Effects of Temperature and Food Concentration on the Population Recruitment of Acartia bifilosa (Copepoda, Calanoida): Implications for the Over-Summering Life History Strategy in Jiaozhou Bay
, Xiaotong Gao 6, Lei Wang 6 and Song Sun 1,2,4,5,*Round 1
Reviewer 1 Report
This study is of interest, however, the methodology has some lags.
The essence of the field study and laboratory study is not clear.
the other abiotic factors which must have contributed to the production of resting eggs were not measure, such as pH and Oxygen.
Sediment samples were not collected from the bay during summer, to ascertain the present of resting eggs, which did not provide proof of summering strategy especially when population reduced in summer.
temperature change in the environment is not abrupt, its gradual, hence, the process of producing resting eggs is possible when temperature begins to increase. However, in the laboratory test, samples collected from the field were just placed in the different temperature. this method would have stressed the copepod, in a way that might affect reproduction. they would have been placed in the same temperature as in the field and gradually increase the temperature.
The manuscript should undergo a major English correction.
Line 88, in summer in particular should be particularly in summer
line 89, instead of versatile, use diverse
Line 278, the were or they were?
Line 282, "population supplementation" or rather, population recruitment
The discussion needs to be worked on and conclusion, does not really capture the result gotten
Author Response
Response to Reviewer 1 Comments
Point 1: This study is of interest, however, the methodology has some lags. The essence of the field study and laboratory study is not clear. the other abiotic factors which must have contributed to the production of resting eggs were not measure, such as pH and Oxygen. Sediment samples were not collected from the bay during summer, to ascertain the present of resting eggs, which did not provide proof of summering strategy especially when population reduced in summer. Temperature change in the environment is not abrupt, its gradual, hence, the process of producing resting eggs is possible when temperature begins to increase. However, in the laboratory test, samples collected from the field were just placed in the different temperature. this method would have stressed the copepod, in a way that might affect reproduction. they would have been placed in the same temperature as in the field and gradually increase the temperature.
Response 1: Our present study was aimed to comment the results of Zhong and Xiao (1999) that the over-summering life history strategy A. bifilosa in the Jiaozhou Bay was resting eggs. The field survey was to confirm that adults can still maintain a high population abundance in high seawater temperature summer season. And the laboratory experiments were conducted to confirm that A. bifilosa could not produce the resting eggs at high temperatures with different food levels. The distributions of A. bifilosa in the Jiaozhou Bay from June to August are shown in new Figure 2, which shows that the A. bifilosa population could distribute in the Jiaozhou bay during the summer period, with the highest abundance over 500 ind/m3 in August. Combined with the field survey and laboratory simulated culture experiments, we found the A. bifilosa female could live at 28 °C (the highest temperature in the Jiaozhou Bay) with ≥ 5 g/L Chl a food condition. We also confirmed that the population recruitment of A. bifilosa under the extreme conditions of summer temperatures and food levels with laboratory experiments.
Furthermore, according to the results of previous studies, no acidification and hypoxia were found in the Jiaozhou Bay (Sun et al., 2011). As the comment of the reviewer, temperature change in the environment is not abrupt, its gradual. The thermal shock could influence the behavior of A. bifilosa. To solve the problem of temperature shock, we collected the zooplankton from the field for culture experiments in July, when the average seawater temperature was about 24 °C. Additionally, the incubated temperature of A. bifilosa was firstly set to 24 °C. And the culture temperature was increased or lowered by one degree per hour until it reached the designed temperature. These contents have been added in the “Material and Methods” section.
- Sun, S., Sun, X.X., Zhang, G.T., Tang, H.B., Liu, Q, Li, G.M. Long-term changes in major meteorological and hydrological factors in the Jiaozhou bay. Oceanologia et Limnologia Sinica 2011, 42(5), 632-638. (in Chinese with English abstract)
Point 2: Line 88, in summer in particular should be particularly in summer
Response 2: According to the suggestion of the reviewer, this sentence has been revised.
Point 3: line 89, instead of versatile, use diverse
Response 3: According to the suggestion of the reviewer, the word “versatile” has been replaced by “diverse”.
Point 4: Line 278, the were or they were?
