Review Reports - Embryo and Larval Developmental Staging Guides for Striped Bass

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript review report

Embryo and fry developmental staging guides for striped bass to support larval rearing and hatchery management

The following review critically examines the manuscript's conceptual, theoretical, and methodological aspects to improve its quality. This review is conducted with the utmost respect for the authors' intellectual work. The manuscript aims to be a practical, modern guide to the embryo-to-juvenile development of Morone saxatilis. To date, no publication with this focus exists, so this work could help fill the gap in a guide to the early life history of Morone. However, it contains several conceptual, theoretical, and methodological errors that must be clarified before publication.

After careful review, the following suggestions and observations are made:

Line 133. In studies that describe embryonic and larval development, it is important to characterize gonadal maturation in both males and females. The effects of reproductive management on gamete and offspring quality are well documented. It is recommended to accurately describe the feeding strategy for broodstock (e.g., feed composition, feeding frequency, feeding time), the methods of gonadal maturation and spawning, as well as water quality (pH, nitrogen compounds, alkalinity, etc.). The photothermal regime should be described by specifying the times of temperature increase or decrease and the photoperiod.

Line 148. This work does involve one experiment and one observational study. It is suggested that the term "experiment" be changed to "developmental monitoring study."

Line 148. Replace “yolk sac attrition” with “yolk consumption”. The yolk sac is an extraembryonic structure; the yolk is the contents of the yolk sac. In a semantic context, "yolk consumption" is the appropriate phrase.

Line 149. The density experiment is unclear. The densities of embryos or larvae tested are not specified.

Line 152. Water quality conditions are not indicated in the mortality study.

Line 163. It is not mentioned how the yolk consumption assessment was carried out, for example, the number of samples per sampling time, sampling frequency, whether the images taken were of live anesthetized larvae or not.

Line 165. Why was the area of the yolk and oil globule used instead of volume? Area alone tells us little, as does the use of diameters. A better approximation is the volume of the yolk and oil globule, calculated based on the diameters of regular geometric solids. For this topic, it is suggested to review the following chapter: Heming H. and R.K. Buddington. 1988. Yolk absorption in embryonic and larval fishes, 407-446. In: Hoar W.S. and Randall D.J. (Eds.) Fish Physiology Vol. 11. Academic Press, California. 546 p.

Line 171. Replace “p = 0.05” with “p < 0.05”.

Lines 186-187. Why is 18 °C used as the reference temperature for standardization? Does this temperature correspond to the biological zero temperature? If not, what justifies "standardization" with this temperature? In fish, the usefulness of effective growing degree days (GDD) as a metric for documenting developmental progression has already been documented. If 18°C does not correspond to the biological zero temperature, the "standardization proposed by the authors" has no biological basis; at most, it would be a rescaling into dimensionless units with no biological meaning.

Line 199. Avoid using conceptually imprecise terms like fry, fingerling, or alevin. It's better to use the terms embryo, larva, or juvenile.

Line 213. n = 3; only three specimens per stage? If so, what is the reason?

Line 223. To mention a weak correlation, the P-value must be shown.

Line 231. Figure 1. This is not clear: “Data points represent mean + SD from replicate tanks (n = 10) during each hourly interval.” n = number of tanks? In the methods section, it said there were three tanks per density, not 10.

Line 231. Why talk about fry lost instead of mortality percentage?

Line 234. Figure 2. Why talk about fry loss percentage instead of mortality percentage?

Line 234. Do the points on the graph represent a single value or the average? If they represent the average, why was the variation (e.g., standard deviation) omitted?

Line 247. In this section, the level of description of the development is very superficial. If the purpose is to propose a reference guide to embryonic development, the level of detail should be in-depth. For example, it is recommended to consult works such as Kimmel et al. (1995. Stages of embryonic development of the zebrafish. Developmental Dynamics, 203, 253–310. https://doi.org/10.1002/aja.1002030302; Iwamatsu, T. 2004. Stages of normal development in the medaka Oryzias latipes. Mechanisms of Development, 121, 605–618. https://doi.org/10. 1016/j.mod.2004.03.012; Shardo, J.D. 1995. Comparative embryology of teleostean fishes. 1. Development and staging of the American shad, Alosa sapidissima (Wilson, 1811). J. Morphol., 225: 125-167.

Line 265. Figure 4 contains terminological and conceptual errors. At hour 0, a yolk sac is indicated, but this extraembryonic structure forms later. What is indicated is the yolk. 265. The blastoderm is indicated between 1 and 4 hours. There seems to be a conceptual confusion here. What is indicated is a cleaving blastodisc. The blastoderm forms during blastulation when the periderm, deep cell, and periblast layers appear. The blastoderm corresponds to the periderm and the deep cell layer. For terminology at this stage, it is recommended to consult: Carvalho, L., & Heisenberg, C. P. 2010. The yolk syncytial layer in early zebrafish development. Trends in Cell Biology, 20, 586–592. https://doi.org/10.1016/j.tcb.2010.06.009; Kunz, Y. W. (2004). Developmental biology of teleost fishes. Dordrecht, Springer.

Line 265. At 7-8 hours, the term "YSL formation" is mentioned, where the blastoderm fuses with the yolk cytoplasm. This is a misinterpretation. No histomorphological evidence is presented to support this claim. In other species, there is no fusion between the blastoderm and the yolk.

Line 266. At 13-14 hours. The germinal ring continues to invaginate. In fish with meroblastic divisions and stereoblastulae, there is no invagination during gastrulation. The involution movement gives rise to the hypoblast, which forms the germinal ring. The other movements of the blastoderm are extension and convergence.

Lines 265-268. All the images in Figure 4 are very small and lack sufficient magnification and contrast, making it difficult to see what is being described. It is recommended to enlarge the images and improve the contrast. Some images were also distorted during editing, possibly to fit the figure, resulting in the embryos appearing slightly elongated along the horizontal axis.

Line 316. 28 days post-hatching. The image shows the first evidence of scale appearance. However, this cannot be considered conclusive. To study scale development and the progression of scaling (lepidotaxy), alizarine red staining is necessary to visualize this developmental process. The brightness in the scaled region would only indicate guanine crystal deposition during iridophore differentiation. It is very possible that at this stage, there are already patches of scales in other regions of the body.

