviewer#2.

 

Line 165. Critical thermal maximum reference and body size of Melita palmata

Reply: The subtitle has been corrected in accordance with the reviewer’s suggestion. (Line 214)

 

Line 219. Provide reference for calculation of a body condition index or provide more detailed description.

Reply: To best accommodate this request, we have now added to the revised manuscript the following information that now reads: “This approach is a standard, well-established index, widely used in ecological research, and has demonstrated a reliable performance in multiple validation studies [77,78].” (Line 277 - 279). The following references were added to this sentence:

77. 77. Jakob, E.M.; Marshall, S.D.; Uetz, G.W. Nordic Society Oikos Estimating Fitness: A Comparison of Body Condition Indices. Source: Oikos 1996, 77, 61–67.
78. 78. Schulte-Hostedde, A.I.; Zinner, B.; Millar, J.S.; Hickling, G.J. Restitution of Mass-Size Residuals: Validating Body Condition Indices. Ecology 2005, 86, 155–163, doi:10.1890/04-0232

 

Enlarge the fonts in the captions on the drawings, very small, difficult to make out. It may be better to transfer the station names to the captions.

Reply: We thank this suggestion provided by Reviewer #2 and the font size in all three figures has now been increased to improve readability. Station names have also been transferred to figure captions as recommended.

 

Line 372. …. conspecific populations 

Reply: We have revised the text to address the comment; the sentence now reads: “Our study demonstrates that M. palmata amphipods from low-salinity environments exhibit reduced thermal limits compared to conspecific populations from high-salinity sites, indicating that the energetic demands of osmoregulation under hypo-osmotic conditions can constrain their capacity to tolerate higher water temperatures.” (Lines 451 -454).

 

The research has been done at a good level, and after the corrections mentioned above, it can be accepted for publication.

Reply: The authors acknowledge the positive feedback from Reviewer #2 and hope that, in its revised form, the present manuscript can now be recommended for publication.

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript has been significantly improved, but there are still minor edits in the text. I cite them below.

Minor corrections

Line 25 …We investigated the effect of salinity on the termal tolerance of

Line 29 … with different salinity regimes (…

Line 31… salinities, indicating that osmoregulatory costs may restrict thermal resistance.

Lines 34-35 …. and differences in body condition index (BCI) between sites…

Line 41    …aquaculture production, particularly in earthen ponds

Line 43 …. Add “thermal safety margins”

Lines 52-55  Marine invertebrates may undergo enhanced osmotic gradients across body surfaces in response to salinity variations, resulting in…

Line 61. … also important in practical settings.

Line 133. Delete “amphipods”

Line  140… individuals of M. palmata 

Lines 273-276 …  to examine whether individuals of M. palmate….from different sampling …. conditions differ in weight     from a linear regression model fitting the relationship….

Lines 296-301 …showed no abnormal behaviour

….loss of equilibrium, were caused by…

….During the CTmax trials, theamphipods were continuously monitored,

Line 308 …The lowest recorded CTmax values were…

Line 309. … the highest recorded values were

Line 329…were generally higher

Line 332…indicating that specimens of M palmata at this location had higher thermal resistance than those at other sites.

Lines 463. …resilience of M. palmata and other amphipod species. This will help to support their potential use in aquaculture and promote their integration into sustainable production systems. A better understanding of these processes will not only help elucidate species' adaptive responses in natural environments but also facilitate their use in aquaculture.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

We thank Reviewer #2 for the positive reassessment of the manuscript and for the helpful minor suggestions. All comments have been fully addressed, and the manuscript has been revised accordingly to improve clarity, accuracy, and consistency. A detailed, point-by-point response is provided below, and all alterations in the revised manuscript are marked in red.

Line 25 …We investigated the effect of salinity on the thermal tolerance of

Reply: We have revised the text to address the suggestion, and the sentence now reads: “We investigated the effect of salinity on the thermal tolerance of the estuarine amphipod Melita palmata (Montagu, 1804), …” (Lines 25 – 26)

 

Line 29 … with different salinity regimes (…

Reply: We have revised the text to address the suggestion, and the sentence now reads: “The critical thermal maximum (CTmax) was determined for males and females collected from three sites within a temperate coastal lagoon (Ria de Aveiro, Portugal) characterized with different salinity regimes (15, 20, and 30).” (Lines 28 - 30)

 

Line 31… salinities, indicating that osmoregulatory costs may restrict thermal resistance.