Response 4: According to the comment of the reviewer, this sentence has been revised.
Point 5: Line 282, "population supplementation" or rather, population recruitment
Response 5: According to the suggestion of the reviewer, the “population supplementation” in the sentence has been revised to “population recruitment”. Thank you very much.
Point 6: The discussion needs to be worked on and conclusion, does not really capture the result gotten.
Response 6: According to the suggestion of the reviewer, the discussion has been revised, especially the section 4.1.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Review of the manuscript “Effects of Temperature and Food Concentration on the Population Recruitment of Acartia bifilosa (Copepoda, Calanoida): Implications for the Over-Summering Life History Strategy in Jiaozhou Bay”.
The authors aimed to study the distribution and abundance patterns of A. bifilosa in Jiaozhou Bay. They also examined experimentally its reproductive and developmental responses to the variants of temperatures and food concentrations.
In my opinion, this is an interesting paper but the methodology section and results presentation should be improved.
Remarks:
Line 98 – could you provide citations supporting your statement?
Line 125-129 - Have your specimens undergone any acclimation before temperature treatment? Thermal shock could influence your results - it was examined for other calanoids (see eg. Petkeviciute et al., 2015; Low et al., 2018; Nowakowski & Sługocki, 2021).
Petkeviciute, E., Kania, P. W., & Skovgaard, A. (2015). Genetic responses of the marine copepod Acartia tonsa (Dana) to heat shock and epibiont infestation. Aquaculture Reports, 2, 10-16.
Low, J. S., Chew, L. L., Ng, C. C., Goh, H. C., Lehette, P., & Chong, V. C. (2018). Heat shock response and metabolic stress in the tropical estuarine copepod Pseudodiaptomus annandalei converge at its upper thermal optimum. Journal of thermal biology, 74, 14-22.
Nowakowski, K., & Sługocki, Ł. (2021). Short-term heat shock perturbation affects populations of Daphnia magna and Eurytemora carolleeae: a warning to the water thermal pollution. Scientific reports, 11(1), 1-8.
Lines 164-166 - You have chosen the parametric ANOVA test and Pearson correlation. Could you provide support for this decision? Have you checked the distribution of data?
Lines 168-169 - Please provide criteria that led to choosing the RDA method. If you used CANOCO for RDA, maybe a manual for CANOCO will be useful (eg. ter Braak & Šmilauer, 2012), or other references regarding usage of RDA.
ter Braak, C. J. F. & P. Šmilauer, 2012. Canoco Reference Manual and User's Guide: Software for Ordination (version 5.0). Microcomputer Power, Ithaca, New York.
Line 184 - Which values you used for ANOVA calculations? Values from the last day of the experiment or values from each day of the experiment? It seems that comparing survival rates using ANOVA is not very common. For that kind of experiment survival analysis (eg. Kaplan-Meier, Log-rank test) is dedicated. If you don’t want to change the analysis, please support your decision with proper references.
Lines 280-285 - RDA description should be supplemented with additional information. Could you provide information about the percentage of the total variation that is explained by certain variables? Are they significant? Could you provide also a p-value for each variable? It seems that temperature had a negative (not positive) effect on the values of PDT, HR, and EPR.
Lines 286-288 – Could you provide exact R and p values for significant correlations?
Author Response
Response to Reviewer 2 Comments
Point 1: In my opinion, this is an interesting paper but the methodology section and results presentation should be improved.
Response 1: According to the comment of the reviewer, the methodology section and results presentation have been improved the Material and Methods and Results section. Please review the revised manuscript. Thank you very much.
Point 2: Line 98 – could you provide citations supporting your statement?
Response 2: According to the comment of the reviewer, the references about the long-term changes of the environmental factors (chlorophyll-a concentration, primary productivity, and hydrological factors) in the Jiaozhou Bay are added in this sentence.
Point 3: Line 125-129 - Have your specimens undergone any acclimation before temperature treatment? Thermal shock could influence your results - it was examined for other calanoids (see eg. Petkeviciute et al., 2015; Low et al., 2018; Nowakowski & Sługocki, 2021).
Petkeviciute, E., Kania, P. W., & Skovgaard, A. (2015). Genetic responses of the marine copepod Acartia tonsa (Dana) to heat shock and epibiont infestation. Aquaculture Reports, 2, 10-16.
