Effects of the Invasive Fish Species Ameiurus nebulosus on Microbial Communities in Peat Pools
, Wojciech Płaska, Małgorzata Adamczuk, Magdalena Toporowska and Aleksandra BartkowskaRound 1
Reviewer 1 Report
The paper is simple and clear, the theme is interesting, the study of food chain in this type of environments deserves to be continued.
Author Response
Than you for review of my ms.
Reviewer 2 Report
The authors analyze the impact of an alien and invasive fish species functioning of peatlands, more detailed on the on the microbial communities and small metazoa (phycoflora, bacteria, heterotrophic flagellates, ciliates, and crustaceans) in laboratory experiments, considering also the effect of increasing temperature in a scenario of global warming.
The study involves an area with scarce scientific knowledge and it is well conducted in terms of methodology and conclusions are clear. The presence of the exotic species (brown bullhead) shows in fact a decrease in planktonic crustaceans and an increase in ciliates (predators) in the microbial community, whereas increasing temperature reduces body size.
However, I think that some aspects need still some attention:
_In the objectives I don't see a clear difference between the 2 hypotheses
_ In the discussion it is not conveniently treated the relation between environmental variables and their influence on phycoflora and microbial communities, namely based on CANOCO ordination.
_it is not clear from the data obtained with the chemical variables that alien species and climate warming may favor eutrophication. This assumption needs to be supported by data.1801soverc
Author Response
In the objectives I don't see a clear difference between the 2 hypotheses:
- we rejected hypotheses 1 and now is one hypotheses: „The objective of this study was to test the following hypotheses: predation pressure from ‘top predators’ (brown bullhead) significantly influences the abundance and size structure of aquatic organisms. This effect increases with temperature, as according to the ‘metabolic theory’ [14] an increase in temperature enhances the effect of the ‘top-down’ regulation mechanism. “
_ In the discussion it is not conveniently treated the relation between environmental variables and their influence on phycoflora and microbial communities, namely based on CANOCO ordination.
- as suggested we added this information in discussion section: l. 386-397 „The increase in the proportion of cyanobacteria was due to the increased pH and fertility of the water in the +F treatments as well as to the simulated increase in temperature. An increase in the contribution of cyanobacteria following experimental fertilization of Sphagnum peatlands was also observed by Gilbert et al. [40]. The results indicate that the organic matter in the water increases with the temperature increase. In the experimental treatments the abundance of bacteria, phycoflora and protozoa was strongly correlated with organic matter. Therefore it seems that organic matter may affect the abundance of these microorganisms. This is also supported by research by Gilbert et al. [40] and Fischer et al. [41], but according to the authors it was not the content of mineral compounds and organic compounds that influenced microbial abundance, but their availability, primarily determined by the presence of refractory organic compounds utilized after enzymatic decomposition.” and l. 410-417 “The increase in the fertility of the water was most likely due to excretion by brown bullhead, as well as to enrichment of the water with biogenic compounds from sediments as a result of their resuspension by the fish. It appears that the combined effect of the increase in temperature and the concentrations of biogenic compounds may significantly affect the diversity and abundance of protozoans. This is also confirmed by research in lake ecosystems [42]. A study by Zingel et al. [43] showed that in eutrophic and hypertrophic water bodies the abundance and biomass of ciliates is often more than 8 times as high as in oligotrophic water bodies.”
_it is not clear from the data obtained with the chemical variables that alien species and climate warming may favor eutrophication. This assumption needs to be supported by data.1801soverc
- we explained in conclusion that: l. 472-473 „The overlapping effects of alien species and gradual climate warming may intensify eutrophication (increase in the concentrations of nutrients at the highest temperature in +F treatments) of peatland ecosystems and the increase in the proportion of cyanobacteria, thereby affecting the carbon cycle in these ecosystems.”
Reviewer 3 Report
GENERAL COMMENTS
I found this an interesting and well-delineated manuscript about the effects of an invasive fish (A. nebulosus) on the microbial communities of peat pools. Overall, it is well-written and structured, but there some points in the M&M that need clarifications. In the Results, authors should provide the statistics (e.g. p level, F statistic) of their results, or whenever they say that they identified significant differences. Captions to figures and tables also need improvement.
SPECIFIC COMMENTS
L3 – Remove the parenthesis from the title. Suggest instead: “ - an experimental approach.”
L23 – To make the abstract more appealing, I suggest add a final sentence of conclusions/take-home message (i.e. the implications of your results).
L82 – Any picture/map of the study area? This would improve the perception of readers. What is the size/area of the pools?
L103 – How was the temperature increased? A heater? Provide brand and model.
L104 – plural for aquarium is aquaria.
L149 – provide brand and model.
L192 – “was significantly varied”. What you mean? This sentence does not sound well; please re-write clearer.
