Behavioral Responses of Galaxias platei to Salmo trutta: Experimental Evidence of Competition and Predation Risk
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe article proposal, which questions the interactions in the microhabitat preferences of Salmo trutta, an invasive species for South America, and Galaxias platei, a native widespread species in Chile and in the LC category according to IUCN criteria, was tried to be explained through simulations created in tanks.
It is not understood whether the optimum experimental volume in the experiment tank is sufficient for an experiment to understand the adverse impacts of invasive Salmo trutta on native Galaxias platei in the article proposal. It is not known how to support the idea that these species have a clear preference among selected microhabitats in their natural environment.
Biologically, it should be considered whether the widespread Galaxias platei has specific microhabitat preferences, as well as spawning, growth and feeding periods. On the other hand, it is debatable whether the invasive Salmo trutta has microhabitat preferences other than the specific conditions listed above.
It does not seem possible to distinguish between the microhabitat preferences observed in the experimental tanks and the need for forced circulation space.
Apart from the water temperature in the test tanks, pH, dissolved oxygen, salinity, etc., water quality parameters are not known. The quality of the water in the test tanks is also important.
Competition between species due to differences in swimming speed is a biologically expected phenomenon.
Author Response
Comments 1: It is not understood whether the optimum experimental volume in the experiment tank is sufficient for an experiment to understand the adverse impacts of invasive Salmo trutta.
Response to comments 1:
Given the central role that experimental analysis plays in science (Kilkenny et al., 2010), various studies have employed aquaria to examine fish behavior under different conditions (Maierdiyali et al., 2020; Fraser & Lampher, 2013). Such investigations have shown that fish living space can affect growth and feed intake, which tend to increase with tank size (Espmark et al., 2017), and that larger enclosures can induce fish to swim faster than under natural conditions (Tang et al., 1993). We acknowledge the reviewer's concern regarding whether the experimental tank volume used in our study was optimal. Recognizing that tank size can influence fish behavior and physiology, we have added a paragraph in the Discussion section addressing this consideration and highlighting it as an area for future research.
While the present study offers valuable insights into the behavioral responses of G. platei to the presence of the invasive S. trutta, several methodological limitations must be acknowledged. Experimental research is a fundamental tool in ecological science (Kilkenny et al., 2010), yet it is often constrained by logistical and practical challenges that can influence the outcomes and their interpretation. Previous studies have shown that spatial confinement can affect physiological and behavioral traits in fish, with increased tank volume being associated with enhanced growth and feed intake (Espmark et al., 2017), as well as altered swimming behavior, such as increased activity in larger enclosures compared to natural conditions (Tang et al., 1993). In this study, we maintained environmental parameters representative of the native habitats of the species involved (Cussac et al., 2020), and incorporated environmental enrichment following established guide-lines (Williams et al., 2009; Näslund & Johnsson, 2014). Nevertheless, it is possible that tank size and other artificial conditions may have influenced the behavioral patterns observed in G. platei. This possibility should be addressed in future studies.
Comments 2: Biologically, it should be considered whether the widespread Galaxias platei has specific microhabitat preferences, as well as spawning, growth and feeding periods. On the other hand, it is debatable whether the invasive Salmo trutta has microhabitat preferences other than the specific conditions listed above.
Response to comments 2: We appreciate the comment on the microhabitat preferences of Galaxias platei. We have included the following paragraph in the introduction section:
A preference for deep habitats in Patagonian lakes (>40 m) and low water temperatures (<12°C) has been identified in this species, with both factors being positively correlated with sexual maturity (Milano & Barriga, 2018).
Optimal growth for this species has been estimated to occur at temperatures between 10 and 16 °C during the warmer seasons. It exhibits a benthic carnivorous feeding strategy, primarily consuming prey from the littoral zones of lakes. In the presence of invasive trout, G. platei juveniles show slower ontogenetic development and an expansion of their trophic niche toward allochthonous prey. This shift is density-dependent and occurs in reciprocal relation to the trophic position of S. trutta (Cussac et al., 2020).
Comments 3: It does not seem possible to distinguish between the microhabitat preferences observed in the experimental tanks and the need for forced circulation space.