Reply: We have revised the text to address the suggestion, and the sentence now reads: “Individuals from lower-salinity environments exhibited significantly lower CTmax values than those from higher salinities, indicating that osmoregulatory costs may restrict thermal resistance.” (Lines 30 – 32)

 

Lines 34-35 …. and differences in body condition index (BCI) between sites…

Reply: We have revised the text to address the suggestion, and the sentence now reads: “However, thermal safety margins (TSM) increased with salinity, indicating greater thermal tolerance under higher salinity conditions, and differences in body condition index (BCI) between sites suggest salinity-related effects on growth performance.” (Lines 33 – 35)

 

Line 41    …aquaculture production, particularly in earthen ponds

Reply: We have revised the text to address the suggestion, and the sentence now reads: “The species' physiological plasticity under such variable conditions reinforces its suitability for aquaculture production, particularly in earthen ponds in estuarine environments.” (Lines 40 – 41)

 

Line 43 …. Add “thermal safety margins”

Reply: The keyword was added.

 

Lines 52-55 Marine invertebrates may undergo enhanced osmotic gradients across body surfaces in response to salinity variations, resulting in…

Reply: We have revised the text to address the suggestion, and the sentence now reads: “Marine invertebrates may undergo enhanced osmotic gradients across body surfaces in response to salinity variations, resulting in passive water influx and ion loss [9,10].” (Lines 54 – 56)

 

Line 61. … also important in practical settings.

Reply: We have revised the text to address the suggestion, and the sentence now reads: “Beyond their ecological relevance, these processes are also important in practical settings.” (Lines 60 – 61)

 

Line 133. Delete “amphipods”

Reply: The word “amphipods” was deleted from the sentence.

 

Line 140… individuals of M. palmata 

Reply: We have revised the text to address the suggestion, and the sentence now reads: “We further predicted that individuals of M. palmata would be…” (Lines 139 – 140)

 

Lines 273-276 …  to examine whether individuals of M. palmata….from different sampling …. conditions differ in weight from a linear regression model fitting the relationship….

Reply: We have revised the text to address the suggestion, and the sentence now reads “We also calculated a body condition index (BCI) to examine whether individuals of M. palmata from different sampling sites and exposed to site-specific salinity conditions differ in weight from a linear regression model fitting the relationship between log-transformed DW and TL.” (Lines 274 – 277)

 

Lines 296-301 …showed no abnormal behaviour

….loss of equilibrium, were caused by…

….During the CTmax trials, the amphipods were continuously monitored,

Reply: We have revised the text to address the suggestion, and the sentences now read: “Amphipods in the control group showed no abnormal behaviour, mortality, or changes in swimming activity, confirming that behavioural shifts recorded in the experimental groups, such as changes in movement patterns and loss of equilibrium, were caused by increasing water temperature. During CTmax trials, amphipods were continuously monitored…”(Lines 297 – 301)

 

 

 

 

 

 

Line 308 …The lowest recorded CTmax values were…

Reply: We have revised the text to address the suggestion, and the sentence now reads:  “The lowest recorded CTmax values were 31.6 ºC for females and 32.6 ºC for males, both from the MCU site.” (Line 309)

 

Line 309. … the highest recorded values were

Reply: We have revised the text to address the suggestion, and the sentence now reads: “In contrast, the highest recorded values were 35.6 ºC for females from IC and 35.4 ºC for males from MCD (Figure 2).” (Lines 310 – 311)

 

Line 329…were generally higher

Reply: We have revised the text to address the suggestion, and the sentence now reads: “Interindividual differences in CTmax responses were generally higher for females…” (Lines 328 – 329)

 

Line 332…indicating that specimens of M palmata at this location had higher thermal resistance than those at other sites.

Reply: We have revised the text to address the suggestion, and the sentence now reads: “…indicating that specimens of M. palmata at this location had a higher thermal resistance than those at the other sites.” (Lines 332 – 333)

 

Lines 463. …resilience of M. palmata and other amphipod species. This will help to support their potential use in aquaculture and promote their integration into sustainable production systems. A better understanding of these processes will not only help elucidate species' adaptive responses in natural environments but also facilitate their use in aquaculture.

Reply: We have revised the text to address the suggestion, and the sentences now read: “Future research should further examine how environmental variability shapes the physiological performance and resilience of M. palmata and other amphipod species. This will help to support their potential use in aquaculture and promote their integration into sustainable production systems. A better understanding of these processes will not only help elucidate species' adaptive responses in natural environments but also facilitate their use in aquaculture.” (Lines 461 – 466)