Line 343. Discussion, 4.1. Larval mortality and yolk sac attrition. Throughout this discussion section, it is suggested that there is an association between mortality and energy status during the final stage of yolk and oil globule consumption. As interpreted, this appears to be a constant event, meaning its occurrence is predictable. However, there is no discussion of the variation in mortality associated with variations in egg quality, nor of the effects of breeder management on egg quality variation. If the aim is to establish a guide to the developmental events of Morone saxatilis, it is necessary to establish and discuss the starting point of the description, which is the egg quality used in this study.

Line 388. 4.2. Embryo developmental staging guide. This section does not address the biological significance of the developmental events documented in the study. If a guide to embryonic development is intended, the critical developmental events relevant to hatchery management must be discussed in detail.

Line 394. The type of degree-day standardization proposed in this work is not useful for temperature comparisons. For that purpose, effective degree-days should be used. The remaining chapters of the discussion are similar: the guidelines' usefulness and value are mentioned, but the key aspects of development that could serve as critical points or quality criteria for practical application are not explored in depth. The scope of the results in a broader context regarding the early development of Morone or other species with similar ontogeny is not addressed.

Comments for author File: Comments.pdf

Author Response

Comment one: Line 133. In studies that describe embryonic and larval development, it is important to characterize gonadal maturation in both males and females. The effects of reproductive management on gamete and offspring quality are well documented. It is recommended to accurately describe the feeding strategy for broodstock (e.g., feed composition, feeding frequency, feeding time), the methods of gonadal maturation and spawning, as well as water quality (pH, nitrogen compounds, alkalinity, etc.). The photothermal regime should be described by specifying the times of temperature increase or decrease and the photoperiod.

Response one: Thank you for this comment. We acknowledge the importance of oogenesis, egg quality, and broodstock management in influencing larval development. Relevant aspects of egg quality and oogenesis have been included in the manuscript (Reading et al., (2018). Oogenesis and Egg Quality in Finfish: Yolk Formation and Other Factors Influencing Female Fertility. Fishes, 3(4), 45. https://doi.org/10.3390/fishes3040045). Detailed descriptions of broodstock feeding, reproductive management, spawning protocols, and photothermal conditions have been previously published (Andersen et al., (2021a). Methods of domestic Striped Bass (Morone saxatilis) spawning that do not require the use of any hormone induction. Aquaculture, 533, 736025. https://doi.org/10.1016/j.aquaculture.2020.736025) and are also described in the supplementary materials outlining the source of larvae used in this study.

Comment 2: Line 148. This work does involve one experiment and one observational study. It is suggested that the term "experiment" be changed to "developmental monitoring study."

Response 2: Thank you for this suggestion. We agree that “developmental monitoring study” more accurately describes this component of the work. The manuscript has been revised in methods section 2.2 as “One experiment and one developmental monitoring study were conducted to…” in line 150.

Comment 3: Line 148. Replace “yolk sac attrition” with “yolk consumption”. The yolk sac is an extraembryonic structure; the yolk is the contents of the yolk sac. In a semantic context, "yolk consumption" is the appropriate phrase.

Response 3: Thank you for pointing this out. We agree that “yolk consumption” is the more appropriate terminology. This change has been made throughout the manuscript.

Comment 4: Line 149. The density experiment is unclear. The densities of embryos or larvae tested are not specified.

Response 4: Thank you for pointing this out. We agree that the description of the density treatments was unclear. The range of densities was moved to section 2.2 line 152 as stated here: “1) quantify mortality of striped bass larvae in aquaria within the first three (72 hours) days post-hatch (dph) across ten larval densities (larvae/mL) ranging from 1.1 to 6.8 larvae/mL…” and densities were explicitly stated in section 3.1 line 229: “Ten larval rearing densities (1.1, 1.5, 2.0, 2.4, 3.1, 3.2, 3.9, 5.3, 6.3, 6.8 larvae/mL), based on common practice in hatchery settings, were chosen for evaluation.”

Comment 5: Line 152. Water quality conditions are not indicated in the mortality study.

Response 5: Thank you for this comment. We acknowledge that water quality conditions were not originally described. Larvae were maintained in flow-through well water under standard hatchery conditions, and because they were not fed and exposure duration was short, water quality parameters such as ammonia, nitrites, and nitrates were not expected to be limiting (Harrell et al., (1990). Culture and Propagation of Striped Bass and its Hybrids (p. 95,102). Striped Bass Committee, Southern Division, American Fisheries Society, Bethesda, Maryland). We have revised the manuscript to include the relevant temperature information including: “Water temperature was measured throughout the experiment and study.” (Section 2.2, line 171) and “The average hatchery water temperature during the sampling period was 18.9 ± 1.2 °C.” (Section 3.1, line 237).

Comment 6: Line 163. It is not mentioned how the yolk consumption assessment was carried out, for example, the number of samples per sampling time, sampling frequency, whether the images taken were of live anesthetized larvae or not.

Response 6: Thank you for this comment. We agree that additional methodological detail was needed. The manuscript has been revised to clarify the sampling approach in section 2.2 lines 166-170 as: “Yolk consumption of larvae reared solely in aquaria was assessed daily from pre-hatch through 15 dph. At each sampling time point, three live larvae were imaged once movement ceased. Yolk and lipid droplet area were measured from acquired images using a stage micrometer and calibrated measurement tool in ImageJ.”

Comment 7: Line 165. Why was the area of the yolk and oil globule used instead of volume? Area alone tells us little, as does the use of diameters. A better approximation is the volume of the yolk and oil globule, calculated based on the diameters of regular geometric solids. For this topic, it is suggested to review the following chapter: Heming H. and R.K. Buddington. 1988. Yolk absorption in embryonic and larval fishes, 407-446. In: Hoar W.S. and Randall D.J. (Eds.) Fish Physiology Vol. 11. Academic Press, California. 546 p.

Response 7: Thank you for this comment. Yolk and oil globule area were measured from two-dimensional microscopy images, and direct estimation of 3D volume was not feasible. While the yolk exists in three dimensions, all larvae were imaged and analyzed under identical conditions, allowing area to serve as a consistent proxy for relative comparisons of yolk utilization over time. The objective of this study was to evaluate changes through time rather than estimate absolute yolk volume.

Comment 8: Line 171. Replace “p = 0.05” with “p < 0.05”.

Response 8: Thank you for pointing this out. The manuscript has been revised in line 173.

Comment 9: Lines 186-187. Why is 18 °C used as the reference temperature for standardization? Does this temperature correspond to the biological zero temperature? If not, what justifies "standardization" with this temperature? In fish, the usefulness of effective growing degree days (GDD) as a metric for documenting developmental progression has already been documented. If 18°C does not correspond to the biological zero temperature, the "standardization proposed by the authors" has no biological basis; at most, it would be a rescaling into dimensionless units with no biological meaning.