Response to comments 3:
We appreciate the reviewer’s concern regarding the potential confounding effect between observed microhabitat preferences and the need for forced circulation space in the experimental tanks. This issue has been previously addressed in experimental fish ecology, and several studies support the use of aquaria as valid settings for identifying habitat preferences, provided that tank design minimizes directional flow constraints and offers spatial heterogeneity (Näslund & Johnsson, 2016). In our experiments, the tank was structured with discrete and contrasting microhabitat options (e.g., refuges with cobble substrate versus open areas), and water flow was evenly distributed to avoid directional bias. To strengthen this point, we have clarified these methodological details in the revised manuscript and discussed the implications for extrapolating experimental findings to natural settings.
Comments 4: Apart from the water temperature in the test tanks, pH, dissolved oxygen, salinity, etc., water quality parameters are not known. The quality of the water in the test tanks is also important.
Response to comments 4: We agree with the observation, we have included a paragraph in the methodology:
Environmental variables were maintained under controlled conditions: water temperature at 14 °C, dissolved oxygen at 8 ppm, and pH at 7. A 12:12 h light-dark photoperiod was implemented using fluorescent lighting.
Comments 5: Competition between species due to differences in swimming speed is a biologically expected phenomenon.
Responser to comments 5: We agree with the reviewer that interspecific competition mediated by differences in swimming performance is a well-established and biologically expected phenomenon. Swimming speed directly influences key ecological processes such as territory acquisition, foraging efficiency, predator avoidance, and access to preferred microhabitats. In particular, faster or more maneuverable species often outcompete slower species in spatially limited or structurally complex environments (Plaut, 2001). In the context of our study, the observed spatial displacement of G. platei in the presence of S.trutta can be interpreted as a behavioral response to competitive pressure, where differences in swimming ability may underlie asymmetries in habitat use and dominance. We have incorporated this interpretation into the discussion to better link our findings with established ecological theory.
References
Kilkenny, C.; Browne, W.J.; Cuthill, I.C.; Emerson, M.; Altman, D.G. Improving Bioscience Research Reporting: The ARRIVE Guidelines for Reporting Animal Research. Plos. Biol. 2010, 8, 1–5
Maierdiyali, A., Wang, L., Luo, Y., & Li, Z. Effect of Tank Size on Zebrafish Behavior and Physiology. Animals, 2020 10(12), 2353.
Fraser, D., Lamphere, D. Experimental evaluation of predation as a facilitator of success in a stream fish. 2013 Ecol. 94 (3), 640-649.
Espmark, A.M.; Kolarevic, J.; Asgard, T.; Terjesen, B.F. Tank size and fish management history matters in experimental design. Aquac. Res. 2017, 48, 2876–2894.
Tang, M.; Boisclair, D. Influence of the Size of Enclosures on the Swimming Characteristics of Juvenile Brook Trout (Salvelinus-Fontinalis). Can. J. Fish. Aquat. Sci. 1993, 50, 1786–1793.
Williams, T.D.; Readman, G.D.; Owen, S.F. Key issues concerning environmental enrichment for laboratory-held fish species. Lab. Anim. 2009, 43, 107–120.
Naslund, J.; Johnsson, J.I. Environmental enrichment for fish in captive environments: Effects of physical structures and substrates. Fish. Fish. 2016, 17, 1–30.
Plaut, I. Critical swimming speed: its ecological relevance. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2001 131(1), 41–50.
Cussac, V. E., Barrantes, M. E., Boy, C. C., Górski, K., Habit, E., Lattuca, M. E., Rojo, J. H. New insights into the distribution, physiology and life histories of South American galaxiid fishes, and potential threats to this unique fauna. Diversity 2020 12(5), 178.
Milano, D., Barriga, J. Reproductive aspects of Galaxias platei (Pisces, Galaxiidae) in a deep lake in North Patagonia. Mar. Freshw. Res. 2018 69 (9), 1379-1388.
Reviewer 2 Report
Comments and Suggestions for AuthorsThis study was well-done and the writing was mostly clear and well-organized. I have some line-by-line comments that may help further clarify aspects of the paper.
Title: Although the subject of this could be considered "sublethal effects", I feel like behavior change or response is more representative of what was done. When I think sublethal effects I think more about aggression and nipping, or the effect of chemicals on animal health. I recommend modifying the title to more precisely describe the experiments, which were geared more towards evaluating the behavioral response of G. platei to Salmo trutta.
