Muscle Transcriptome Analysis Reveals Molecular Mechanisms of Superior Growth Performance in Kuruma Shrimp, Marsupenaeus japonicus

Round 1

Reviewer 1 Report

In this paper, the authors employ RNA-Seq to compare global gene expression in muscle of fast- and slow-growing shrimp, M. japonicus. The RNA-Seq outcome was then complemented by qPCR analysis and in silico analysis for SNPs and the specifics of a gene(s) encoding cuticle proteins. The approaches generally seem methodological sound, but the experimental design is entirely missing and there are concerns that the “muscle” tissue is contaminated by epidermal cells that are involved in expression of moulting-related genes, that only indirectly relate to growth 9and at the very least, are not muscle-derived); in addition, sample sizes are an issue. It is not clear why the authors zoomed in on annotating cuticle protein gene(s). The ms is nicely laid out and easy to follow, and the quality of English is commendable.

 

Specific Comments

Title: reviewer suggests a small change: RNA-Seq analysis reveals molecular mechanisms of superior growth performance in shrimp, Marsupenaeus japonicus

 

Abstract

L17: the meaning of ‘bait conditions’ is not clear

L30: replace ~ the cuticle genes of Litopenaeus vannamei, Fenneropenaeus chinensis, and Penaeus monodon ~  with ~ penaeid cuticle genes ~

 

Introduction

L47: the meaning of anhydrous transportation is not clear – do authors mean that shrimps can be transported out-of-water, perhaps in a humid environment?

L53: authors are describing culture conditions, then relay that these shrimps live at depths of 10-100 m; this is presumably the wild situation – how does that compare to shrimps in culture?

L58, onwards: this ms is missing some key detail, in my view: WHAT IS GROWTH? How is this defined, and what do we associate this with? More cells? Larger cells? How does this translate to genes that one might expect to be upregulated? In my view, this MUST be worked into the Introduction, and can then be reflected upon in the Discussion

L65: what is FFRC?

 

Materials & methods

L96: there is a notable omission in this paper in that there is NO experimental design (!) – it is not clear what was done; L98: larvae were grown – how many, and in how many conical-bottom tanks? What was the scale of the company (tonnes/year) and the scale of the larval rearing? Most importantly, what was the experimental design? Did the authors “simply” chose some big shrimp and some (how many?) small shrimp after 70 days? Were animals graded during the culture period? If not, then does difference in size reflect difference in growth, or in growth potential? Once a larva is slightly smaller than its neighbour, is it then doomed to remain smaller? Is a difference in BW a true reflection of ‘reduced growth’? Or did some smaller individuals start growing later and once weaned onto feed had the same specific growth rates as larger individuals. These are all relevant questions that are likely to impact on the outcome of the study – but none of these are addressed, let alone described; a detailed section on experimental design that addresses these, and other relevant issues, needs to be included and evaluated by review, as this is absolutely KEY to the study and its implications.

L106: please provide detail on anaesthesia

L107: the authors collected “muscle” – from where was this muscle collected? Given that so many moulting-related genes seem to be identified, it appears that not only muscle, but also epidermis was collected. This is a notable issue that confounds the study.

L115, 118: n=3 is not insufficient, both for RNA-Seq and qPCR

L138: the cloning steps seem incomplete; which vector was used, how was the insert subcloned and which E. coli strain was used?

L138: genes were considered differentially expressed for P < 0.05 – was a false discovery rate used, or some other measure to take into account multiple statistical testing? If not, the used approach is not appropriate.

 

Results

L196: Many of the genes in Table 2 seem associated with the moulting cycle, and hence, with epidermal gene expression, rather than muscle. As timing of the moulting cycle may affect moulting gene-related expression, the suspected inclusion of epidermal cells may well dramatically affect the insight into gene expression in muscle tissue.

L203: the comment for L196 is reinforced by identification of “chitin metabolic process” as the biological pathway – this does not seem to reflect muscle tissue gene expression….

L214: the legend for Fig 2 needs much more detail, including species of interest, detail on expt design and the abbreviations used under ‘Category’

L217: in my view, the samples used for RNA-Seq should have been included in the qPCR analyses. A sample size of n=3 is insufficient, and again, correction for multiple statistical testing is required.

L225: more detail is needed on what the symbols represent, how the graphs are generated, etc. Is this mean and SE? Sample size..?

L249: it is entirely obscure why this gene was chosen for annotation – is this a muscle tissue-associated gene, or an epidermis-associated gene involved in moulting…? Presumably the latter, which makes this an unsuitable candidate for ‘growth-related’ RNA-Seq.

 

Discussion

See introduction: it is not clear what “growth” is – presumably, some of this is realised during the intermoult, which makes me question why all these moulting-related genes are appearing, and especially, why they would appear in muscle.

 

Typographical

L31: ~ this study provides valuable data ~

L45: ~ so it is very much loved ~

L47 ~ high fecundity ~

L60: ~ shed their exoskeleton ~

L62 ~ Chinese perch, Siniperca ~

L69: ~ mantles of oyster, Pinctada ~

L72: ~ sizes of abalone, Haliotis discus ~

L76, and elsewhere: please add trivial names, as done for the 3 examples above, for all animal names in this paper

L132: featurerts?

L138: when When?

N/A

Author Response

Dear Editor and reviewers,

 

Thank you for your letter and the reviewers’ comments concerning our manuscript entitled “Comparing transcriptomes reveals molecular mechanisms in superior growth performance Marsupenaeus japonicus” (fishes-2432032). Those comments are valuable and very helpful for revising and improving our manuscript as well as important for guiding the significance of our research. We have read through the comments carefully and made corrections. Based on the instructions provided in your letter, we uploaded the file of the revised manuscript. The revised portions are indicated in red in the manuscript. The responses to the reviewer’s comments are as follows.