Low, J. S., Chew, L. L., Ng, C. C., Goh, H. C., Lehette, P., & Chong, V. C. (2018). Heat shock response and metabolic stress in the tropical estuarine copepod Pseudodiaptomus annandalei converge at its upper thermal optimum. Journal of thermal biology, 74, 14-22.
Nowakowski, K., & Sługocki, Ł. (2021). Short-term heat shock perturbation affects populations of Daphnia magna and Eurytemora carolleeae: a warning to the water thermal pollution. Scientific reports, 11(1), 1-8.
Response 3: As the comment of the reviewer and results of the former papers, thermal shock could influence the behavior of A. bifilosa. To solve the problem of temperature shock, we collected the zooplankton from the field for culture experiments in July, when the average seawater temperature was about 24 °C. Additionally, the incubated temperature of A. bifilosa was firstly set to 24 °C. And the culture temperature was increased or lowered by one degree per hour until it reached the designed temperature.
Point 4: Lines 164-166 - You have chosen the parametric ANOVA test and Pearson correlation. Could you provide support for this decision? Have you checked the distribution of data?
Response 4: According to the data shown in Table 1 and the figures (4, 5, and 6), the distribution of data is continuous and relatively linear. With reference to other studies, we chosen the one-way ANOVA test and Pearson correlation.
Point 5: Lines 168-169 - Please provide criteria that led to choosing the RDA method. If you used CANOCO for RDA, maybe a manual for CANOCO will be useful (eg. ter Braak & Šmilauer, 2012), or other references regarding usage of RDA.
ter Braak, C. J. F. & P. Šmilauer, 2012. Canoco Reference Manual and User's Guide: Software for Ordination (version 5.0). Microcomputer Power, Ithaca, New York.
Response 5: Thank you for your suggestion and the reference of CANOCO. PCA and RDA are both ordination analysis based on linear model. PCA is an unconstrained ordination, which generally uses only ordination of population data. The environmental factors can also be added to PCA, but the number of environmental factors has no influence on the PCA sorting results. However, RDA is a constrained ordination, which is a PCA under the constraints of selected environmental factors. So, selecting different combinations of environmental factors in RDA will produce different sorting analysis results. Using CANOCO software, the RDA results are further analysed with environmental factors after the PCA results of population parameters. As the comment of the reviewer, we have modified the analysis method to PCA.
Point 6: Line 184 - Which values you used for ANOVA calculations? Values from the last day of the experiment or values from each day of the experiment? It seems that comparing survival rates using ANOVA is not very common. For that kind of experiment survival analysis (eg. Kaplan-Meier, Log-rank test) is dedicated. If you don’t want to change the analysis, please support your decision with proper references.
Response 6: The temperature and food affect the population recruitment of A. bifilosa at the same time. Different combinations of the two factors may have the same or different effects. According to the four temperatures (20, 22, 25, and 28 °C) and three food levels (0, 5, 10, and 15 μg/L Chl a), there were 16 treatment in our experiments. The differences of Female Survival rates (FSRs) among the treatments under different temperatures and food concentrations were examined using one-way ANOVA. The analysis method is also often used in the culture experiments of other dominant zooplankton species, Calanus sinicus and Euphausia pacifica (Pu et al., 2004; Tao et al., 2015). If the survival analysis (Kaplan-Meier or Log-rank test) is necessary for the publication of our study, please comment us. We will do further Kaplan-Meier analysis with IBM SPSS Statistics V20. Thank you very much.
- Pu XM, Sun S, Yang B, Ji P, Zhang YS, et al. (2004) The combined effects of temperature and food supply on Calanus sinicus in the southern Yellow Sea in summer. Journal of Plankton Research 26: 1049-1057.
- Tao Z C, Li C L,Sun S. (2015). Grazing and Metabolism of Euphausia pacifica in the Yellow Sea. Plos One, 10(2): e0115825.
Point 7: Lines 280-285 - RDA description should be supplemented with additional information. Could you provide information about the percentage of the total variation that is explained by certain variables? Are they significant? Could you provide also a p-value for each variable? It seems that temperature had a negative (not positive) effect on the values of PDT, HR, and EPR.
