Differential Metabolic Changes in Zebrafish Embryos Are Induced by Discontinued Citalopram Exposure

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Cooper et al. studied the effects of the antidepressant citalopram on developing zebrafish. The study employed a metabolomics approach to assess developmental disruption in the zebrafish and report that glycine, serine, and threonine metabolism, as well as putrescine pathways were altered in one or more concentrations of citalopram.  Heart rates were also assessed and increased with exposure. The manuscript is well written although the writing can be more succinct.

Specific comments.

The introduction is too long and can be shorted by 1 page.  For example, Ln 67 to 89 is not overly relevant to the study. The  introduction contains extraneous information.

Please add hypothesis to the introduction (last paragraph).

Details on zebrafish husbandry are needed, including the strain of Danio (and where derived), salinity, dissolved oxygen, pH, and conductivity. Add size of tanks and age of fish used to generate eggs.

In section 2.1, please add  justification for chemical concentrations and time points assessed.

Please add some details on how heart rate was measured.

Figure 2 captions state it is a box and whisker plot, but this is not true. Panel B please check. Why the use of violin plots – these are not typical and are less intuitive than box plots. In figure caption, remove p values, and add sample sizes. Add significance (asterisk to groups different than control (Panel A).

Add statistical output for body length and heart rate (degrees of freedom, test statistic, actual p-value). This is good statistical reporting.

Can remove reference to figures in discussion – more appropriate to retain in results only.

Resolution on Figure 5 is low.

Author Response

Specific comments.

1) The introduction is too long and can be shorted by 1 page.  For example, Ln 67 to 89 is not overly relevant to the study. The  introduction contains extraneous information.

Thank you for this suggestion. The introduction has been shorted and information regarding the role of serotonin in the fetus has been removed. We do believe however, there was information relevant to the study in the suggested lines, particularly the information regarding the role of serotonin during the embryonic period (Lines 58-74). This information is essential to proving readers with a foundation for the significance of this study and why use of an SSRI during pregnancy could have untargeted effects on embryonic development. It also sets the stage for our requisite use of an embryonic model. This section has now been significantly revised to retain only the minimal required information.

2) Please add hypothesis to the introduction (last paragraph).

Thank you for this suggestion. We have now clarified our central hypothesis by adding the following on lines 159-161:

“We hypothesized that a transient, low dose citalopram exposure to zebrafish embryos would result in a metabolic shift similar to that observed in the aforementioned transcriptomic analysis.”

3) Details on zebrafish husbandry are needed, including the strain of Danio (and where derived), salinity, dissolved oxygen, pH, and conductivity. Add size of tanks and age of fish used to generate eggs.

Thank you for this suggestion, details on zebrafish husbandry have been included on lines 168-171:

“Four to six month old adult wild type adult Danio rerio (zebrafish) (Carolina Biologics) were maintained under standard conditions in fish water at a temperature of 28(+/- 1 ℃) in 10 gallon tanks. Dissolved oxygen was maintained at 7.8 mg/L. Water pH ranged from 7-7.6 and conductivity from 400-800 µS/cm.”

Salinity was not independently determined since it correlates with conductivity.

4) In section 2.1, please add  justification for chemical concentrations and time points assessed.

Thank you for this suggestion. The following has been added to lines 175-178 for justification of the citalopram concentrations:

                “At 0-2 hpf, clutches were divided into one control group and four citalopram exposure groups: 0.03 ng/mL (environmental surface water concentration), 0.9 ng/mL (wastewater treatment plant effluent), 50 ng/mL (therapeutic human serum concentration), and 250 ng/mL (supertherapeutic human serum concentration) [48].”

The following has been added to lines 185-187 for justification of the time points assessed:

“The first 24 hours of zebrafish embryo development correspond to the first month of human development. 48 hpf is analogous to the first 8 weeks of human gestation (ref 49).“

5)Please add some details on how heart rate was measured.

Thank you for this suggestion, the following information on how heart rate was measured has been added.

Line 197-198:

“Heart rate was measured using live video feed by counting the number of beats in 30 seconds and multiplying by two [50].”

6) Figure 2 captions state it is a box and whisker plot, but this is not true. Panel B please check. Why the use of violin plots – these are not typical and are less intuitive than box plots. In figure caption, remove p values, and add sample sizes. Add significance (asterisk to groups different than control (Panel A).

Thank you for this comment.

Figure 2, panel A on line 280 has been updated to report that it is a violin plot.

