Ionic Stress Prompts Premature Hatching of Zebrafish (Danio rerio) Embryos
Round 1
Reviewer 1 Report
As a consequence of a number of factors, including rising sea levels (climate change) and road salt run-off, there is increasing concern over the ecological consequences of the salination of freshwater habitats. This is especially true for the more vulnerable juvenile stages (eggs and larvae) of aquatic organisms, including fish. This study investigates the consequences of increased salinity on embryo development and hatching success in a freshwater fish, the zebrafish.
Before this paper can be considered for publication the author needs to address the below comments.
- In the Introduction (Line 29) the author states that the salinity of FW habitats is exacerbated by both rising sea levels as a consequence of global climate change, and by road salt run-off. The author should indicate the range of reported salinity levels as a result of road salt run-off. Also, the author could mention that an additional cause for the increased salinity of FW habitats is saltwater intrusion as a consequence of intensive aquaculture. This is especially prominent in SE Asia as a consequence of intensive shrimp farming.
- Line 40. Change ‘mature’ to ‘adult’
- The author should have discussed the possible influence of the strain of ZF used in this study. ZF are highly domesticated and many different strains are available commercially. As the common husbandry practice is to rear fish under low salinity conditions (0.25-0.75 ppt), has domestication influenced salinity tolerance. It would have been interesting to compare with a more ‘wild-type’ strain.
- Along the same lines, as current evidence indicates that ZF do display some salinity tolerance, it would also have been interesting to compare embryo responses between ZF and a FW species that is truly stenohaline.
- From Figure 1, the author predicted that the LC50 was 5.6 ppt. Given the very wide spread in mortality at 5 ppt (for the 3 replicates), the author should have repeated the study focusing on exposing embryos to a salinity range around the predicted LC50 value (for example, 4, 5, 6 and 7 ppt). Figure 1 needs to be cited in the text.
- Figure 2. The author needs to check the statistical analysis of this figure, particularly the best fit line for the 5 ppt group. As it stands, the curve shows that some hatching occurs immediately after 24 hours, reaching 25% hatching at 36 hours. I suspect that this best fit line is incorrect between 24 and 48 hours. To verify advanced hatching in the 5 ppt group, earlier observations (for hatching) should have been made (ie at 36 and 42 hours).
- The MS would have been improved if the author had recorded the incidence of larval deformities following hatching.
Author Response
I would like to thank the reviewer for their time and their insightful comments on the manuscript. Please see responses to the comments below.
1) A sentence has been added regarding the range of salinities resulting from roadside salt run-off, and another regarding aquaculture as an additional source of salt contamination to FW ecosystems, both citing published literature.
2) Mature changed to adult
3 and 4) A paragraph has been added about the possible influence of domestication and the insight that could be gained from inter-strain and inter-species comparative studies
5 and 6) Although I agree that further insight could have been gained from repeating the experiments, for instance as suggested by the reviewer: examining a finer range of salinities around 5ppt and examining a finer range of hatching timepoints around 48hpf, unfortunately this is not possible as I no longer have access to the facilities and resources. I have, however, added an analysis of some additional data which was not initially included because it is not directly comparable with the data originally presented (slightly different experimental design in that embryos were exposed at a later dev. stage). However, this additional analysis complements the existing data as it again appears to show that hatching occurs earlier in response to 5ppt salinity.
7) In response to concerns that the curve fits were inappropriate for Fig. 2, the display format for the data has been changed to mean+/- SEM of each treatment with each time point connected by lines.
8) Some deformities were noticed in the 5ppt group, but were not formally recorded or quantified.
Reviewer 2 Report
This study investigated the effects of increasing salinity on the mortality and hatching time of freshwater model fish Dania rerio. The 24-h LC50 was 5.6 ppt, and 5 ppt caused earlier hatching. The manuscript is well written and easy to follow. The results, in general, correspond with the research aims. The reviewer has a major concern on the biological replicates used in the exposure. Line 61 and line 86 indicate that N = 3 dishes of 30 embryos per treatment level. However, Line 121 says 3 clutches of fertilized eggs were derived from 3 separate sets of mixed-sex mature fish and each clutch was considered to be a biological replicate. From my understanding, each clutch/brood has only one dish of 30 embryos per treatment level (i.e., N =1). This could be problematic because as shown in the both Fig.1 and Fig.2, at 5 ppt, there were huge variations of data, which could be due to genetic differences among different clutches/broods, as pointed out in line 81. Therefore, the experiment should be better designed to have 3 dishes of 30 embryos per brood per treatment level. Alternatively, the whole exposure with original design could be repeated, at least for the 5 ppt treatment group. Another major concern is regarding the use of the term ‘sublethal’. The authors considered 5 ppt as sublethal, but in fact 5 ppt killed half (very high) of the embryos in one replicate. Based on the date in Fig. 1, a concentration between 2-5 ppt looks more reasonable to be sublethal. Better judgement should be given on the reason of using of ‘sublethal’. The above comments need to be addressed before this article to be considered for publishing in Fishes. See other minor comments below.
Line 34 References are missing.
Line 79-80 ‘…misidentified as an earlier developmental stage?’ Weren’t 4‐64‐cell stage embryos < 2 hpf selected for exposure? 4-64 stages are easy to identify.
Author Response
I would like to tank the reviewer for their time and for their useful and insightful comments on the manuscript. Please see my responses, below.
Line 34: Additional literature has been cited in this paragraph regarding anthropogenic sources of environmental salinity
The reviewer is correct that there was no 'within-brood' replication As I considered each brood to be biologically independent, this is why I considered it to be N=3. However, I realise that this may have been unclear. Therefore I have 1) made it clear in the figure legends what each datapoint represents (e.g. a single dish or the mean of three dishes), and 2) acknowledged the lack of within-brood replication and conceded that this renders the variation in response at 5ppt difficult to explain.
Unfortunately, I no longer have access to the animals or facility where the experiment was carried out, and so I am unable to repeat the experiment with additional replicates as suggested by the reviewer. I have, however, added an analysis of some additional data which was not initially included because it is not directly comparable with the data originally presented (slightly different experimental design in that embryos were exposed at a later dev. stage). However, this additional analysis complements the existing data as it again shows that hatching occurs earlier in response to 5ppt salinity.
Use of the term sublethal has been removed as the reviewer is correct that 5 ppt cannot be considered sublethal when mortality did indeed occur. For the title, I have replaced this with 'ionic stress' which I hope would be considered a more broadly encompassing term.
Line 79-80: Indeed, it is just one possible explanation and in hindsight it is unlikely.
Round 2
Reviewer 1 Report
Reviewer 2 Report
The author has provided additional data and addressed all previous comments and questions from the reviewer.