Response 7: As we have explained in Response 3, a principal components analysis (PCA) was carried out using IBM SPSS Statistics V20 to analyse the impact of the temperature and food level on the A. bifilosa population recruitment. As the suggestion of the reviewer, the PCA loading values of the components were supplemented in the section 3.6. The temperature showed significant correlations with the PDT, HR, and EPR of A. bifilosa. Due to our carelessness, the words “positive” in this sentence of original manuscript should be revised to “negative”. Thank you very much.
Point 8: Lines 286-288 – Could you provide exact R and p values for significant correlations?
Response 8: According to the comment of the reviewer, the exact R and p values have been supplemented in Table 2.
Author Response File: Author Response.pdf
Reviewer 3 Report
The paper is dealing with an interesting subject important for understanding ecosystem functioning, and providing crucial information on the biology of a species
The authors have done an interesting approach combining field and experimental data, making this paper worth publishing
However, there are several points that need to be addressed before being ready for publication
A comment referring to the whole ms is English language that has to be corrected by a native speaker or somebody knowing the language fluently and the same time is an expert in the subject
Another comment referring to the whole ms is the rule of using past tenses when you are talking about your results (in the M&Ms, result and discussion section) and present tenses when talking about other peoples’ findings (introduction and discussion section). This makes it easy to follow especially the discussion and identify the significance of your findings. I have made some corrections but in no way can they be considered exhaustive
Introduction
The introduction and the following discussion will be assisted by providing basic biological information concerning A. bifilosa and the resting eggs formation (e.g. what are the major favourable conditions, which ones drive the resting egg production etc)
Lines 47-51 are not clear to me. I think that the sentence has to spilt in order for the reader to be able to follow all these interactions you try to squeeze in here
Line 56-57 not clear what a ‘key species for ecosystem dynamics’ means
Line 62 what do you mean when you say ‘autochthonous’. Is this being used in relation to invasive species, so A. fibilosa is in some areas invasive?
Line 67 what do you mean when you say ‘use of resting eggs’. They are used for what?
Line 69-70 not appropriate use of ‘According to a fixed site’ It could be improved for example like ‘During the study of a fixed site in …..’
Line 74 you say ‘Since then’. When are you referring to?
Line 92-93 not clear to me what you mean ‘…thus the potential life history strategies under high temperature’
Material and Methods
In 2.1 please specify, how many time, how many stations, how many depths and type of nets were used. Moreover, provide information regarding the distribution of the stations
Line 116 when you say ’counted’ you mean that you counted all individuals? Please define
In 2.2 you have to improve the description of the experimental process. Define how many experimental set ups were used and eventually how many replicate treatments. Better describe the reproduction experiments. Also define what you mean when you say ‘filtered water’
Line 127 you say ‘filled with surface water’. Was this filtered? How did you make sure that no extra individuals were present?
Line 128 how did you define ‘healthy’ individuals?
Line 129 ‘batches’ what do you mean? Define
Line 29 write something like ‘The reproduction experiments were performed under four different temperatures’
Line 134 did the females you used possess specific characteristics in order to be peaked e.g same size?
Line 135 define what you mean by ‘desired food’
Line 143 define how you selected and collected the microalgae provided as food
The results were treated as totals?
In 2.3 did you check for normality and collinearity and inflation factors and statistical significance? Please provide the necessary information
Results
Please provide the results presented in 3.1 also graphically
Line 191-192 not clear what you mean ‘in the end several days’
Line 199-200 what do you mean?
Line 208 what do you mean ‘batches of five females could not produce’?
Line 226 use square brackets
Table 1 explain what is the number following the average
Figure 3 explain what are the line bars
Line 235-237 how is this connected to resting eggs?
Line 238 not clear what you mean ‘incubated beyond 3 days’
Line 240 what do you mean ‘late experimental period’
Line 255-256 it is not common to use references in the results section
Line 263 these results are not clearly seen in the figure
Line 276 and figure 6 not clear to me what does the cluster show… please explain the data used to perform it
Table 2 please provide the actual values of the correlations
Discussion
A general comment about the discussion is that in extended areas it seems that sentences are simply written with obvious connection in order to provide a logical meaning
In the discussion it would be interesting to discuss the spatial variation recorded from the field sampling. The sentences need to be connected in order to lead to a reasonable explanation. When doing so, I wonder would there be an explanation-discussion relating the oversummering of A. fibilosa with it residing in deeper colder waters?