Abstract: I recommend softening the last sentence, as there are behaviors that could potentially happen outside of an experimental microcosm. "could be insufficient" would be better.
Line 41: I recommend using a scientific study as this reference rather than an essay.
Line 43: Reference 6 is about two sympatric species, so it seems less relevant in this context.
Lines 90- 94: These are more like predictions rather than hypotheses.
Lines 122-127: Do you have references for your experimental design? Why every 10 minutes? Why didn't you have any replication for the different densities? Do you think this is a limitation that should be included in the discussion?
Figure 2: Add a y-axis label
Discussion: I recommend adding some discussion of the limitations of your study.
Author Response
Comments 1:
This study was well-done and the writing was mostly clear and well-organized. I have some line-by-line comments that may help further clarify aspects of the paper.
Title: Although the subject of this could be considered "sublethal effects", I feel like behavior change or response is more representative of what was done. When I think sublethal effects I think more about aggression and nipping, or the effect of chemicals on animal health. I recommend modifying the title to more precisely describe the experiments, which were geared more towards evaluating the behavioral response of G. platei to S. trutta.
Response 1:
We sincerely thank for the valuable observation regarding the specificity and accuracy of the manuscript’s title. We agree that the term “sublethal effects” can encompass a broad range of biological phenomena, including physiological impacts, chemical exposures, and aggressive interactions. In the context of our study, the primary focus was indeed on the behavioral responses of Galaxias platei to the presence of Salmo trutta, with particular attention to microhabitat use and spatial displacement in controlled experimental conditions. In response, we have modified the manuscript’s title to better reflect the experimental objectives and nature of the findings.
New Title: Behavioral Responses of Galaxias platei to Salmo trutta: Experimental Evidence of Competition and Predation Risk.
Comments 2:
Abstract: I recommend softening the last sentence, as there are behaviors that could potentially happen outside of an experimental microcosm. "could be insufficient" would be better.
Response 2:
We agree with this observation which we welcome.
Comments 3:
Line 41: I recommend using a scientific study as this reference rather than an essay.
Response 3:
We have kept the reference, considering that it is a recognized article and highly cited in similar studies.
Comments 4:
Line 43: Reference 6 is about two sympatric species, so it seems less relevant in this context.
Response 4:
We welcome the suggestion
Comments 5:
Lines 90- 94: These are more like predictions rather than hypotheses.
Response 5:
(i) G. platei increases its use of microhabitats with available refuges in the presence of juvenile brown trout to reduce competition; (ii) G. platei increases its use of refuge microhabitats in the presence of predatory brown trout to mitigate predation risk and (iii) G. platei exhibits lower critical swimming performance than S. trutta limiting its capacity to escape from predatory encounters.
Comments 6:
Lines 122-127: Do you have references for your experimental design? Why every 10 minutes? Why didn't you have any replication for the different densities? Do you think this is a limitation that should be included in the discussion?
Response 6:
The research of Sobenes et al., 2013 was reviewed, where Vanderpham et al., 2012, Araky & Tokeshi 2012, Davey et al., 2005 were previously reviewed. In this study, during preliminary observations, a low level of displacement activity was observed in G. platei between five-minute periods, so it was defined as observing changes in use every 10 minutes. To improve this definition in materials and methods we include that observation procedure was according to Sobenes et al, (2013).
The lack of replication across density treatments is a limitation of our study. This was primarily due to logistical and resource constraints commonly encountered in controlled experiments involving live fish. However, the use of non-parametric statistics can be used in these cases in order to make inferences without the problems of parametric statistics. We agree that an experiment with a larger number of replications does not require greater safeguards in the use of its conclusions. We include this in the discussion as a limitation of the present study:
Moreover, the absence of replication across certain density treatments presents an additional limitation, potentially reducing the generalizability of our findings. This issue, commonly encountered in behavioral experiments with live fish, raises concerns about pseudoreplication (Hurlbert, 1984). Although such experimental designs are frequently used to explore interspecific interactions under controlled conditions, we acknowledge that increased replication would improve the robustness and statistical power of the study. In this context, the use of non-parametric statistical analyses allowed us to draw conservative inferences without relying on assumptions of data normality (Anderson et al., 2008), thus partially mitigating the constraints imposed by the experimental design.
Comments 7: Figure 2: Add a y-axis label.