Response 9: Thank you for this comment. We acknowledge that 18 °C does not represent a biological zero temperature. Instead, it was selected as a biologically relevant reference, as striped bass are known to spawn and undergo early development at approximately 18 °C under both natural and culture conditions (Andersen et al., (2021a). Methods of domestic Striped Bass (Morone saxatilis) spawning that do not require the use of any hormone induction. Aquaculture, 533, 736025. https://doi.org/10.1016/j.aquaculture.2020.736025; Harrell et al., (1990). Culture and Propagation of Striped Bass and its Hybrids (p. 95,102). Striped Bass Committee, Southern Division, American Fisheries Society, Bethesda, Maryland). As fertilization, spawning, and early embryonic development occur at this temperature, it provides a practical and consistent baseline for comparison. This clarification has been added to the manuscript (Section 2.3, lines 193–194): “A reference temperature of 18 °C was used as it reflects typical ambient spawning and early developmental conditions for striped bass under both natural and culture settings.”

Comment 10: Line 199. Avoid using conceptually imprecise terms like fry, fingerling, or alevin. It's better to use the terms embryo, larva, or juvenile.

Response 10: Thank you for pointing this out. We have applied the revision throughout the entire manuscript accordingly.

Comment 11: Line 213. n = 3; only three specimens per stage? If so, what is the reason?

Response 11: Thank you for this comment. Three larvae were measured per developmental stage to obtain an average total length for each time point. This clarification has been added to the manuscript (Section 2.4, lines 221-222): “Each image includes mean total length (mm) calculated from three larvae per developmental stage (n = 3) and a description highlighting key morphological changes.”

Comment 12: Line 223. To mention a weak correlation, the P-value must be shown.

Response 12: Thank you for this comment. We agree that the p-value should be reported alongside the correlation. The manuscript has been revised to include the corresponding p-value and clarify the strength of the relationship. This has been added to the manuscript (Figure 2, lines 245–246): “A weak, non-significant correlation was observed between density and mortality (r² = 0.0657, p = 0.47).”

Comment 13: Line 231. Figure 1. This is not clear: “Data points represent mean + SD from replicate tanks (n = 10) during each hourly interval.” n = number of tanks? In the methods section, it said there were three tanks per density, not 10.

Response 13 : Thank you for this comment. We agree that clarification was needed regarding the reported sample size. A total of 10 tanks were used, each representing a different density as described in the Results and shown in Figure 2. Figure 1 presents the average values across these 10 tank replicates. The manuscript and figure caption have been revised accordingly to improve clarity (Figure 1 description, line 243): “Bars represent average values of 10 tanks; brackets represent standard deviation.”

Comment 14: Line 231. Why talk about fry lost instead of mortality percentage?

Response 14: Thank you for pointing this out. We agree that mortality percentage more accurately describes the work instead of fry lost. Figure 1 has been revised on line 240.

Comment 15: Line 234. Figure 2. Why talk about fry loss percentage instead of mortality percentage?

Response 15: We agree that mortality percentage better fits the work rather than fry loss percentage. Figure 1 and 2 and corresponding descriptions have been revised accordingly on lines 240 through 245.

Comment 16: Line 234. Do the points on the graph represent a single value or the average? If they represent the average, why was the variation (e.g., standard deviation) omitted?

Comment 16: Thank you for this comment. The values shown represent the average across 10 tanks, and variation was already indicated by brackets representing standard deviation. To improve clarity, the figure caption has been revised to explicitly state this (Figure 1 description, line 243): “Bars represent average values of 10 tanks; brackets represent standard deviation.”

Comment 17: Line 247. In this section, the level of description of the development is very superficial. If the purpose is to propose a reference guide to embryonic development, the level of detail should be in-depth. For example, it is recommended to consult works such as Kimmel et al. (1995. Stages of embryonic development of the zebrafish. Developmental Dynamics, 203, 253–310. https://doi.org/10.1002/aja.1002030302; Iwamatsu, T. 2004. Stages of normal development in the medaka Oryzias latipes. Mechanisms of Development, 121, 605–618. https://doi.org/10. 1016/j.mod.2004.03.012; Shardo, J.D. 1995. Comparative embryology of teleostean fishes. 1. Development and staging of the American shad, Alosa sapidissima (Wilson, 1811). J. Morphol., 225: 125-167.

Response 17: Thank you for this comment and for the suggested references. We agree that highly detailed embryological staging frameworks exist for model and well-characterized teleost species. However, the objective of this study was to develop a qualitative, visually based staging guide for striped bass that is directly applicable to hatchery operations, rather than to provide a comprehensive mechanistic description of embryogenesis. In this context, the guide is intended to assist hatchery personnel in estimating developmental stage and predicting hatching timing when spawning events are not directly observed. For example, during batch spawning (Andersen et al., (2021a). Methods of domestic Striped Bass (Morone saxatilis) spawning that do not require the use of any hormone induction. Aquaculture, 533, 736025. https://doi.org/10.1016/j.aquaculture.2020.736025), embryos may be collected after fertilization has already occurred, and staging must be inferred from morphology to estimate time to hatch. Thus, the key features highlighted in this guide are those that can be readily observed under standard hatchery microscopy to support practical decision-making, rather than detailed embryological processes or pathologies. We note that early developmental timepoints (e.g., 4–6 hours post-fertilization) are particularly important for assessing embryo viability (Chapman RW, Reading BJ, Sullivan CV (2014) Ovary Transcriptome Profiling via Artificial Intelligence Reveals a Transcriptomic Fingerprint Predicting Egg Quality in Striped Bass, Morone saxatilis. PLoS ONE 9(5): e96818. doi:10.1371/journal.pone.0096818) in the discussion, whereas later stages are primarily used operationally to track developmental progression and predict hatching timeframe. Accordingly, the guide focuses on clear, observable morphological transitions that enable consistent stage identification across users. To address the reviewer’s concern, the manuscript text has been revised to incorporate additional developmental terminology and improve the level of detail while maintaining this applied focus. The revised description (Section 3.2, lines 262–275) now reads: “Embryonic development progressed through a series of morphologically distinct stages, beginning with meroblastic cleavage and blastula formation (1–10 hours), during which repeated cell divisions produced a blastodisc that expanded into a blastoderm overlying the yolk. This was followed by epiboly and early gastrulation (11–16 hours), characterized by progressive spreading of the blastoderm over the yolk and the onset of germ ring formation at the blastoderm margin. The embryonic shield subsequently became visible, indicating establishment of the dorsal axis. Head fold and tail bud formation (17–20 hours) marked the transition toward organogenesis [27]. Continued growth and differentiation were observed through 30–36 hours, including the appearance of melanophores (26 hours), formation of pigmented eyespots (30 hours), elongation of the tail bud and body axis (32 hours), and progressive compression of the abdominal yolk during organogenesis (44 hours). Development of body structures became increasingly apparent by approximately 32–36 hours with completion of epiboly, somite formation, and separation of the tail from the yolk.”