Line 300-302 Try and write this like: ‘It is well known that higher temperatures increase the energy cost of both females and larvae regarding copepod metabolism. Several studies have shown that the upper temperature tolerance limits of A. bifilosa are different in different habitats in the world, while the combined effects of ocean acidification and global warming predicted for the year 2100 on A. bifilosa, showed that higher temperatures could decrease egg viability and nauplii development of A. bifilosa. More specifically, …..’
Line 305 what is Mundaka?
Line 354-359 Did you do such an analysis?
Line 365-367 My question would probably be answered if in the introduction (as I mentioned above) basic information will be provided. However, I wonder are resting eggs produced when unfavourable conditions are coming or are they produced when conditions are excellent. At least is the way things work in rotifers which are much more dependent on the production of resting eggs.
Finally, it would be in favour of the article if the ecological implications of yours results are clearly discussed
Comments for author File: Comments.pdf
Author Response
Response to Reviewer 3 Comments
Introduction
Point 1: The introduction and the following discussion will be assisted by providing basic biological information concerning A. bifilosa and the resting eggs formation (e.g. what are the major favourable conditions, which ones drive the resting egg production etc)
Response 1: According to the suggestion of the reviewer, the resting eggs formation has supplemented in the Introduction section. As some studies have reported, the resting eggs of A. bifilosa were commonly spiny and crinkle (Castro-Longoria and Williams, 1999; Choi et al., 2021). It was reported that the smooth eggs and “spiny resting eggs” were found in the Jiaozhou Bay (Zhong and Xiao,1992).
Point 2: Lines 47-51 are not clear to me. I think that the sentence has to spilt in order for the reader to be able to follow all these interactions you try to squeeze in here
Response 2: According to the comment of the reviewer, this sentence has been revised.
Point 3: Line 56-57 not clear what a ‘key species for ecosystem dynamics’ means
Response 3: In this sentence, “key species” should be “keystone species”. Keystone species has a much greater impact on ecosystem processes than would be expected from its biomass (Paine 1966, 1969). The number of individuals of keystone species may be few or large. The disappearance or weakening of keystone species can cause fundamental changes in the entire community and ecosystem, which plays an important role in maintaining the community structure of marine ecosystems.
- Paine R. T. Food web complexity and species diversity [J]. The American Naturalist, 1966, 100 (910): 65-75.
- Paine R. T. A note on trophic complexity and community stability [J]. The American Naturalist, 1969, 103(929): 91-93
Point 4: Line 62 what do you mean when you say ‘autochthonous’. Is this being used in relation to invasive species, so A. fibilosa is in some areas invasive?
Response 4: According to the comment of the reviewer, this word has been deleted in this sentence.
Point 5: Line 67 what do you mean when you say ‘use of resting eggs’. They are used for what?
Response 5: According to the comment of the reviewer, this sentence has been revised. “Acartia bifilosa has long been recognized to produce the resting eggs in June for the over-summering”
Point 6: Line 69-70 not appropriate use of ‘According to a fixed site’ It could be improved for example like ‘During the study of a fixed site in …..’
Response 6: According to the suggestion of the reviewer, this sentence has been revised.
Point 7: Line 74 you say ‘Since then’. When are you referring to?
Response 7: According to the comment of the reviewer, “Since then” has been revised to “Over the past several decades” in this sentence.
Point 8: Line 92-93 not clear to me what you mean ‘…thus the potential life history strategies under high temperature’
Response 8: This sentence is too long and confusing. According to the comment of the reviewer, this sentence has been revised.
Material and Methods
Point 9: In 2.1 please specify, how many time, how many stations, how many depths and type of nets were used. Moreover, provide information regarding the distribution of the stations
Response 9: According to the comment of the reviewer, zooplankton sampling information has been added in the section.
Point 10: Line 116 when you say ’counted’ you mean that you counted all individuals? Please define
Response 10: All species/taxa in zooplankton samples were identified and counted with a stereoscopic microscope in the laboratory.