Response: We back to the figure but the y-axis is D, and in the title of the figure it is the meaning of Jacob´s index (D)
Comments 8:
Discussion: I recommend adding some discussion of the limitations of your study.
Response:
We agree with this suggestion and have incorporated a discussion of the study’s limitation in the revised manuscript. In the improved manuscript there is a paragraph that points out the limitations of the study
References
Sobenes, C., Link, O., Habit. E. Selección denso – dependiente de microhábitat en Galaxias platei: un estudio experimental. Gayana 2013 77, 35-42.
Vanderphan, J.P., Nakagawa, S, Closs, G.P. Diel variation in use cover and feeding activity of a benthic freshwater fish in response to olfactory cues of a diurnal predatory. Env. Biol. Fishes, 2012 93:547-556.
Araki, S. Tokeshi, M. Species and size matter: An experimental study of microhabitat use under the influence of competitive interactions in intertidal gobiids. J. Exp. Mar. Biol. Ecol. 2012 418-419, 59-68.
Davey, A.J., Hawkins, S.J., Turner, G.F., Doncaster, C.P. Size-dependent microhabitat use and intraspecific competition in Cottus gobio. J. Fish Biol. 2005 67:428-443.
Hurlbert, S. Pseudoreplication and the Design of Ecological Field Experiments. Ecol. Monogr. 1984 54 (2) 187-211.
Anderson, M.J., Gorley, R.N., Clarke, K.R. PERMANOVA+ for PRIMER. Guide to software and Statistical Methods.2008 PRIMER-E. Plymouth, UK.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript entitled ‘Unravelling the Sub-Lethal Effects of Salmo trutta on Galaxias platei: an Experimental Study on Competition and Predation Risk’ is well designed and generally well written. the authors provided very useful data. I think it can be accepted for publish after some minor revision. One of my concerns is that the body size of brown trout is larger than G. platei, which cannot be fixed at this stage. However, I think it should have negatable effect on behavioral observation.
Specific comment
Abstract line 27-30, it is ok, however, I suggest the authors change ‘do not’ to a more proper way
Material and method
The direct observation is ok. It would be nice if you can video the behavior without disturbance
Fish were repeatedly used. Did the author consider the possible effect?
Table 1. The body sizes are important information. Can the authors provide the data of body size along with the sample size in the Table 1?
Swimming performance, it is ok to use Ucrit, as a matter of fact fast-start and Ucat are more relevant for prey-predator interaction
Table 2 is no need to be provided. The data in figure 5 is sufficient.
Line 277, sentence need revision.
Author Response
Comments 1:
The manuscript entitled ‘Unravelling the Sub-Lethal Effects of Salmo trutta on Galaxias platei: an Experimental Study on Competition and Predation Risk’ is well designed and generally well written. the authors provided very useful data. I think it can be accepted for publish after some minor revision. One of my concerns is that the body size of brown trout is larger than G. platei, which cannot be fixed at this stage. However, I think it should have negatable effect on behavioral observation.
Response:
We sincerely thank for the positive evaluation of our manuscript and for recognizing the value of the data and experimental design.
Regarding the concern raised about the size discrepancy between S. trutta and G.platei, we agree that body size is a critical factor in predator-prey dynamics and interspecific interactions.
In our study, size differences were deliberately included to reflect ecologically realistic scenarios. Predatory adult brown trout were expected to be larger than G. platei, as this reflects the natural predator-prey relationships observed in natural freshwater systems. Similarly, juvenile brown trout were selected within a size range to simulate early competitive interactions without immediate predation risk. While size asymmetry could influence behavior, we ensured that interactions were observed within structured microhabitats where both refuge use and swimming responses could be evaluated independently of direct size-based dominance.
As correctly noted, the size difference is a design feature that cannot be altered post-experiment, but we concur that it should have minimal impact on the validity of the behavioral observations, especially given the spatial complexity and the focus on indirect, trait-mediated responses.
We include in point 2.3 a new paragraph:
The selection of G. platei and S. trutta individuals for the experimental trials was based on naturally occurring size classes. Juvenile and adult brown trout were chosen to simulate realistic competitive and predatory interactions, respectively. While adult S. trutta individuals were larger than G. platei, the experimental design included structured microhabitats and refuge zones to prevent direct aggressive encounters and allow for behavioral responses to be evaluated independently of size-based dominance.