 

Point 1: In this paper, the authors employ RNA-Seq to compare global gene expression in muscle of fast- and slow-growing shrimp, M. japonicus. The RNA-Seq outcome was then complemented by qPCR analysis and in silico analysis for SNPs and the specifics of a gene(s) encoding cuticle proteins. The approaches generally seem methodological sound, but the experimental design is entirely missing and there are concerns that the “muscle” tissue is contaminated by epidermal cells that are involved in expression of moulting-related genes, that only indirectly relate to growth and at the very least, are not muscle-derived); in addition, sample sizes are an issue. It is not clear why the authors zoomed in on annotating cuticle protein gene(s). The ms is nicely laid out and easy to follow, and the quality of English is commendable.

Response 1: We sincerely appreciate the valuable comments. During the cultivation of M. japonicus of the same batch, there are often obvious differences in the growth of different larvae, but the underlying growth regulation mechanism is not yet clear. To explore the molecular mechanism of this growth difference, this study used RNA-seq technology to compare M. japonicus individuals with significant growth differences from the same family. The exoskeletal cuticle and muscles constitute the arthropod musculoskeletal system and function jointly to enable animal movements and locomotion (Mark et al., 2017). The establishment of accurate structural connections between cuticle and muscles via epidermal tendon cells is an essential part of exoskeletal cuticle morphogenesis at the organism level. In crustaceans, reports on the microscopic architecture of cuticle-muscle connections refer to different body regions in adult specimens of several taxa, including Branchiopoda, Balanidae, Ostracoda, Brachyura, Mystacocarida, and Isopoda. The fibers that are anchored to the apical membrane of the tendon cell and extend deeply into the cuticle are also interesting and were defined as muscle attachment fibers. Given the special relationship between skin and muscle tissue, it may be difficult for the average researcher to isolate the two absolutely, especially when studying smaller shrimp. We will increase the sampling level in future experiments. Cesar et al established a cDNA library of shrimp juvenile abdominal muscle by PCR-based SMART^TM cDNA technology. The high identity-matched ESTs included multiple cuticle protein genes (Cesar et al., 2008). It is also possible that cuticle proteins are not specific to the epidermal tissue. Tissue distribution analysis revealed that a novel cuticle protein gene, LvCPAP1, was predominantly expressed in the epidermis, stomach, and muscle. In Litopenaeus vannamei, 13,000 DEGs, including genes encoding the cuticle, chitin, ecdysteroids, and muscle proteins, were identified in families with higher and lower growth performances (Santos et al., 2021). Cuticular proteins are important in the formation of new cuticles before and after molting (Vincent et al., 2002). Different types of cuticular proteins bind to long-chain chitin and affect the structure and function of the cuticle (Willis et al., 2012). In this paper, the real research results are analyzed and discussed. In future studies, we will consider conducting comparative transcriptome analysis on the epidermal tissue of the same family of M. japonicus, as well as comparative analysis with the muscle transcriptome data, hoping to screen for more potential growth regulatory genes.

References:

• Mrak P, Bogataj U, Štrus J, et al. Cuticle morphogenesis in crustacean embryonic and postembryonic stages[J]. Arthropod Structure & Development, 2017, 46(1): 77-95.
• Cesar J R, Zhao B, Yang J. Analysis of expressed sequence tags from abdominal muscle cDNA library of the pacific white shrimp Litopenaeus vannamei[J]. animal, 2008, 2(9): 1377-1383.
• Santos C A, Andrade S C S, Teixeira A K, et al. Transcriptome differential expression analysis reveals the activated genes in Litopenaeus vannamei shrimp families of superior growth performance[J]. Aquaculture, 2021, 531: 735871.
• Vincent, J.F. Arthropod cuticle: a natural composite shell system. Composites Part A: Applied Science and Manufacturing 2002, 33, 1311-1315.
• Willis, J.H.; Papandreou, N.C.; Iconomidou, V.A.; Hamodrakas, S.J. Cuticular proteins. In Insect Molecular Biology and Biochemistry; Elsevier: 2012; pp. 134-166.

 

Specific Comments

Point 2: Title: reviewer suggests a small change: RNA-Seq analysis reveals molecular mechanisms of superior growth performance in shrimp, Marsupenaeus japonicus.

Response 2: We really appreciate your suggestions. We have made changes in the revised manuscript (L2-4).

Point 3: Abstract, L17: the meaning of ‘bait conditions’ is not clear.

Response 3: Thanks for your friendly reminder. We have made changes in the revised manuscript (L18).

Point 4: Abstract, L30: replace ~ the cuticle genes of Litopenaeus vannamei, Fenneropenaeus chinensis, and Penaeus monodon ~  with ~ penaeid cuticle genes ~

Response 4: We really appreciate your professional comments. We have made changes in the revised manuscript (L31).

 

Introduction

Point 5: L47: the meaning of anhydrous transportation is not clear – do authors mean that shrimps can be transported out-of-water, perhaps in a humid environment?

Response 5: Thanks for your friendly reminder. Due to multiple merits of M. japonicus, such as good reproductive performance, fast growth rate, and the capability of being transported live without water, the culture areas of which expands rapidly. In the shrimp-grade culture system, live shrimps are removed from the water and exposure in the air for transport between ponds. The shrimp must be alive in order to gain the highest prices. After harvest, shrimp are chilled to around 14 °C until torpid, which reduces activity sufficiently to enable them to survive the packing and transport processes. Torpid shrimp are then packed in 1-kg net-weight cardboard boxes without water, but with a covering of damp sawdust, and transported to the aquatic product market, a process that may take 24 to 36 hours.

Point 6: L53: authors are describing culture conditions, then relay that these shrimps live at depths of 10-100 m; this is presumably the wild situation – how does that compare to shrimps in culture?