Point 11: In 2.2 you have to improve the description of the experimental process. Define how many experimental set ups were used and eventually how many replicate treatments. Better describe the reproduction experiments. Also define what you mean when you say ‘filtered water’
Response 11: According to the comment of the reviewer, the section 2.2 “laboratory experiments” has been revised and the ‘filtered water’ has been defined.
Point 12: Line 127 you say ‘filled with surface water’. Was this filtered? How did you make sure that no extra individuals were present?
Response 12: The “surface water” is used for the temporary breeding of zooplankton samples collected during field survey. It was collected from the surface seawater of the field investigation sea area.
Point 13: Line 128 how did you define ‘healthy’ individuals?
Response 13: Individuals with active swimming, complete body and appendages are considered as healthy individuals.
Point 14: Line 129 ‘batches’ what do you mean? Define
Response 14: The “batches” originally means the experiment treatments. According to the comment of the reviewer, the word “batches” have been deleted and replaced with “treatments” in the section.
Point 15: Line 129 write something like ‘The reproduction experiments were performed under four different temperatures’
Response 15: According to the suggestion of the reviewer, this sentence has been revised.
Point 16: Line 134 did the females you used possess specific characteristics in order to be peaked e.g same size?
Response 16: YES. All the females incubated in the egg production experiments are mature individuals with active swimming, complete body and similar size, and carefully identified and selected under a stereoscopic microscope.
Point 17: Line 135 define what you mean by ‘desired food’
Response 17: According to the comment of the reviewer, the ‘desired food’ was revised to “the mixed algal diet”. The description of “the mixed algal diet” for feeding Acartia bifilosa were added in this section.
Point 18: Line 143 define how you selected and collected the microalgae provided as food
The results were treated as totals?
Response 18: According to the comment of the reviewer, the description of “the mixed algal diet” for feeding Acartia bifilosa were added in this section.
Point 19: In 2.3 did you check for normality and collinearity and inflation factors and statistical significance? Please provide the necessary information
Response 19: According to the comment of the reviewer, the description of statistical significance was added in the section and Results.
Results
Point 20: Please provide the results presented in 3.1 also graphically
Response 20: According to the comment of the reviewer, the distribution of Acartia bifilosa abundance in the Jiaozhou Bay from June to August is shown in new Figure 2.
Point 21: Line 191-192 not clear what you mean ‘in the end several days’
Response 21: ‘in the end several days’ means “during the experiment period”. This sentence is confusing. According to the comment of the reviewer, this sentence has been revised.
Point 22: Line 199-200 what do you mean?
Response 22: This sentence is confusing and useless. According to the comment of the reviewer, this sentence has been deleted.
Point 23: Line 208 what do you mean ‘batches of five females could not produce’?
Response 23: The “batches” originally means the experiment treatments. According to the comment of the reviewer, this sentence has been revised.
Point 24: Line 226 use square brackets
Response 24: According to the suggestion of the reviewer, this sentence has been revised.
Point 25: Table 1 explain what is the number following the average
Response 25: The number following the average is error bar of the six replicates. According to the comment of the reviewer, this sentence has been revised.
Point 26: Figure 3 explain what are the line bars
Response 26: The line bar is the error bar of the six replicates in each treatment.
Point 27: Line 235-237 how is this connected to resting eggs?
Response 27: The resting eggs of A. bifilosa are spiny or crinkle (Castro-Longoria and Williams, 1999). All eggs produced in our experiments are smooth.
Point 28: Line 238 not clear what you mean ‘incubated beyond 3 days’
Response 28: This sentence means that the unhatched eggs were hatched for three more days after six days incubation. The “incubated beyond 3 days” has been revised to “hatched for 3 more days”.
Point 29: Line 240 what do you mean ‘late experimental period’
Response 29: According to the comment of the reviewer, the sentence containing ‘late experimental period’ has been revised. Meanwhile, new figure 5 (Original Figure 4) has also been redrawn.
Point 30: Line 255-256 it is not common to use references in the results section
Response 30: According to the suggestion of the reviewer, this sentence with reference has been moved to the Material and Methods section.