L194 – “highest values were noted in the +4°C treatments and the lowest in the control treatments”. If you increased the temperature in 4C this would be more than expected, right? Suggest to remove.
Table 1 – provide description of the acronyms, either in the caption or as footnote to the table. Same comment for figure 1.
L206-234 – “significantly higher”….. Provide test statistics (P value, F) for this and other results marked as significant throughout this section.
Figure 1 – I suggest you marked the significant differences referred in the previous paragraph, with an asterisk (*) above the columns, for a better perception (* P< 0.05).
L243 – what was the increase? %? Was significant? Be specific for this and other findings.
L244 – same comment here. How more???
L245 – What was the increase?? Please be specific in your results, this is too vague.
L247-248 – Same comments as above.
L267 and throughout the manuscript – “Changes in biomass of..”. Changes in response to what? Please give complete captions in all you figures and tables.
L272-273 – Details here too, please (increased, decreased,..; how increased/decreased? Significant? %?).
L274- How sharply? Was this decrease significant? Provide details/statistics.
L277 – Increased substantially? In what way? Details please!
L270-283 – “Figures 2a-g”. There are 7 graphs here. I suggest that for each of your results, you specify the letter to which it applies.
L290-291 – “The first two CCA axes explained 31.8%”. On L287 you referred 81.2%. So once again, which concern a and which ones concern b in “Figure 3a-b”. What represents each of the axes?
L303 – P> 0.05? Please check.
L302-303 – Perhaps elucidate what represents the upper values and lower values.
L323-331 – Please provide statistics (P level) for significant variations/differences.
L336-419- This is a very long section and difficult to read at once. Please make at least 3 paragraphs of this section!
L376 – add comma after “bodies”.
L395 – add comma after “temperature”.
Author Response
As suggested we added results of statistical analyses p and F.
SPECIFIC COMMENTS
L3 – Remove the parenthesis from the title. Suggest instead: “ - an experimental approach.”
- we removed in the title „an experimental approach”
L23 – To make the abstract more appealing, I suggest add a final sentence of conclusions/take-home message (i.e. the implications of your results).
- we added o final sentence of conclusion „The overlapping effects of alien species and gradual climate warming may intensify eutrophication of peatland ecosystems and the increase in the proportion of cyanobacteria, thereby affecting the carbon cycle in these ecosystems.”
L82 – Any picture/map of the study area? This would improve the perception of readers. What is the size/area of the pools?
- we added map of study area – now Figure 1, information on ponds size is in l. 85 (area 0.5 ha, max. depth 2.2 m)
L103 – How was the temperature increased? A heater? Provide brand and model.
- we added information: l. 414-416 „Temperature manipulation was made possible by a TX-30 temperature modification system placed under the surface of the water in each enclosure. Mercury thermometers were additionally used to control the temperature.”
L104 – plural for aquarium is aquaria.
- we corrected on „aquaria”
L149 – provide brand and model.
- we added model: l. 159 „Nikon SMZ800”
L192 – “was significantly varied”. What you mean? This sentence does not sound well; please re-write clearer.
- we corrected on: l. 202 „The water temperature varied between treatments (ANOVA, F1.33 = 63.0-64.2, p < 0.001).
L194 – “highest values were noted in the +4°C treatments and the lowest in the control treatments”. If you increased the temperature in 4C this would be more than expected, right? Suggest to remove.
- we removed this sentence
Table 1 – provide description of the acronyms, either in the caption or as footnote to the table. Same comment for figure 1.
- we added information : Table 1. Changes in physical and chemical parameters in six experimental mesocosms. Key: reaction (pH), dissolved oxygen (DO), total phosphorus (Ptot), phosphates (P-PO43-), total nitrogen (Ntot), ammonia nitrogen (N-NH4), nitrite nitrogen (N-NO2-), chemical oxygen demand (COD), biological oxygen demand (BOD), total suspended solids (TSS) and total organic carbon (TOC), treatments −FCT (control), −FT+2°C (treatment without fish with temperature increased by +2°C relative to the control), and −FT+4°C (treatment without fish with temperature increased by +4°C relative to the control), and treatments with fish: +FCT (control), +FT+2°C (treatment with temperature increased by +2°C relative to the control), and +FT+4°C (treatment with temperature increased by +4°C relative to the control). And in Figure 1 (now figure 2): Figure 2. Numbers of (a) phycoflora; (b) bacteria; (c) heterotrophic flagellates; (d) testate amoebae; (e) ciliates; (f) cladocerans; (g) copepods in experimental mesocosms; -F treatments without fish, +F treatments with fish, CT – control treatments, +2oC - treatment with temperature increased by +2°C relative to the control, +4oC - treatment with temperature increased by +4°C relative to the control.