Response 6: We really appreciate your professional comments. In this study, we used kurama shrimp, a popular farmed shrimp species with great economic value. The sand substrates are traditionally considered as an essential requirement for kurama shrimp farming. Under normal conditions, kuruma shrimp dives in the sand during the daytime and go out for food at night. Under the wild situation, M. japonicus often inhabits sandy or sandy-muddy sea areas with a water depth of 10-100 meters and has a clear habit of burrowing in the sand.

Point 7: L58, onwards: this ms is missing some key detail, in my view: WHAT IS GROWTH? How is this defined, and what do we associate this with? More cells? Larger cells? How does this translate to genes that one might expect to be upregulated? In my view, this MUST be worked into the Introduction, and can then be reflected upon in the Discussion.

Response 7: Thanks for your friendly reminder. The growth and development of animals is a process in which a large number of cells continuously grow, divide and differentiate to make tissues, organs, and systems perfect and mature in structure and function. Zhao et al. performed a comparative transcriptome analysis between a fast-growth group and a slow growth group at different stages by SMRT (single molecule real-time) and NGS. The shrimp came from three full-sib families (Family 1, Family 2, and Family 3). The culture densities in the three ponds were the same, with 40 individuals/m². Starting at 30 days of age, the body length and weight of the kuruma shrimp were monitored every 10 days until the end of the culture experiment, and a total of 8 measurements were performed during this period. To measure, thirty shrimps were randomly captured in each pond, and then the weight and length of each shrimp were measured with an electronic balance and a scale. Starting at 40 days of age, the specific growth rate (SGR) of the kuruma shrimp was calculated every 10 days. The SGR was calculated as described previously, with slight modification, namely SGR (%/day) = 100 × (ln N-days-of-age body weight − (N − 10)-days-of-age body weight) / 10 days (N = 40, 50, 60, 70, 80, 90, 100). The above information is clearly recorded in the literature (Zhao et al. 2021). We have made changes in the revised manuscript (L61-64).

Point 8: L65: what is FFRC?

Response 8: Thanks for your friendly reminder. Wang et al. conducted a comparative transcriptome analysis of the muscle tissue of a 6-month-old Cyprinus carpio in the same family with different growth rates. The FFRC represents the freshwater fisheries research center of China. We have made changes in the revised manuscript (L69).

 

Materials & methods

Point 9: L96: there is a notable omission in this paper in that there is NO experimental design (!) – it is not clear what was done; L98: larvae were grown – how many, and in how many conical-bottom tanks? What was the scale of the company (tonnes/year) and the scale of the larval rearing? Most importantly, what was the experimental design? Did the authors “simply” chose some big shrimp and some (how many?) small shrimp after 70 days? Were animals graded during the culture period? If not, then does difference in size reflect difference in growth, or in growth potential? Once a larva is slightly smaller than its neighbour, is it then doomed to remain smaller? Is a difference in BW a true reflection of ‘reduced growth’? Or did some smaller individuals start growing later and once weaned onto feed had the same specific growth rates as larger individuals. These are all relevant questions that are likely to impact on the outcome of the study – but none of these are addressed, let alone described; a detailed section on experimental design that addresses these, and other relevant issues, needs to be included and evaluated by review, as this is absolutely KEY to the study and its implications.

Response 9: We really appreciate your professional comments. During the cultivation of M. japonicus of the same batch, there are often obvious differences in the growth of different larvae, but the underlying growth regulation mechanism is not yet clear. Previous studies on comparative transcriptomics of individuals with different growth sizes in the same family of other species have identified potential growth regulatory genes, which is helpful to further explore the mechanism of species growth regulation and the breeding of new varieties. Muscle growth in crustaceans is intermittent and closely associated with the molt cycle due to the presence of the rigid calcified exoskeleton. Increases in muscle mass are restricted to the ecdysial period when the old exoskeleton is shed and the new exoskeleton expands in size (Whiteley et al., 1997). Tissue growth in Crustacea occurs at specific stages of the molt cycle and is influenced by a number of physical, hormonal, and environmental factors (EI et al., 1997). Consequently, growth in Crustacea is closely associated with the molt cycle, in particular the stages surrounding ecdysis when there is a considerable increase in the rate of water uptake and a subsequent increase in hydrostatic pressure causing the

new uncalcified exoskeleton to expand providing space for tissue growth (Mykles, 1980). Typically, the larger individuals at the beginning have a stronger competitive advantage and will have access to more food. Whether it's behavioral or genetic.

To further reveal the molecular mechanism of the obvious growth differences in the culture of M. japonicus, this study used comparative transcriptome sequencing technology to analyze the transcriptome of individuals with different growth characteristics in the same batch and screened out potential growth trait–related genes. The functional research on the up-and downregulated genes lays the foundation for molecular marker-assisted breeding of M. japonicus. M. japonicus was derived from a laboratory-constructed full-sib family. About 4,000 larvae were grown in conical-bottomed tanks at a density of 200 individuals/m² and fed twice a day with continuous aeration. The entire cultivation lasted for 70 days. We randomly selected 200 individuals from the same family, measured their body weight, and selected the top 30 individuals as the large individual group, and the last 30 individuals as the small individual group. Then 18 individuals were selected from the large individual group and the small individual group as experimental samples. The average weight of individuals in the fast-growing group was 2.241 ± 0.54 g, and the average weight of individuals in the slow-growing group was 0.733 ± 0.32 g. We have made changes in the revised manuscript (L102-104, 107-111).

References:

• Whiteley N M, El Haj A J. Regulation of muscle gene expression over the moult in crustacea[J]. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 1997, 117(3): 323-331.
• El Haj A J, Whiteley N M. Molecular regulation of muscle growth in Crustacea[J]. Journal of the Marine Biological Association of the United Kingdom, 1997, 77(1): 95-106.
• Mykles D L. The mechanism of fluid absorption at ecdysis in the American lobster, Homarus americanus[J]. Journal of Experimental Biology, 1980, 84(1): 89-102.

Point 10: L106: please provide detail on anaesthesia

Response 10: Thanks for your friendly reminder. We have made changes in the revised manuscript (L114-115).