Comment 18: Line 265. Figure 4 contains terminological and conceptual errors. At hour 0, a yolk sac is indicated, but this extraembryonic structure forms later. What is indicated is the yolk. 265. The blastoderm is indicated between 1 and 4 hours. There seems to be a conceptual confusion here. What is indicated is a cleaving blastodisc. The blastoderm forms during blastulation when the periderm, deep cell, and periblast layers appear. The blastoderm corresponds to the periderm and the deep cell layer. For terminology at this stage, it is recommended to consult: Carvalho, L., & Heisenberg, C. P. 2010. The yolk syncytial layer in early zebrafish development. Trends in Cell Biology, 20, 586–592. https://doi.org/10.1016/j.tcb.2010.06.009; Kunz, Y. W. (2004). Developmental biology of teleost fishes. Dordrecht, Springer.

Response 18: Thank you for these clarifications and for the recommended references. The terminology in Figure 4 has been revised to improve accuracy and align with established developmental descriptions. Specifically, “yolk sac” at 0 hours has been corrected to “yolk,” as this stage represents the unfertilized egg prior to formation of extraembryonic structures. In addition, early cleavage stages (1–4 hours) have been revised from “blastoderm” to “blastodisc” to accurately reflect the structure present during meroblastic cleavage. The term “blastoderm” is now reserved for later stages following blastulation.

Comment 19: Line 265. At 7-8 hours, the term "YSL formation" is mentioned, where the blastoderm fuses with the yolk cytoplasm. This is a misinterpretation. No histomorphological evidence is presented to support this claim. In other species, there is no fusion between the blastoderm and the yolk.

Response 19: Thank you for this clarification. We agree that the previous wording implied a specific cellular mechanism that was not directly assessed in this study. The description in Figure 4 has been revised to remove any reference to fusion between the blastoderm and yolk cytoplasm.

Comment 20: Line 266. At 13-14 hours. The germinal ring continues to invaginate. In fish with meroblastic divisions and stereoblastulae, there is no invagination during gastrulation. The involution movement gives rise to the hypoblast, which forms the germinal ring. The other movements of the blastoderm are extension and convergence.

Response 20: Thank you for this clarification. We agree that the use of “invagination” was not appropriate in this context and may imply a specific developmental mechanism that was not directly assessed in this study. The description has been revised to remove this terminology and instead reflect observable morphological progression. Specifically, the text at 13–14 hours has been updated to describe germ ring formation progressing at the blastoderm margin.

Comment 21: Lines 265-268. All the images in Figure 4 are very small and lack sufficient magnification and contrast, making it difficult to see what is being described. It is recommended to enlarge the images and improve the contrast. Some images were also distorted during editing, possibly to fit the figure, resulting in the embryos appearing slightly elongated along the horizontal axis.

Response 21: Thank you for this comment. Figure 4 has been revised to improve clarity and visualization of developmental features. Specifically, image size has been increased to enhance visibility of key morphological structures. In addition, all images were re-exported and reformatted to maintain their original aspect ratios and prevent distortion.

Comment 22: Line 316. 28 days post-hatching. The image shows the first evidence of scale appearance. However, this cannot be considered conclusive. To study scale development and the progression of scaling (lepidotaxy), alizarine red staining is necessary to visualize this developmental process. The brightness in the scaled region would only indicate guanine crystal deposition during iridophore differentiation. It is very possible that at this stage, there are already patches of scales in other regions of the body.

Response 22: Thank you for this comment. We acknowledge that more detailed assessment of scale development can be achieved using techniques such as alizarin red staining. However, the objective of this study was to document observable developmental milestones using a dissecting microscope. In this context, scaling was defined as the stage at which scales were visibly present and detectable under dissecting microscopy and/or by touch. This clarification has been added to the manuscript (Section 3.3, lines 316–321): “Metamorphosis progressed with development of dorsal fin spines and initial scale formation on the anterior portion of the body (28 dph), with scales identified based on visible presence under dissecting microscopy and/or by touch.”

Comment 23: Line 343. Discussion, 4.1. Larval mortality and yolk sac attrition. Throughout this discussion section, it is suggested that there is an association between mortality and energy status during the final stage of yolk and oil globule consumption. As interpreted, this appears to be a constant event, meaning its occurrence is predictable. However, there is no discussion of the variation in mortality associated with variations in egg quality, nor of the effects of breeder management on egg quality variation. If the aim is to establish a guide to the developmental events of Morone saxatilis, it is necessary to establish and discuss the starting point of the description, which is the egg quality used in this study.

Response 23: Thank you for this comment. While we understand the reviewer’s interest in egg quality and breeder effects, detailed evaluation of these factors was beyond the scope of the present study. In this study, embryos were controlled for spawn quality in that they were obtained from a single spawning cohort from a group spawn, involving multiple maternal and paternal lineages of a domesticated striped bass population (Andersen et al.,(2021c). The status of striped bass, Morone saxatilis, as a commercially ready species for U.S. marine aquaculture. Journal of the World Aquaculture Society, 52(3), 710–730. https://doi.org/10.1111/jwas.12812). We do not anticipate substantial variation in fertility or developmental rates associated with successive generations in this population. Abnormal embryo development and egg quality variation have been previously characterized in this system (Chapman RW, Reading BJ, Sullivan CV (2014) Ovary Transcriptome Profiling via Artificial Intelligence Reveals a Transcriptomic Fingerprint Predicting Egg Quality in Striped Bass, Morone saxatilis. PLoS ONE 9(5): e96818. doi:10.1371/journal.pone.0096818;30 and Reading et al., (2018). Oogenesis and Egg Quality in Finfish: Yolk Formation and Other Factors Influencing Female Fertility. Fishes, 3(4), 45. https://doi.org/10.3390/fishes3040045). For this study, only robust, swim-up larvae representative of those selected in standard hatchery practices were used for subsequent analyses. This clarification has been added to the manuscript (Section 4.1, lines 369–372): “Embryos were obtained from a single group spawning event involving multiple parental lineages of a domesticated striped bass population [28], and only viable, swim-up larvae representative of standard hatchery selection practices were used for subsequent analyses.”