Violin plots were utilized to show the full distribution of the data. Box and whisker plots only show summary statistics, but violin plots provide this data in addition to showing the distribution pattern of the data in an intuitively interpretable way. We do appreciate that violin plots are a more recent addition to visualization approaches for statistical analyses, however, they are now well accepted tool in omics analyses. Therefore, we respectfully request to retain the figure in the current form.

In the figure caption, the p-values have been removed and the sample sizes have been added. Line 282-283:

“ANOVA shows significant differences between heart rates for control and citalopram exposed embryos at 24 hpf (n=5) at 48 hpf (n=5), and at 72 hpf (n=5).”

Thank you for suggesting the addition of asterisks to groups significantly different from the control. We had also considered this, but we elected not to add them because it makes the graph rather busy, therefore taking away from the message. Additionally, asterisks were not added to groups different from the control in panel A because the comparison was an ANOVA. To address your point that this information is still valuable and should be included, statistical output for the body length and heart rate data has been added to the supplemental information in the form of tables (see next point below) and referred to in the main text on lines 277-278 and 290-291.

“Complete statistical outputs for all HR are included in Supplemental Table 1.”

“Complete statistical outputs for body lengths are included in Supplemental Table 2.”

7) Add statistical output for body length and heart rate (degrees of freedom, test statistic, actual p-value). This is good statistical reporting.

Thank you for this, we value the importance of transparent and complete statistical reporting. A supplemental table with the complete statistical outputs has been added to the supplemental information and referred to in the manuscript on lines 277-278 and 290-291.

 

	p-value	Control mean	0.03 ng/mL mean	Standard error of difference	Degrees of freedom	Test statistic
24 hpf	0.90	57.6	58.0	3.3	8	0.11
48 hpf	0.0083	111.2	135.2	6.9	8	3.4
72 hpf	0.00015	159.2	128.8	4.5	8	6.7
	p-value	Control mean	0.9 ng/mL mean	Standard error of difference	Degrees of freedom	Test statistic
24 hpf	0.59	57.60	59.2	2.83	8	0.57
48 hpf	0.014	111.2	134.8	7.57	8	3.12
72 hpf	0.029	159.2	147.2	4.53	8	2.65
	p-value	Control mean	50 ng/mL mean	Standard error of difference	Degrees of freedom	Test statistic
24 hpf	0.013	57.60	68.00	3.25	8	3.200
48 hpf	0.031	111.2	126.4	5.80	8	2.62
72 hpf	0.011	159.2	144.0	4.63	8	3.28
	p-value	Control mean	250 ng/mL mean	Standard error of difference	Degrees of freedom	Test statistic
24 hpf	>0.99	57.60	57.6	2.59	8	0.000
48 hpf	0.31	111.2	116.0	4.45	8	1.078
72 hpf	0.0044	159.2	134.4	6.33	8	3.92

Supplemental Table 1: Complete statistical output for analysis of heart rate data.

 

 

	p-value	Control mean	0.9 ng/mL mean	Standard error of difference	Degrees of freedom	Test statistic
72 hpf	0.062	2.6	2.4	0.099	14	2.02

Supplemental Table 2: Complete statistical output for analysis of body length.

 

8) Can remove reference to figures in discussion – more appropriate to retain in results only.

Thank you for this suggestion. We respectfully request to retain references to the figures in the discussion to support our arguments with the visual evidence. In our opinion, this enables the reader to connect the data and interpretations without ambiguity.

9) Resolution on Figure 5 is low.

Thank you for noticing this. We have replaced Figure 5 with a higher resolution image per journal requirements of 300 dpi.

Reviewer 2 Report

Comments and Suggestions for Authors

1. The manuscript mixes units (ng/mL vs µg/L) across sections and figures. Standardize units throughout the manuscript
2. The rationale for selecting a 24 h exposure followed by 24 h drug-free development should be strengthened.
3. For zebrafish exposure and maintenance methods cite this reference for strengthing the studies. https://doi.org/10.1016/j.fct.2021.112587 
4. How was the heart rate was calculated, mention it in methods

5.  Mention the ethical clearance number

    

6.  Grouping of zebrafish for this experimental studies should be clearly mentioned in the methods

    

7. The result of the present study should be interpreted with recent relevant studies

    

8. The clarity of the figures should be improved

    

9. Throughout the manuscript the grammar and spacing error should be checked in the manuscript

    

10.  The hypothesis of the study should be clearly mentioned in the introduction 

Author Response

1. The manuscript mixes units (ng/mL vs µg/L) across sections and figures. Standardize units throughout the manuscript

Thank you for catching this. We have standardized units to ng/mL throughout the manuscript.

2. The rationale for selecting a 24 h exposure followed by 24 h drug-free development should be strengthened.

Thank you for this suggestion as this exposure protocol is a key point of our study.