Point 11: L107: the authors collected “muscle” – from where was this muscle collected? Given that so many moulting-related genes seem to be identified, it appears that not only muscle, but also epidermis was collected. This is a notable issue that confounds the study.

Response 11: We really appreciate your professional comments. The exoskeletal cuticle and muscles constitute the arthropod musculoskeletal system and function jointly to enable animal movements and locomotion (Mark et al., 2017). The establishment of accurate structural connections between cuticle and muscles via epidermal tendon cells is an essential part of exoskeletal cuticle morphogenesis at the organism level. In crustaceans, reports on the microscopic architecture of cuticle-muscle connections refer to different body regions in adult specimens of several taxa, including Branchiopoda, Balanidae, Ostracoda, Brachyura, Mystacocarida, and Isopoda. The fibers that are anchored to the apical membrane of the tendon cell and extend deeply into the cuticle are also interesting and were defined as muscle attachment fibers. Given the special relationship between skin and muscle tissue, it may be difficult for the average researcher to isolate the two absolutely, especially when studying smaller shrimp. We will increase the sampling level in future experiments. Cesar et al established a cDNA library of shrimp juvenile abdominal muscle by PCR-based SMART^TM cDNA technology. The high identity-matched ESTs included multiple cuticle protein genes (Cesar et al., 2008). It is also possible that cuticle proteins are not specific to the epidermal tissue. Tissue distribution analysis revealed that a novel cuticle protein gene, LvCPAP1, was predominantly expressed in the epidermis, stomach, and muscle. In Litopenaeus vannamei, 13,000 DEGs, including genes encoding the cuticle, chitin, ecdysteroids, and muscle proteins, were identified in families with higher and lower growth performances (Santos et al., 2021). Cuticular proteins are important in the formation of new cuticles before and after molting (Vincent et al., 2002). Different types of cuticular proteins bind to long-chain chitin and affect the structure and function of the cuticle (Willis et al., 2012). The first abdominal muscle tissue of 18 shrimp from each group was snap-frozen in liquid nitrogen and then transferred to -80°C for RNA extraction. We have made changes in the revised manuscript (L115).

Point 12: L115, 118: n=3 is not insufficient, both for RNA-Seq and qPCR

Response 12: We really appreciate your professional comments. The first abdominal muscle tissue of 18 shrimp from two groups was snap-frozen in liquid nitrogen and then transferred to -80°C for RNA extraction. The muscle RNA of nine shrimp in the fast-growing group and the slow-growing group, respectively, were randomly divided into three groups. Equal amounts of total RNA from each group (containing three individuals) were pooled. Finally, each group had three RNA samples for transcriptome and real-time quantitative PCR analysis, which met the basic requirements of statistical analysis. Muscle tissue is a shrimp's largest tissue, so the sample is able to satisfy the transcriptome and real-time quantitative PCR analysis, which has been demonstrated in our previous studies.

Point 13: L138: the cloning steps seem incomplete; which vector was used, how was the insert subcloned and which E. coli strain was used?

Response 13: We really appreciate your suggestions. The PCR-amplified product was ligated into the pMD19-T vector (Takara, Dalian, China), and transformed into E. coli DH5α cells (Takara, Dalian, China) and sequenced by paired-end sequencing. We have made changes in the revised manuscript (L152-154).

Point 14: L138: genes were considered differentially expressed for P < 0.05 – was a false discovery rate used, or some other measure to take into account multiple statistical testing? If not, the used approach is not appropriate.

Response 14: Thanks for your friendly reminder. Genes were considered to be significantly differentially expressed when the padj < 0.05 and log2(Fold change) > 1. We have made changes in the revised manuscript (L144-146).

 

Results

Point 15: L196: Many of the genes in Table 2 seem associated with the moulting cycle, and hence, with epidermal gene expression, rather than muscle. As timing of the moulting cycle may affect moulting gene-related expression, the suspected inclusion of epidermal cells may well dramatically affect the insight into gene expression in muscle tissue.

Response 15: We really appreciate your professional comments. The exoskeletal cuticle and muscles constitute the arthropod musculoskeletal system and function jointly to enable animal movements and locomotion (Mark et al., 2017). The establishment of accurate structural connections between cuticle and muscles via epidermal tendon cells is an essential part of exoskeletal cuticle morphogenesis at the organism level. In crustaceans, reports on the microscopic architecture of cuticle-muscle connections refer to different body regions in adult specimens of several taxa, including Branchiopoda, Balanidae, Ostracoda, Brachyura, Mystacocarida, and Isopoda. The fibers that are anchored to the apical membrane of the tendon cell and extend deeply into the cuticle are also interesting and were defined as muscle attachment fibers. Given the special relationship between skin and muscle tissue, it may be difficult for the average researcher to isolate the two absolutely, especially when studying smaller shrimp. We will increase the sampling level in future experiments. Cesar et al established a cDNA library of shrimp juvenile abdominal muscle by PCR-based SMART^TM cDNA technology. The high identity-matched ESTs included multiple cuticle protein genes (Cesar et al., 2008). It means that cuticle proteins are not specific to the epidermal tissue. Tissue distribution analysis revealed that a novel cuticle protein gene, LvCPAP1, was predominantly expressed in the epidermis, stomach, and muscle. In Litopenaeus vannamei, 13,000 DEGs were identified in muscle tissue, including genes encoding the cuticle, chitin, ecdysteroids, and muscle proteins (Santos et al., 2021). Cuticular proteins are important in the formation of new cuticles before and after molting (Vincent et al., 2002). Different types of cuticular proteins bind to long-chain chitin and affect the structure and function of the cuticle (Willis et al., 2012). This study identified 1375 differentially expressed genes between fast-growing and slow-growing M. japonicus. Transcriptome and quantitative PCR analysis identified several genes associated with molting or muscle growth. The dynamic expression characteristics of these genes in the molting process and different developmental stages of M. japonicus need to be further studied.