Comment 24: Line 388. 4.2. Embryo developmental staging guide. This section does not address the biological significance of the developmental events documented in the study. If a guide to embryonic development is intended, the critical developmental events relevant to hatchery management must be discussed in detail.

Response 24: Thank you for this comment. We have revised Section 4.2 to more explicitly connect key developmental stages with their biological and practical relevance in hatchery settings. While early timepoints (e.g., 4–6 hours post-fertilization) remain the most critical for assessing embryo viability (Chapman RW, Reading BJ, Sullivan CV (2014) Ovary Transcriptome Profiling via Artificial Intelligence Reveals a Transcriptomic Fingerprint Predicting Egg Quality in Striped Bass, Morone saxatilis. PLoS ONE 9(5): e96818. doi:10.1371/journal.pone.0096818;30), later developmental stages are primarily used to track progression and estimate time to hatch. In practice, embryos are often staged after fertilization has already occurred, and observable morphological features provide the basis for estimating developmental timing. To address this, additional text has been included to clarify how specific developmental features correspond to hatchery decision-making. The revised text (Section 4.2, lines 433–446) now reads: “Additionally, the staging guide provides practical reference points for hatchery management by linking visible developmental features to operational decision-making. Early cleavage and blastula stages confirm successful fertilization and initial development, while progression through epiboly and germ ring formation provides a clear indicator of normal developmental progression during gastrulation. The appearance of the embryonic shield and subsequent head fold and tail bud formation mark key transitions into organogenesis, allowing hatchery personnel to estimate proximity to hatch. Later-stage features such as pigmentation, somite formation, and tail elongation provide additional indicators of developmental timing and remaining time to hatching. These observable morphological benchmarks are particularly valuable when spawning events are not directly observed, as they enable hatchery managers to stage embryos under microscopy and predict hatching windows with greater accuracy. Thus, the guide serves not only as a descriptive framework, but as a practical tool for synchronizing hatchery operations with developmental timing.”

Comment 25: Line 394. The type of degree-day standardization proposed in this work is not useful for temperature comparisons. For that purpose, effective degree-days should be used. The remaining chapters of the discussion are similar: the guidelines' usefulness and value are mentioned, but the key aspects of development that could serve as critical points or quality criteria for practical application are not explored in depth. The scope of the results in a broader context regarding the early development of Morone or other species with similar ontogeny is not addressed.

Response 25: Thank you for this comment. We acknowledge that effective degree-day approaches can be useful for temperature-based comparisons when parameters such as Tmin, Tmax, and Tbase are defined. However, these parameters are not currently established for striped bass and were not the focus of this study. The objective of this work was to develop a practical developmental staging guide under typical spawning and culture conditions, rather than to define temperature-dependent growth relationships. Accordingly, a simplified degree-day approach based on a biologically relevant reference temperature (18 °C) was used to normalize developmental progression. This clarification has been added to the manuscript (Discussion, lines 353–355): “The degree-day approach used in this study was intended to provide a practical framework for normalizing developmental progression under typical spawning conditions, rather than to define temperature-dependent growth relationships.”

Reviewer 2 Report

Comments and Suggestions for Authors

Comments to the authors

・The title should be shorten by deleting “to support larval rearing and hatchery management”

・change the word fry to larva throughout the manuscript

・lipid drops should be changed the word as oil globule.

・How far is this fish and the results obtained current study from the referenced European sea bass? The authors should discuss more about this point.

・Why did the authors take a focus on stocking density as the factor causing mortality? The physical factors may influence mortality rather than density.

・I have confused that the first experiment (quantify mortality within 72h was carried out by using different tank volume (10-90L) or not? If the author used different volumes of tanks, why? I thought this experiment should be conducted with same tank volume with different number of stocking larvae.

・I could not find out any cleavage from these pictures, why?

・Pictures should be shown more bigger and the explanations smaller.

・Are these pictures “high-resolution”?

・The authors should explain more about line 378-380. “Rearing densities between 2-5 larva / ml may promote consistency in survival” Which result support this?

・Discussion part 4.2 is not necessary or move it to introduction.

・The authors are advised to discuss more about obtained results. The benefit of this study is all depending on potential readers. Please do not overate own work.

Author Response

Comment 1: The title should be shorten by deleting “to support larval rearing and hatchery management”

Response 1: Thank you for pointing this out. We have revised the manuscript title to “Embryo and larval developmental staging guides for striped bass”.

Comment 2: change the word fry to larva throughout the manuscript

Response 2: Thank you for this comment. We have applied the revision throughout the entire manuscript accordingly.

Comment 3: lipid drops should be changed the word as oil globule.

Response 3: Thank you for this comment. We acknowledge that “oil globule” is commonly used terminology; however, the terms “lipid droplet” and “oil globule” are used interchangeably in the literature to describe this feature (Hiramatsu et al., (2015). Ovarian yolk formation in fishes: Molecular mechanisms underlying formation of lipid droplets and vitellogenin-derived yolk proteins. General and Comparative Endocrinology, 221, 9–15. https://doi.org/10.1016/j.ygcen.2015.01.025 and Reading et al., (2018). Oogenesis and Egg Quality in Finfish: Yolk Formation and Other Factors Influencing Female Fertility. Fishes, 3(4), 45. https://doi.org/10.3390/fishes3040045 and Reading et al., (2011, 2017) THE REPRODUCTIVE ORGANS AND PROCESSES, Vitellogenesis in Fishes, Editor(s): Anthony P. Farrell, Encyclopedia of Fish Physiology, Academic Press, Pages 635-646; Vitellogenesis in Fishes, Reference Module in Life Sciences, Elsevier). At our first introduction we define it in line 120: “The objectives of this study were to: 1) Quantify larval mortality and yolk sac/lipid droplet (oil globule)...”

Comment 4: How far is this fish and the results obtained current study from the referenced European sea bass? The authors should discuss more about this point.

Response 4: Thank you for this comment. We acknowledge that Dicentrarchus labrax is a related species within the Moronidae family and provides useful developmental context. This was already noted in the manuscript, where we state (Introduction, line 58): “Additionally, embryonic development has been described in detail for related finfish within the family Moronidae, like European sea bass (Dicentrarchus labrax), and these studies provide useful reference points for interpreting developmental progression.” At the same time, the objective of this study was to address the lack of a contemporary staging guide for striped bass specifically, as reflected in the manuscript statement (Introduction, line 60): “However, comparable developmental guides for striped bass remain limited in the literature.” Accordingly, the manuscript emphasizes species-specific developmental progression in striped bass rather than a direct comparison with European sea bass.