In the discussion section we include:
One limitation of the experimental design is the difference in body size between S. trutta and G. platei. However, this size asymmetry is ecologically realistic and expected to occur in natural predator-prey and competitive contexts. Previous studies have shown that size-structured interactions are common in freshwater systems and that body size plays a fundamental role in shaping behavioral and spatial dynamics (Werner & Gilliam, 1984). In our experiments, refuge availability and habitat heterogeneity allowed for the assessment of behavioral responses without direct physical dominance.
Comments 2:
Abstract line 27-30, it is ok, however, I suggest the authors change ‘do not’ to a more proper way.
Response:
we back to the manuscript and we improve the paragraph in the first line of the paragraph replaced by “fails to increase”; and in the last sentence of this paragraph “G. platei does not display”..
Comments 3:
Material and method
The direct observation is ok. It would be nice if you can video the behavior without disturbance.
Response:
A video recording was made for review only. We included this observation in the methodology. In point 2.2 of the paper we include a sentence”….over a total period of 48 hours, supported with videos for the observer's review and consultation, according to Sobenes et al. (2013).
Comments 4:
Fish were repeatedly used. Did the author consider the possible effect?
Response:
In our experimental design, we took specific measures to minimize the potential influence behavioral response due to learning, habituation or stress, effects associated with repeated use. First, individuals were randomly assigned to treatments, ensuring that no systematic bias could influence the results across experimental conditions. Second, adequate recovery periods were implemented between trials for the tested group of fish to reduce potential behavioral conditioning or fatigue. Third, measured behaviors, such as microhabitat use, were observed in short-term trials where novelty and initial response patterns tend to predominate, reducing the likelihood of significant learning effects over time.
To address this concern more explicitly, we have added a statement in the Methods and Discussion sections.
In methods section point 2.2 we include: To minimize potential side effects from the repeated use of individuals, each fish had a rest period between trials, and all test sequences were randomized. Behavioral trials were short and designed to capture immediate responses to environmental cues, rather than long-term conditioned behaviors.
In Discussion section: Although some individuals were used more than once, we implemented randomized test sequences and adequate rest periods to mitigate potential carryover effects. Furthermore, the observed behavioral responses were consistent across treatments, supporting the validity of our findings despite potential individual habituation. Similar approaches have been successfully used in other experimental studies focusing on short-term behavioral responses in fish (Lucon-Xiccato & Bisazza, 2017).
Comments 5: Table 1. The body sizes are important information. Can the authors provide the data of body size along with the sample size in the Table 1?
Response:
We back to table 1 and include the data of body size.
Comments 6:
Swimming performance, it is ok to use Ucrit, as a matter of fact fast-start and Ucat are more relevant for prey-predator interaction.
Response: we agree that Ucrit are particularly relevant when evaluating a prey’s immediate escape response from predators. This velocity provides ecologically meaningful information on sustained swimming performance and overall locomotor ability under prolonged flow conditions (Plaut, 2001).
Comments 7:
Table 2 is no need to be provided. The data in figure 5 is sufficient.
Response:
We appreciate the suggestion; however we prefer to keep Table 2 because it provides accurate values for the speeds of S. trutta and allows for a better understanding of the comparative analysis.
Comments 7:
Line 277, sentence need revision.
Response:
We back to the manuscript and we change the sentence: The presence of the predator significantly reduced the preference of G. platei for with-in-cobble and vegetated microhabitats, while simultaneously increasing their use of slab stone habitat.
References
Werner, E. E., Gilliam, J.F. The ontogenetic niche and species interactions in size-structured populations. Ann. Rev. Ecol. Syst. 1984. 15:393-425.
Luccon – Xicatto,T., Bisazza, A. Individual differences in cognition among teleost fishes. 2017 Behav. Process. 141 82), 184-195.
Plaut, I. Critical swimming speed: its ecological relevance. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2001 131(1), 41–50.
Sobenes, C., Link, O., Habit. E. Selección denso – dependiente de microhábitat en Galaxias platei: un estudio experimental. Gayana 2013 77, 35-42.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Authors,
I have previously written that the experimental environment, especially the tank volume, which is the basis of the article, is not sufficient. I have also written that the parameters regarding the experimental conditions are controversial. On the other hand, I have concerns about the contributions of the selected invasive species and the selected native species to the scientific world.
However, reasonable responses have been given to my recommendations.
Sincerely