Point 16: L203: the comment for L196 is reinforced by identification of “chitin metabolic process” as the biological pathway – this does not seem to reflect muscle tissue gene expression….

Response 15: We really appreciate your professional comments. Crustaceans, such as shrimp and crabs, constitute the group with the largest chitin output, and their growth and development are majorly associated with the biosynthesis and modification of chitin (Zhang et al., 2021). Chitin metabolism is the result of the joint activity of the chitin synthase system and the chitin hydrolase system, both of which comprise a dynamic equilibrium process that is regulated by hormones and multiple signal pathways. In insects, the chitinase genes were originally divided into eight groups, all of which play different roles in growth and development (Arakane et al., 2010). Crustacean chitinases perform three main functions. Namely, crustacean chitinases participate in the molting and growth process, the digestion of chitin-containing food, and the immune response/disease prevention. Chitinases with different functions are mainly expressed in different tissue sites. Moreover, some chitinase genes are only detected in one tissue, whereas others are found in multiple tissues (Zhang et al., 2021). The dynamic expression characteristics of these genes in different tissues and developmental stages of M. japonicus need to be further studied.

References:

• Zhang X, Yuan J, Li F, et al. Chitin synthesis and degradation in crustaceans: a genomic view and application[J]. Marine Drugs, 2021, 19(3): 153.
• Arakane Y, Muthukrishnan S. Insect chitinase and chitinase-like proteins[J]. Cellular and molecular life sciences, 2010, 67: 201-216.

Point 17: L214: the legend for Fig 2 needs much more detail, including species of interest, detail on expt design and the abbreviations used under ‘Category’

Response 17: Thanks for your friendly reminder. We have made changes in the revised manuscript (L222-223).

Point 18: L217: in my view, the samples used for RNA-Seq should have been included in the qPCR analyses. A sample size of n=3 is insufficient, and again, correction for multiple statistical testing is required.

Response 18: Thanks for your friendly reminder. We really appreciate your professional comments. The first abdominal muscle tissue of 18 shrimp from two groups was snap-frozen in liquid nitrogen and then transferred to -80°C for RNA extraction. The muscle RNA of nine shrimp in the fast-growing group and the slow-growing group, respectively, were randomly divided into three groups. Equal amounts of total RNA from each group (containing three individuals) were pooled. Finally, each group had three RNA samples for transcriptome and real-time quantitative PCR analysis, which met the basic requirements of statistical analysis. Muscle tissue is a shrimp's largest tissue, so the sample is able to satisfy the transcriptome and real-time quantitative PCR analysis, which has been demonstrated in our previous studies.

Point 19: L225: more detail is needed on what the symbols represent, how the graphs are generated, etc. Is this mean and SE? Sample size..?

Response 19: Thanks for your friendly reminder. Each bar represents the mean ± S.D (n = 3). A significant difference between groups at p < 0.05 (n = 3, ANOVA) is indicated by different letters above the bars. We have made changes in the revised manuscript (L237-239).

Point 20: L249: it is entirely obscure why this gene was chosen for annotation – is this a muscle tissue-associated gene, or an epidermis-associated gene involved in moulting…? Presumably the latter, which makes this an unsuitable candidate for ‘growth-related’ RNA-Seq.

Response 20: We really appreciate your professional comments. The growth and development of crustaceans are represented by a discontinuous process of molting. Crustacean growth is mainly concentrated in the molting stage. Crustaceans have a hard outer cuticle whose organic matter is mainly chitin and cuticular proteins. Different types of cuticular proteins bind to long-chain chitin and affect the structure and function of the cuticle. Crustaceans, such as shrimp and crabs, constitute the group with the largest chitin output, and their growth and development are majorly associated with the biosynthesis and modification of chitin (Zhang et al., 2021). Chitin metabolism is the result of the joint activity of the chitin synthase system and the chitin hydrolase system, both of which comprise a dynamic equilibrium process that is regulated by hormones and multiple signal pathways. In insects, the chitinase genes were originally divided into eight groups, all of which play different roles in growth and development (Arakane et al., 2010). Crustacean chitinases perform three main functions. Namely, crustacean chitinases participate in the molting and growth process, the digestion of chitin-containing food, and the immune response/disease prevention. Chitinases with different functions are mainly expressed in different tissue sites. In crustaceans, reports on the microscopic architecture of cuticle-muscle connections refer to different body regions in adult specimens of several taxa, including Branchiopoda, Balanidae, Ostracoda, Brachyura, Mystacocarida, and Isopoda. The dynamic expression characteristics of these genes in different tissues and developmental stages of M. japonicus need to be further studied.

 

Discussion

Point 21: See introduction: it is not clear what “growth” is – presumably, some of this is realised during the intermoult, which makes me question why all these moulting-related genes are appearing, and especially, why they would appear in muscle.

Response 21: We really appreciate your professional comments. Crustaceans, such as shrimp and crabs, constitute the group with the largest chitin output, and their growth and development are majorly associated with the biosynthesis and modification of chitin (Zhang et al., 2021). Chitin metabolism is the result of the joint activity of the chitin synthase system and the chitin hydrolase system, both of which comprise a dynamic equilibrium process that is regulated by hormones and multiple signal pathways. In insects, the chitinase genes were originally divided into eight groups, all of which play different roles in growth and development (Arakane et al., 2010). Crustacean chitinases perform three main functions. Namely, crustacean chitinases participate in the molting and growth process, the digestion of chitin-containing food, and the immune response/disease prevention. Chitinases with different functions are mainly expressed in different tissue sites. Moreover, some chitinase genes are only detected in one tissue, whereas others are found in multiple tissues. Cesar et al established a cDNA library of shrimp juvenile abdominal muscle by PCR-based SMART^TM cDNA technology. The high identity-matched ESTs included multiple cuticle protein genes (Cesar et al., 2008). It means that cuticle proteins are not specific to the epidermal tissue. Tissue distribution analysis revealed that a novel cuticle protein gene, LvCPAP1, was predominantly expressed in the epidermis, stomach, and muscle. In Litopenaeus vannamei, 13,000 DEGs were identified in muscle tissue, including genes encoding the cuticle, chitin, ecdysteroids, and muscle proteins (Santos et al., 2021). Cuticular proteins are important in the formation of new cuticles before and after molting (Vincent et al., 2002). The dynamic expression characteristics of these genes in different tissues and developmental stages of M. japonicus need to be further studied.