Comment 5: Why did the authors take a focus on stocking density as the factor causing mortality? The physical factors may influence mortality rather than density.

Response 5: Thank you for this comment. The focus on stocking density was motivated by long-term hatchery observations suggesting that aquaria with higher numbers of larvae often exhibited greater total losses, leading to the hypothesis that density could influence mortality. However, the results of this study did not support that assumption, as no significant relationship between density and mortality percentage was observed across the tested range. Instead, mortality was more closely associated with developmental timing and endogenous energy availability, as stated in the manuscript: “Together, these findings demonstrate that early survival is closely tied to developmental timing and endogenous energy availability rather than stocking density alone.” This interpretation suggests that factors other than stocking density are the primary drivers of early larval mortality under the conditions examined.

Comment 6: I have confused that the first experiment (quantify mortality within 72h was carried out by using different tank volume (10-90L) or not? If the author used different volumes of tanks, why? I thought this experiment should be conducted with same tank volume with different number of stocking larvae.

Response 6: Thank you for this comment. All mortality experiments were conducted using ten aquaria of approximately 90 L volume. While minor variation in volume existed among tanks due to differences in water column height, the volume of each tank was measured and accounted for when estimating larval numbers and densities. Therefore, differences in mortality were evaluated based on stocking density rather than tank volume. We have revised the manuscript to clarify this (Section 2.2, lines 154–155): “For the mortality experiments, striped bass larvae were contained in ten approximately 90 L aquaria.”

Comment 7: I could not find out any cleavage from these pictures, why?

Response 7: Thank you for this comment. Figure 4 has been revised to improve image size and clarity. Cleavage is observable in the 1–2 hour images, where early cell divisions forming the blastodisc are visible. The increased image size enhances the visibility of these features.

Comment 8: Pictures should be shown more bigger and the explanations smaller.

Response 8: Thank you for this comment. Figure 4 has been revised to increase image size and improve visibility of developmental features.

Comment 9: Are these pictures “high-resolution”?

Response 9: Thank you for this comment. We agree that the term “high-resolution” may be misleading in this context. The manuscript has been revised to remove this wording and instead clarify that images were obtained using a digital camera mounted on a dissecting microscope.

Comment 10: The authors should explain more about line 378-380. “Rearing densities between 2-5 larva / ml may promote consistency in survival” Which result support this?

Response 10: Thank you for this comment. We agree that this statement required clarification. Although no significant differences were observed across densities, the following sentence has been revised to better reflect this: “Although not significantly different, commercial hatcheries should probably consider stocking larvae at 2–5 larvae/mL.” (Discussion section 4.1, lines 406-407).

Comment 11: Discussion part 4.2 is not necessary or move it to introduction.

Response 11: Thank you for this comment. We respectfully disagree that Section 4.2 should be removed or moved to the Introduction. As noted by Reviewer 1, additional discussion of the biological and practical significance of the embryonic staging guide was warranted. Accordingly, Section 4.2 has been retained and expanded to more clearly link developmental stages with their relevance to hatchery management. The revised section now emphasizes how observable morphological features can be used to assess developmental progression and predict hatching timing, which represents a primary application of the staging guide in operational settings. These additions strengthen the interpretation of the results and provide necessary context for their practical use, and therefore are more appropriately presented in the Discussion rather than the Introduction. The text in the manuscript is (Section 4.2, lines 434–446): “Additionally, the staging guide provides practical reference points for hatchery management by linking visible developmental features to operational decision-making. Early cleavage and blastula stages confirm successful fertilization and initial development, while progression through epiboly and germ ring formation provides a clear indicator of normal developmental progression during gastrulation. The appearance of the embryonic shield and subsequent head fold and tail bud formation mark key transitions into organogenesis, allowing hatchery personnel to estimate proximity to hatch. Later-stage features such as pigmentation, somite formation, and tail elongation provide additional indicators of developmental timing and remaining time to hatching. These observable morphological benchmarks are particularly valuable when spawning events are not directly observed, as they enable hatchery managers to stage embryos under microscopy and predict hatching windows with greater accuracy. Thus, the guide serves not only as a descriptive framework, but as a practical tool for synchronizing hatchery operations with developmental timing.”

Comment 12: The authors are advised to discuss more about obtained results. The benefit of this study is all depending on potential readers. Please do not overate own work.

Response 12: Thank you for this comment. The discussion has been revised to better emphasize interpretation of the results while avoiding overstatement, with additional clarification of how the findings relate to hatchery application.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript offers a robust contribution to the developmental biology of Morone saxatilis. By integrating high‑resolution imaging with degree‑day standardization, the authors present contemporary and operationally useful staging guides for embryos and early fry. The work linked quantifiable indicators of fry loss and endogenous energy depletion. Together, these elements provide a coherent framework that directly supports hatchery decision‑making and early‑life‑stage management. Below are a few suggestions for improvement and better understanding by the readers.

Line 29: The keywords are a useful tool for readers to find your published paper. Use different words from those already used in the paper title.

Line 127-130: Seems like a discussion leading to a conclusion, not objective.

Line 135: Batch spawns used just one male and one female? How many?

Line 154: Each aquarium had the fry from one couple (one female and one male)?

Line 181: “Images were captured for recently fertilized eggs through hatching over.” Is the development synchronized, or is it an average?

Line 242 Figure 3: “The data points on the graph reflect the mean of triplicate measurements.” And the images, were they from an average development?

The manuscript succeeds in delivering a scientifically and practically relevant contribution. The methodological clarity, high‑quality imaging, and integration of physiological metrics collectively enhance its value for both researchers and hatchery practitioners. Only minor considerations remain and do not detract from the overall quality of the work.

Author Response

Comment 1: Line 29: The keywords are a useful tool for readers to find your published paper. Use different words from those already used in the paper title.

Response 1: Thank you for this comment. The keywords have been revised to avoid repetition of terms used in the title and to improve searchability.

Response 2: Line 127-130: Seems like a discussion leading to a conclusion, not objective.

Comment 2: Thank you for this comment. We agree that this section previously read more like a discussion statement than an objective. The text has been revised to better reflect the study objectives and remove interpretive language. The revised text now reads (Introduction, lines 128–132): “We evaluated larval mortality and rearing density that coincided with rapid depletion of endogenous energy reserves. Integrating digital imaging with degree-day standardization clarifies how developmental timing and internal energy use define periods of vulnerability and inform feeding transitions in hatchery-reared striped bass.”