L206-234 – “significantly higher”….. Provide test statistics (P value, F) for this and other results marked as significant throughout this section.
- as suggested we added this information: l. 224 (F1.33 = 62.71, p < 0.001)., l. 232 (F1.33 = 61.21, p < 0.001), l. 238 (F1.32 = 61.41, p < 0.001), l. 241 (F1.33 = 59.24, p < 0.001), l. 246 temperature (F1.33 = 63.34, p < 0.001)
L243 – what was the increase? %? Was significant? Be specific for this and other findings.
- Now l. 263 we added „(from 34% to 58% in the total abundance of ciliates)”
L244 -245– same comment here. How more???
- now l. 266 – we added information „(from 8 ind. ml-1 to 12 ind. ml-1); it was highest in the +4°C treatment - cladocerans: to 12 ind. ml-1 and in the +2°C treatment - copepods: to 320 ind. ml-1).”
L247-248 – Same comments as above.
- now l. 279 we added informaton „In the experiments without fish (–F), the highest biomass of phycoflora was noted in the control sample and the +4°C treatment (26 mg ml-1)
L267 and throughout the manuscript – “Changes in biomass of..”. Changes in response to what? Please give complete captions in all you figures and tables.
- we corrected on Biomass of….and added: Biomass of (a) phycoflora; (b) bacteria; (c) heterotrophic flagellates; (d) testate amoebae; (e) ciliates; (f) cladocerans; (g) copepods in experimental mesocosms; -F treatments without fish, +F treatments with fish, CT – control treatments, +2oC - treatment with temperature increased by +2°C relative to the control, +4oC - treatment with temperature increased by +4°C relative to the control.
L272-273 – Details here too, please (increased, decreased,..; how increased/decreased? Significant? %?). and 274-277
- we added this information – l. 292-297 „The reverse trend was noted for copepods (Figures 3g). In the +F experiments, the highest biomass of phycoflora was noted in the control sample, and it significantly decreased as the temperature increased (F1.33 = 61.21, p < 0.001). Biomass of bacteria, flagellates and ciliates significantly increased with temperature (F1.33 = 62.74, p < 0.001), while that of testate amoebae significantly decreased (F1.23 = 52.31, p < 0.05). Biomass of crustaceans was highest in the control sample at the start of the experiment and dropped sharply in the warmer temperature treatments (Figures 3f-g).”
L270-283 – “Figures 2a-g”. There are 7 graphs here. I suggest that for each of your results, you specify the letter to which it applies.
- we coorrected in l. 279-297
L290-291 – “The first two CCA axes explained 31.8%”. On L287 you referred 81.2%. So once again, which concern a and which ones concern b in “Figure 3a-b”. What represents each of the axes?
- as suggested we corrected on l. 308-314: „Canonical correspondence analyses showed that environmental variables were more significant for the development of aquatic communities in fishless environments. In these environments the first two axes explained 64.2% of the variance in the aquatic communities (Figure 4a). Although some communities showed clear relationships with individual environmental parameters, only pH, temperature and nutrients had a significant effect (Monte Carlo permutation test, λ = 0.84, F = 5.94, p = 0.012). In +F treatments the first two CCA axes explained 21.6% of the variance in aquatic communities in the environment (Figure 4b).
L303 – P> 0.05? Please check
We corrected on p>0.05
L302-303 – Perhaps elucidate what represents the upper values and lower values.
We corrected: Table 2. Linear correlation coefficients between microbial components and metazoan in the investigated treatments, Phy – phycoflora; B – bacteria; HNF – heterotrophic flagellates; TA – testate amoebae; C – ciliates; Cr – crustaceans (cladocerans + copepods). p > 0.05, treatments −FCT (control), −FT+2°C (treatment without fish with temperature increased by +2°C relative to the control), and −FT+4°C (treatment without fish with temperature increased by +4°C relative to the control), and treatments with fish: +FCT (control), +FT+2°C (treatment with temperature increased by +2°C relative to the control), and +FT+4°C (treatment with temperature increased by +4°C relative to the control).
L323-331 – Please provide statistics (P level) for significant variations/differences.
- we added: p values in l. 352-359
L336-419- This is a very long section and difficult to read at once. Please make at least 3 paragraphs of this section!
- we corrected on 3 section: 1. Diet of Ameiurus nebulosus; 2. Functioning of the food webs in treatments with Ameiurus nebulosus and 3. Functioning of the food webs in fishless treatments
L376 – add comma after “bodies”. and L395 – add comma after “temperature”.
- we corrected and added comma
Round 2
Reviewer 3 Report
After went through the revised version as well as the authors comments to my previous concerns, the manuscript has improved significantly over the previous submission. It can now be accepted on its present state.