 

Typographical

Point 22: L31: ~ this study provides valuable data ~

Response 22: Thanks for your friendly reminder. We have made changes in the revised manuscript (L31).

Point 23: L45: ~ so it is very much loved ~

Response 23: Thanks for your friendly reminder. We have made changes in the revised manuscript (L45).

Point 24: L47 ~ high fecundity ~

Response 24: Thanks for your friendly reminder. We have made changes in the revised manuscript (L46-47).

Point 25: L60: ~ shed their exoskeleton ~

Response 25: Thanks for your friendly reminder. We have made changes in the revised manuscript (L58).

Point 26: L62 ~ Chinese perch, Siniperca ~

Response 26: Thanks for your friendly reminder. We have made changes in the revised manuscript (L65).

Point 27: L69: ~ mantles of oyster, Pinctada ~

Response 27: Thanks for your friendly reminder. We have made changes in the revised manuscript (L72).

Point 28: L72: ~ sizes of abalone, Haliotis discus ~

Response 28: Thanks for your friendly reminder. We have made changes in the revised manuscript (L76).

Point 29: L76, and elsewhere: please add trivial names, as done for the 3 examples above, for all animal names in this paper

Response 29: Thanks for your friendly reminder. We have made changes in the revised manuscript (L80).

Point 30: L132: featurerts?

Response 30: Thanks for your friendly reminder. We have made changes in the revised manuscript (L140).

Point 31: L138: when When?

Response 31: Thanks for your friendly reminder. We have made changes in the revised manuscript (L145).

Reviewer 2 Report

 The study "Comparing transcriptomes reveals molecular mechanisms in superior growth performance Marsupenaeus japonicus" by Panpan Wang with colleagues utilizes RNA-seq and qRT-PCR analyses to uncover muscle gene expression differences between fast-growing and slow-growing individuals of M. japonicus from the same family. The study highlights key genes and pathways associated with crustacean growth, contributing to a deeper understanding of the molecular regulatory mechanisms underlying growth differences.   First of all, I would like to point out the badly formulated title. I propose that it be written as "Comparison of muscle transcriptomes reveals molecular mechanisms underlying superior growth performance in Marsupenaeus japonicus". The proposed title also specifies that the transcriptome in the paper was not multi-tissue, but tissue-specific, based on muscle tissue only. Of course, the commercial weight of shrimp is formed solely by muscle, but the lack of specificity may misinform the potential reader. The introduction is mainly an enumeration of results obtained in other works in a related field. And practically does not describe the expected results. For example, it is not at all clear why additional analysis of SNPs is needed and how the authors intend to eventually use these results and the results of muscle transcriptome sequencing for marker-assisted selection. There is uncertainty between the results and the application. For example, it is not clear whether the trait is inherited if the breeding work is started to isolate the fast-growing and slow-growing individuals from each other, and whether this again splits them into large and small individuals in the progeny. Methodologically, it is not at all clear how, with muscular tissue available, without specifying the specifics of the collecting, the authors speculate about cuticle transcripts. It would be nice to disclose in the text how muscle tissue transcripts are related to molting. As such, the paper cannot be published in this journal and requires substantial revision. Furthermore, even a quick look at the authors' publication https://www.sciencedirect.com/science/article/pii/S2352513423001199 one can find almost the same work, only better proofread and on a different subject.   The following are more specific comments on the text: Line 103. Which anaesthetic was used to euthanise the shrimp? Line 135. when When Line 107 and 139. What are the reasons for the different ways of isolating RNA? Line 154. There is no Table S1 in the attached supplementary files. Line 175. It would be nice to decipher FRKM. Table 2. On what basis are these particular genes chosen to be displayed as examples? Figure 2. The category names in the caption to the picture should be deciphered. Line 217. The indicators are visually similar, but no correlation is shown here. Line 241. What's "Liew's research"? 

Author Response

Dear Editor and reviewers,

 

Thank you for your letter and the reviewers’ comments concerning our manuscript entitled “Comparing transcriptomes reveals molecular mechanisms in superior growth performance Marsupenaeus japonicus” (fishes-2432032). Those comments are valuable and very helpful for revising and improving our manuscript as well as important for guiding the significance of our research. We have read through the comments carefully and made corrections. Based on the instructions provided in your letter, we uploaded the file of the revised manuscript. The revised portions are indicated in red in the manuscript. The responses to the reviewer’s comments are as follows.

 

Point 1: The study "Comparing transcriptomes reveals molecular mechanisms in superior growth performance Marsupenaeus japonicus" by Panpan Wang with colleagues utilizes RNA-seq and qRT-PCR analyses to uncover muscle gene expression differences between fast-growing and slow-growing individuals of M. japonicus from the same family. The study highlights key genes and pathways associated with crustacean growth, contributing to a deeper understanding of the molecular regulatory mechanisms underlying growth differences. First of all, I would like to point out the badly formulated title. I propose that it be written as "Comparison of muscle transcriptomes reveals molecular mechanisms underlying superior growth performance in Marsupenaeus japonicus". The proposed title also specifies that the transcriptome in the paper was not multi-tissue, but tissue-specific, based on muscle tissue only. Of course, the commercial weight of shrimp is formed solely by muscle, but the lack of specificity may misinform the potential reader.