Comment 3: Line 135: Batch spawns used just one male and one female? How many?

Response 3: Thank you for this comment. Batch spawning did not involve a single male and female; rather, each spawning event included 25 males and 25 females. This information has been clarified in the manuscript (Section 2.1, lines 136–138): “Batch spawns used 25 three-year-old males (SB 2021 F7.2 generation; 3.72 ± 0.56 kg weight, 628 ± 88 mm total length, all values given as mean ± standard deviation) and 25 four-year-old females (SB 2020 F7.1 generation; 6.92 ± 0.84 kg weight, 741 ± 32 mm total length).”

Comment 4: Line 154: Each aquarium had the fry from one couple (one female and one male)?

Response 4: Thank you for this comment. Larvae were obtained from a group spawning event involving 25 males and 25 females, rather than from individual parental pairs. Larvae from this mixed spawning cohort were used for all aquaria studies. This has been clarified in the manuscript (Section 2.1, lines 135–138).

Comment 5: Line 181: “Images were captured for recently fertilized eggs through hatching over.” Is the development synchronized, or is it an average?

Response 5: Thank you for this comment. Embryos were obtained from a batch spawning event, and fertilization occurred over a short interval due to external fertilization rather than at a single synchronized time point. As such, fertilization timing was treated as a representative (average) time based on the spawning event. Because the staging guide is based on hourly developmental progression, minor variation in fertilization timing is negligible relative to the temporal resolution of the observations. The manuscript has been revised to clarify this point (Section 2.3, lines 186–188): “Images were captured for embryos from fertilization through hatching at hourly intervals, with fertilization time defined as a representative (average) time based on the spawning event.”

Comment 6: Line 242 Figure 3: “The data points on the graph reflect the mean of triplicate measurements.” And the images, were they from an average development?

Response 6: Thank you for this comment. Yes, the images used for yolk sac and lipid droplet measurements represent average developmental progression. At each time point, multiple larvae were sampled, and measurements were obtained from triplicate individuals, with values reported as means. Images were selected to reflect typical morphology for each stage rather than individual variation. The manuscript has been revised to clarify this (Figure 3 line 253-255): “The data points on the graph reflect the mean of triplicate measurements taken from yolk sac and lipid droplet areas in 2D digital images of striped bass larvae, with images selected to represent typical morphology at each developmental stage.”

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has shown substantial improvement in quality, and most of the previous recommendations have been successfully incorporated. However, the following aspects still require attention:

Response 12. The term "weak, non-significant correlation" is conceptually incorrect. Statistically, a correlation must be significant to be recognized as such; otherwise, the existence of any correlation cannot be asserted. It is requested that this interpretation be corrected in the text.

Response 13. The methodological clarification offered by the authors reveals a fundamental problem: the described experiment lacks experimental replications, which reduces its statistical validity and experimental rigor. It is recommended that this section be reformulated to present the procedure only as an exploratory density test, without attributing experimental scope. This decision should be justified in the manuscript. As the authors themselves point out, the guidelines have a practical purpose oriented toward monitoring hatchery operations. However, to reliably achieve this purpose, it is essential that they be supported by rigorous experimental work that adequately reflects the variation observed under real-world conditions. The use of ten tanks with different stocking densities, without replicates and with averaged results, does not offer sufficient experimental support or practical utility.

Response 25. The proposed standardization using degree-days lacks biological meaning and is not a true standardization, as it operates in arbitrary units of limited practical utility and does not allow reliable predictions. In contrast, the use of effective degree-days, while presenting its own limitations, does allow for standardization with greater biological meaning: it eliminates the effect of temperature differences and generates a standardized thermal summation scale. The authors' proposal is not suitable for the stated objective.

Image quality and label accuracy. The images presented are low-resolution and lack sufficient contrast and sharpness. Given that the stated objective is to develop a practical reference guide, their image quality needs to be substantially improved. Furthermore, inaccuracies persist in the labels: for example, the image on line 335 refers to "3 dph, pigment lens," which is anatomically incorrect, since the lens does not become pigmented; it must remain transparent for its optical function. The structure that acquires pigmentation is the retinal pigment epithelium, responsible for the eye's characteristic black color. Although the guide's focus is practical and does not delve into early developmental processes, maintaining anatomical accuracy in its descriptions is essential. We request that the terminology used in all images be reviewed and corrected.

Author Response

Comment 1: The term "weak, non-significant correlation" is conceptually incorrect. Statistically, a correlation must be significant to be recognized as such; otherwise, the existence of any correlation cannot be asserted. It is requested that this interpretation be corrected in the text.

Response 1: Thank you for this clarification. We agree that describing a “weak, non-significant correlation” is not appropriate. Accordingly, we have revised the text to remove this phrasing and instead report that no relationship was detected between the variables within the tested range in section 3.1 line 234 and line 246: “There was no correlation between (R² = 0.0657, p = 0.47) mortality percentage and rearing density, at least within the range of densities evaluated.”

Comment 2: The methodological clarification offered by the authors reveals a fundamental problem: the described experiment lacks experimental replications, which reduces its statistical validity and experimental rigor. It is recommended that this section be reformulated to present the procedure only as an exploratory density test, without attributing experimental scope. This decision should be justified in the manuscript. As the authors themselves point out, the guidelines have a practical purpose oriented toward monitoring hatchery operations. However, to reliably achieve this purpose, it is essential that they be supported by rigorous experimental work that adequately reflects the variation observed under real-world conditions. The use of ten tanks with different stocking densities, without replicates and with averaged results, does not offer sufficient experimental support or practical utility.

Response 2: Thank you for this important clarification. We have revised the manuscript to clarify the scope of this work and to avoid presenting it as a fully replicated experiment. This has been addressed in Section 2.2 (lines 150–151): “An exploratory density assessment and a developmental monitoring study were conducted to: 1) characterize mortality patterns…”

Comment 3: The proposed standardization using degree-days lacks biological meaning and is not a true standardization, as it operates in arbitrary units of limited practical utility and does not allow reliable predictions. In contrast, the use of effective degree-days, while presenting its own limitations, does allow for standardization with greater biological meaning: it eliminates the effect of temperature differences and generates a standardized thermal summation scale. The authors' proposal is not suitable for the stated objective.