Point 31: Line 263 these results are not clearly seen in the figure
Response 31: The developmental times of A. bifilosa at four different temperatures and three food concentrations are similar in new Figure 6. Please check the clear data from Table 1.
Point 32: Line 276 and figure 6 not clear to me what does the cluster show… please explain the data used to perform it.
Response 32: At all four experimental temperatures, A. bifilosa in the 0 g/L Chl a treatment could not produce eggs throughout the experimental period. The corresponding hatching success and post-embryonic development experiments were not conducted. So, according to the four temperatures (20, 22, 25, and 28 °C) and three food levels (5, 10, and 15 μg/L Chl a), there were 12 treatments in our experiment. The temperature and food affect the population recruitment of A. bifilosa at the same time. Different combinations of the two factors may have the same or different effects. Cluster analysis can be used to analyze and classify the effects of 12 treatments on the population recruitment of A. bifilosa.
Point 33: Table 2 please provide the actual values of the correlations
Response 33: According to the suggestion of the reviewer, the actual values of the correlations have been supplemented in Table 2.
Discussion
Point 34: A general comment about the discussion is that in extended areas it seems that sentences are simply written with obvious connection in order to provide a logical meaning. In the discussion it would be interesting to discuss the spatial variation recorded from the field sampling. The sentences need to be connected in order to lead to a reasonable explanation. When doing so, I wonder would there be an explanation-discussion relating the oversummering of A. fibilosa with it residing in deeper colder waters?
Response 34: Firstly, the Jiaozhou Bay is a semi-closed and shallow bay, with an average depth of less than 10m and maximum depth of 35m. The different of the spatial distribution of seawater temperature in the Jiaozhou Bay is not significant. Additionally, according to the previous studies, the distribution of A. bifilosa abundance was not correlated with the seawater temperature. So, the high-temperature seawater could not limit A. bifilosa residing in the deeper colder water area. We suggest that A. bifilosa showed its own unique population recruitment and over-summering strategy in Jiaozhou Bay.
Point 35: Line 300-302 Try and write this like: ‘It is well known that higher temperatures increase the energy cost of both females and larvae regarding copepod metabolism. Several studies have shown that the upper temperature tolerance limits of A. bifilosa are different in different habitats in the world, while the combined effects of ocean acidification and global warming predicted for the year 2100 on A. bifilosa, showed that higher temperatures could decrease egg viability and nauplii development of A. bifilosa. More specifically, …..’
Response 35: According to the suggestion of the reviewer, these sentences have been revised. Thank you very much.
Point 36: Line 305 what is Mundaka?
Response 36: Here should be “in the small temperate estuary of Mundaka, Spain”. This sentence has been revised.
Point 37: Line 354-359 Did you do such an analysis?
Response 37: In our study, the shapes of the A. bifilosa eggs have been examined during counting the number of eggs. The resting eggs of A. bifilosa are generally spiny or crinkle (Castro-Longoria and Williams, 1999). All eggs produced in the egg production experiments are smooth. These results were supplemented in Section 3.4.
Point 38: Line 365-367 My question would probably be answered if in the introduction (as I mentioned above) basic information will be provided. However, I wonder are resting eggs produced when unfavourable conditions are coming or are they produced when conditions are excellent. At least is the way things work in rotifers which are much more dependent on the production of resting eggs. Finally, it would be in favour of the article if the ecological implications of yours results are clearly discussed
Response 38: Our present study was aimed to comment the results of Zhong and Xiao (1999) that the over-summering life history strategy A. bifilosa in the Jiaozhou Bay was resting eggs. Combined with the field investigation and laboratory simulated culture experiments, we found the A. bifilosa female could live at 28 °C (the highest temperature in the Jiaozhou Bay) with ≥ 5 mg/L Chl a food condition. Furthermore, we also confirmed that the population recruitment of A. bifilosa under the extreme conditions of summer temperatures and food levels with laboratory experiments.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
you have addressed some concerns
Reviewer 2 Report
The authors improved the manuscript and satisfactorily replied to reviewer comments. Three minor remarks.
Line 175 – Acartia bifilosa should be written italic
Line 201 – “m3” please correct the index
Line 317 – it is not necessary to write “negative” if you have “-“ with the r-value.