Response 1: We sincerely appreciate the valuable comments. We have made changes in the revised manuscript (L2-4).

Point 2: The introduction is mainly an enumeration of results obtained in other works in a related field. And practically does not describe the expected results. For example, it is not at all clear why additional analysis of SNPs is needed and how the authors intend to eventually use these results and the results of muscle transcriptome sequencing for marker-assisted selection.

Response 2: We really appreciate your professional comments. In the introduction, We first introduced the general situation of M. japonicus. During the culture of M. japonicus, under the same food, water qualities, and environments, the individual differences within the same family of shrimp seeds are obvious, and the growth rate of some shrimp seeds is slow. The authors describe a number of researchers who have used transcriptomics techniques to explore the molecular mechanism behind this phenomenon in other species. At present, the in-depth research on this aspect is relatively few. Most studies use transcriptomic techniques to screen potential functional genes. To further reveal the molecular mechanism of the obvious growth differences in the culture of M. japonicus, this study used comparative transcriptome sequencing technology to analyze the transcriptome of individuals with different growth characteristics in the same batch and screened out potential growth trait–related genes. The functional research on the up- and downregulated genes lay the foundation for molecular marker-assisted breeding of M. japonicus. A single nucleotide polymorphism (SNP) marker is a genetic marker of a single nucleotide mutation that occurs at a specific site of the genome. It is characterized by high abundance, high density, and easy genotyping. It is widely used in plant and animal breeding, disease resistance gene marker, dominant variety screening, and identification of disease-related genes. For species that currently do not have full genomes, transcriptome techniques are a good way to develop SNP markers, which have been recognized by academics for more than a decade. As the most ideal marker for molecular marker-assisted selection breeding, SNP has been widely used in breeding new varieties of large yellow croaker (Ding et al., 2022; Wang et al., 2023), grouper (Yang et al., 2020; Ai et al., 2023) and other species. In future studies, we will consider conducting comparative transcriptome analysis on the epidermal tissue of the same family of M. japonicus, as well as comparative analysis with the muscle transcriptome data, hoping to screen for more potential growth regulatory genes. In future studies, we will develop more SNP markers and apply them to the breeding process of new varieties of M. japonicus.

References:

• Ding J, Zhang Y, Wang J, et al. Genome-wide association study identified candidate SNPs and genes associated with hypoxia tolerance in large yellow croaker (Larimichthys crocea)[J]. Aquaculture, 2022, 560: 738472.
• Wang J, Miao L, Chen B, et al. Development and evaluation of liquid SNP array for large yellow croaker (Larimichthys crocea)[J]. Aquaculture, 2023, 563: 739021.
• Yang Y, Wu L, Wu X, et al. Identification of candidate growth-related SNPs and genes using GWAS in brown-marbled grouper (Epinephelus fuscoguttatus)[J]. Marine Biotechnology, 2020, 22: 153-166.
• Ai C H, Zhu Z X, Huang D D, et al. Identification of SNPs and candidate genes associated with early growth in orange-spotted grouper (Epinephelus coioides) by a genome-wide association study[J]. Aquaculture, 2023, 565: 739129.

 

Point 3: There is uncertainty between the results and the application. For example, it is not clear whether the trait is inherited if the breeding work is started to isolate the fast-growing and slow-growing individuals from each other, and whether this again splits them into large and small individuals in the progeny.

Response 3: We really appreciate your professional comments. Muscle growth in crustaceans is intermittent and closely associated with the molt cycle due to the presence of the rigid calcified exoskeleton. Increases in muscle mass are restricted to the ecdysial period when the old exoskeleton is shed and the new exoskeleton expands in size (Whiteley et al., 1997). Tissue growth in Crustacea occurs at specific stages of the molt cycle and is influenced by a number of physical, hormonal, and environmental factors (EI et al., 1997). Consequently, growth in Crustacea is closely associated with the molt cycle, in particular the stages surrounding ecdysis when there is a considerable increase in the rate of water uptake and a subsequent increase in hydrostatic pressure causing the new uncalcified exoskeleton to expand providing space for tissue growth (Mykles, 1980). Typically, the larger individuals at the beginning have a stronger competitive advantage and will have access to more food. Whether it's behavioral or genetic. Traditional selective breeding methods are also used to select growth-advantaged individuals in a family or population as parents.

References:

• Whiteley N M, El Haj A J. Regulation of muscle gene expression over the moult in crustacea[J]. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 1997, 117(3): 323-331.
• El Haj A J, Whiteley N M. Molecular regulation of muscle growth in Crustacea[J]. Journal of the Marine Biological Association of the United Kingdom, 1997, 77(1): 95-106.
• Mykles D L. The mechanism of fluid absorption at ecdysis in the American lobster, Homarus americanus[J]. Journal of Experimental Biology, 1980, 84(1): 89-102.

 

Point 4: Methodologically, it is not at all clear how, with muscular tissue available, without specifying the specifics of the collecting, the authors speculate about cuticle transcripts. It would be nice to disclose in the text how muscle tissue transcripts are related to molting. As such, the paper cannot be published in this journal and requires substantial revision.