Response 3: We do not have the parameters needed for Tmin, Tmax, and Tbase. Tbase for striped bass larvae is unknown. We did collect AM and PM temperatures, however, there is no way to verify to measure if those qualify for Tmax or Tmin. We disagree with the reviewer in the sense that the use of this equation is not proper, as we are unable to actually use the correct parameters. At best it would be plugging incorrect values into an equation. For example, the only thing we could do with this specific equation would be to use the AM as Tmin and PM as Tmax and more or less arbitrarily determine some value for Tbase which is not experimental data. Since this requires many assumptions, the authors are not comfortable doing that as the result would be spurious and speculative.

Comment 4: Image quality and label accuracy. The images presented are low-resolution and lack sufficient contrast and sharpness. Given that the stated objective is to develop a practical reference guide, their image quality needs to be substantially improved. Furthermore, inaccuracies persist in the labels: for example, the image on line 335 refers to "3 dph, pigment lens," which is anatomically incorrect, since the lens does not become pigmented; it must remain transparent for its optical function. The structure that acquires pigmentation is the retinal pigment epithelium, responsible for the eye's characteristic black color. Although the guide's focus is practical and does not delve into early developmental processes, maintaining anatomical accuracy in its descriptions is essential. We request that the terminology used in all images be reviewed and corrected.

Response 4: Thank you for this careful and helpful evaluation. We have thoroughly reviewed and corrected the terminology used across all embryo and fry images to ensure anatomical accuracy and consistency. In particular, references to incorrect structures (e.g., “pigmented lens”) have been revised to reflect appropriate anatomical descriptions, and all labels have been updated to use observational, morphology-based language aligned with what is directly visible.

Reviewer 2 Report

Comments and Suggestions for Authors

Comment 3: lipid drops should be changed the word as oil globule.

・Then, the authors are advised to explain the similarity of droplet and globule before using these words as same meaning. Within my knowledge, oil globule is used as a technical word in biology and droplet is not.

Comment 4: How far is this fish and the results obtained current study from the referenced European sea bass? The authors should discuss more about this point.

・It is better to discuss the specialty of this species when the authors conclude and emphasize species-specific development in striped bass. Thus, I have suggested to compare with European sea bass. This will bring discussion deeper based on obtained results and attract potential readers.

Comment 5: Why did the authors take a focus on stocking density as the factor causing mortality? The physical factors may influence mortality rather than density.

・These facts and background with concrete examples should be clearly stated in manuscript. In general hatchery management, egg incubation is of primary importance and normally pays great attention to maintain proper physical factors not to occur mortality. Thus, examined range of density would not induce heavy mortality. In my small experience, 15million larvae per 1000L is still capable.

Author Response

Comment 1: Then, the authors are advised to explain the similarity of droplet and globule before using these words as same meaning. Within my knowledge, oil globule is used as a technical word in biology and droplet is not.

Response 1: Thank you for this clarification. We have revised the manuscript to explicitly define the terminology and avoid potential confusion. Specifically, we now clarify that “lipid droplet” and “oil globule” refer to the same endogenous lipid reserve structure and are used interchangeably in this study. This has been added at section 2.2 lines 170-172: “In this study, the term ‘lipid droplet’ refers to the endogenous lipid reserve structure and is equivalent to the ‘oil globule’ terminology commonly used in fish developmental biology; these terms are used interchangeably here.”

Comment 2: It is better to discuss the specialty of this species when the authors conclude and emphasize species-specific development in striped bass. Thus, I have suggested to compare with European sea bass. This will bring discussion deeper based on obtained results and attract potential readers.

Response 2: Thank you for this helpful suggestion. We have expanded the Discussion to include reference to European sea bass (Dicentrarchus labrax) and to more clearly emphasize the species-specific aspects of striped bass development. In particular, we highlight how our detailed and consistent imaging approach provides a more complete and species-specific developmental sequence based on our observations, and we note the potential influence of long-term captive breeding on developmental timing and morphology. This addition has been incorporated as follows in section 4.2, lines 462-472: “While developmental staging has been described in related Moronidae species such as European sea bass (Dicentrarchus labrax) [9], this reference primarily serves as a comparative framework rather than a direct template for striped bass development. In the present study, the use of consistent imaging across development allowed for detailed documentation of striped bass embryonic stages, providing a more complete and species-specific staging sequence than those available from related species. Additionally, the striped bass used in this study have been maintained in captivity and selectively bred over multiple generations, which may further influence developmental timing and morphology relative to other Moronidae species, further emphasizing the importance of establishing a striped bass specific staging framework to support accurate hatchery interpretation and application.”

Comment 3: These facts and background with concrete examples should be clearly stated in manuscript. In general hatchery management, egg incubation is of primary importance and normally pays great attention to maintain proper physical factors not to occur mortality. Thus, examined range of density would not induce heavy mortality. In my small experience, 15million larvae per 1000L is still capable.

Response 3: Thank you for this helpful clarification. We agree that careful control of physical and environmental factors during egg incubation plays a primary role in preventing early larval mortality in typical hatchery conditions. Accordingly, we acknowledge that the range of densities examined in this study would not be expected to induce substantial mortality when these parameters are maintained within acceptable limits. We have revised the manuscript to more clearly state the rationale for evaluating stocking density based on hatchery observations, and to explicitly contextualize our findings within standard hatchery practices. This clarification has been incorporated as follows in Section 4.1, lines 376–386: “Stocking density was evaluated based on common hatchery observations suggesting that aquaria stocked with higher numbers of larvae may experience greater total losses, consistent with reported striped bass hatchery practices [14]. Physical and environmental parameters (e.g., water quality, oxygen, and flow) are generally maintained within acceptable limits during this period. While higher stocking densities may increase total losses in hatchery settings, the results of this study indicate that mortality rates within the tested range were not directly driven by density, but instead aligned with the timing of endogenous energy depletion. Mortality coincided with the period of rapid yolk and lipid depletion, indicating that survival is more closely linked to internal energy availability rather than crowding effects.” Section 4.1 lines 376-386: Stocking density was evaluated based on common hatchery observations suggesting that aquaria stocked with higher numbers of larvae may experience greater total losses, consistent with reported striped bass hatchery practices [14]. Physical and environmental parameters (e.g., water quality, oxygen, and flow) are generally maintained within acceptable limits during this period. While higher stocking densities may increase total losses in hatchery settings, the results of this study indicate that mortality rates within the tested range were not directly driven by density, but instead aligned with the timing of endogenous energy depletion. Mortality coincided with the period of rapid yolk and lipid depletion, indicating that survival is more closely linked to internal energy availability rather than crowding effects.