The rationale for the discontinued protocol can be found on lines 134-136 of the introduction:

“Most of these studies have relied on constant exposures, unable to differentiate between acute changes (due presence of the toxicant at the point of harvest) and changes that persist after exposure has ceased. Such persistent changes after discontinued chemical exposure have the potential to cause lasting developmental perturbations [46-48].”

Additionally, the rationale is reinforced in the Materials and Method section on lines 182-189:

“To investigate the impact of a discontinued citalopram exposure on embryonic development, at 24 hpf the embryos from each dish were washed with Danieau three times and placed into a new dish with fresh 0.3x Danieau without citalopram. The embryos were then returned to the incubator for another 24 hours. At 48 hpf, the embryos were collected for morphological analyses or were harvested for metabolomics. The first 24 hours of zebrafish embryo development correspond to the first month of human development. 48 hpf is analogous to the first 8 weeks of human gestation [49].”

3. For zebrafish exposure and maintenance methods cite this reference for strengthening the studies. https://doi.org/10.1016/j.fct.2021.112587 

Thank you for your suggestion; however, this suggested reference does not seem applicable to our study. More information on zebrafish husbandry and the exposure protocols has been added to strengthen this section of the manuscript. Please see the lines below.

Details on zebrafish husbandry have been included on lines 169-172:

“Four to six month old adult wild type adult Danio rerio (zebrafish) (Carolina Biologics) were maintained under standard conditions in fish water at a temperature of 28(+/- 1 ℃) in 10 gallon tanks. Dissolved oxygen was maintained at 7.8 mg/L. Water pH ranged from 7-7.6 and conductivity from 400-800 µS/cm.”

Lines 176-179:

                “At 0-2 hpf, clutches were divided into one control group and four citalopram exposure groups: 0.03 ng/mL (environmental surface water concentration), 0.9 ng/mL (wastewater treatment plant effluent), 50 ng/mL (therapeutic human serum concentration), and 250 ng/mL (supertherapeutic human serum concentration) [48].”

The following has been added to lines 183-189 for justification of the time points assessed:

“To investigate the impact of a discontinued citalopram exposure on embryonic development, at 24 hpf the embryos from each dish were washed with Danieau three times and placed into a new dish with fresh 0.3x Danieau without citalopram. The embryos were then returned to the incubator for another 24 hours. At 48 hpf, the embryos were collected for morphological analyses or were harvested for metabolomics. The first 24 hours of zebrafish embryo development correspond to the first month of human development. 48 hpf is analogous to the first 8 weeks of human gestation [49].”

4. How was the heart rate was calculated, mention it in methods

Thank you for this suggestion, the following information on how heart rate was measured has been added.

Line 199-201:

“Heart rate of randomly selected embryos (n=5 per group) was measured using live feed by counting the number of beats in 30 seconds and multiplying by two [49].”

5. Mention the ethical clearance number

Thank you for this suggestion, we agree that all information regarding use of animals in research is important. All information regarding IRB approval can be found on lines 638-639:

Institutional Review Board Statement: The animal study protocol was approved by the IACUC Institutional Review Board of Montana State University (protocol number 2022-125-IA, approval date 10 August 2022).

6. Grouping of zebrafish for this experimental studies should be clearly mentioned in the methods

 Thank you for this suggestion. Grouping of the zebrafish has been clarified on lines 176-179.

                “At 0-2 hpf, clutches were divided into one control group and four citalopram exposure groups: 0.03 ng/mL (environmental surface water concentration), 0.9 ng/mL (wastewater treatment plant effluent), 50 ng/mL (therapeutic human serum concentration), and 250 ng/mL (supertherapeutic human serum concentration) [48].”

7. The result of the present study should be interpreted with recent relevant studies

 We agree that the results should be interpreted with recent relevant studies; however, we believe that we have done so accordingly in the discussion. Please see lines 435-437; 441-443; 447-450; 463-466; 486-500; 523-525; 544-556; 586-590; and 600-602.  

8. The clarity of the figures should be improved

 Thank you for this suggestion, the quality of the figures has been improved to the journal requirement of 300 dpi.

9. Throughout the manuscript the grammar and spacing error should be checked in the manuscript

 Thank you for this suggestion, grammar and spacing has been checked throughout.

10. The hypothesis of the study should be clearly mentioned in the introduction 

Thank you for this comment. The following hypothesis was added on line 159-161:

“We hypothesized that a transient, low dose citalopram exposure to zebrafish embryos would result in a metabolic shift similar to that observed in the aforementioned transcriptomic analysis.”