Response 4: We really appreciate your professional comments. We sincerely appreciate the valuable comments. During the cultivation of M. japonicus of the same batch, there are often obvious differences in the growth of different larvae, but the underlying growth regulation mechanism is not yet clear. To explore the molecular mechanism of this growth difference, this study used RNA-seq technology to compare M. japonicus individuals with significant growth differences from the same family. The exoskeletal cuticle and muscles constitute the arthropod musculoskeletal system and function jointly to enable animal movements and locomotion (Mark et al., 2017). The establishment of accurate structural connections between cuticle and muscles via epidermal tendon cells is an essential part of exoskeletal cuticle morphogenesis at the organism level. In crustaceans, reports on the microscopic architecture of cuticle-muscle connections refer to different body regions in adult specimens of several taxa, including Branchiopoda, Balanidae, Ostracoda, Brachyura, Mystacocarida, and Isopoda. The fibers that are anchored to the apical membrane of the tendon cell and extend deeply into the cuticle are also interesting and were defined as muscle attachment fibers. Given the special relationship between skin and muscle tissue, it may be difficult for the average researcher to isolate the two absolutely, especially when studying smaller shrimp. We will increase the sampling level in future experiments. Cesar et al established a cDNA library of shrimp juvenile abdominal muscle by PCR-based SMART^TM cDNA technology. The high identity-matched ESTs included multiple cuticle protein genes (Cesar et al., 2008). It is also possible that cuticle proteins are not specific to the epidermal tissue. Tissue distribution analysis revealed that a novel cuticle protein gene, LvCPAP1, was predominantly expressed in the epidermis, stomach, and muscle. In Litopenaeus vannamei, 13,000 DEGs, including genes encoding the cuticle, chitin, ecdysteroids, and muscle proteins, were identified in families with higher and lower growth performances (Santos et al., 2021). Cuticular proteins are important in the formation of new cuticles before and after molting (Vincent et al., 2002). Different types of cuticular proteins bind to long-chain chitin and affect the structure and function of the cuticle (Willis et al., 2012). In this paper, the real research results are analyzed and discussed. In future studies, we will consider conducting comparative transcriptome analysis on the epidermal tissue of the same family of M. japonicus, as well as comparative analysis with the muscle transcriptome data, hoping to screen for more potential growth regulatory genes.

References:

• Mrak P, Bogataj U, Štrus J, et al. Cuticle morphogenesis in crustacean embryonic and postembryonic stages[J]. Arthropod Structure & Development, 2017, 46(1): 77-95.
• Cesar J R, Zhao B, Yang J. Analysis of expressed sequence tags from abdominal muscle cDNA library of the pacific white shrimp Litopenaeus vannamei[J]. animal, 2008, 2(9): 1377-1383.
• Santos C A, Andrade S C S, Teixeira A K, et al. Transcriptome differential expression analysis reveals the activated genes in Litopenaeus vannamei shrimp families of superior growth performance[J]. Aquaculture, 2021, 531: 735871.
• Vincent, J.F. Arthropod cuticle: a natural composite shell system. Composites Part A: Applied Science and Manufacturing 2002, 33, 1311-1315.
• Willis, J.H.; Papandreou, N.C.; Iconomidou, V.A.; Hamodrakas, S.J. Cuticular proteins. In Insect Molecular Biology and Biochemistry; Elsevier: 2012; pp. 134-166.

 

Point 5: Furthermore, even a quick look at the authors' publication https://www.sciencedirect.com/science/article/pii/S2352513423001199  one can find almost the same work, only better proofread and on a different subject.

Response 5: We really appreciate your professional comments. The research objects of the two articles are Marsupenaeus japonicus and Exopalaemon carinicauda, respectively, which are based on the production situation of individual growth differences in the same family, hoping to explore the mechanism of differences from the molecular level. Aquatic breeding methods include family selection and population selection, in which the traditional selection of large individuals as parents, so as to achieve the cumulative effect of dominant genes, to achieve the purpose of breeding. Our aim was to explore at the molecular level whether large individuals in the family are genetically superior to small individuals. The growth of aquatic animals is not only affected by internal factors such as their own genetic and physiological conditions but also by external factors such as food and water environment. In our previous research, according to the significant differences in growth traits of E. carinicauda from the same family, we performed a comparative transcriptome analysis to identify the key genes involved in weight gain and compared the intestinal flora to screen potential intestinal probiotics.

 

The following are more specific comments on the text:

Point 6:Line 103. Which anaesthetic was used to euthanise the shrimp?

Response 6: Thanks for your friendly reminder. We have made changes in the revised manuscript (L114-115).

Point 7:Line 135. when When Line 107 and 139. What are the reasons for the different ways of isolating RNA?

Response 7: Thanks for your friendly reminder. In the experiment, TRIzol reagent (TaKaRa, Dalian, China) was used to extract RNA from muscle tissue samples of M. japonicus. We have made changes in the revised manuscript (L118-119, 149).

Point 8:Line 154. There is no Table S1 in the attached supplementary files.

Response 8: Thanks for your friendly reminder. We have added the attached materials.

Point 9:Line 175. It would be nice to decipher FRKM.

Response 9: Thanks for your friendly reminder. We have made changes in the revised manuscript (L185-187).

Point 10:Table 2. On what basis are these particular genes chosen to be displayed as examples?

Response 10: We really appreciate your professional comments. A total of 1375 differentially expressed genes were identified in this study. Table 2 shows some growth-related DEGs, such as tubulin α-1 chain, ecdysteroid kinase, myosin heavy chain C, cuticular protein, sarcoplasmic calcium-binding protein beta chain, mitochondrial basic amino acid transporter, and actin. The dynamic expression characteristics and function of these genes in the molting process and different developmental stages of M. japonicus need to be further studied.

Point 11:Figure 2. The category names in the caption to the picture should be deciphered.

Response 11: We really appreciate your professional comments. We have made changes in the revised manuscript (L222-223).

Point 12:Line 217. The indicators are visually similar, but no correlation is shown here.

Response 12: Thanks for your friendly reminder. To verify the reliability of the transcriptome sequencing results, the expression levels of ten differentially expressed genes between the fast-growing group and the slow-growing group were detected by real-time PCR. There was a good correlation between the RNA-seq data expression of candidate genes and the results of real-time PCR, and the expression trends of the same gene between the two groups were consistent. The results showed that the differential gene expression patterns in transcriptome sequencing were reliable.

Point 13:Line 241. What's "Liew's research"? 

Response 13: Thanks for your friendly reminder. Liew et al used engineered plasmids to study all possible SNP base changes by high-resolution melting analysis (Liew et al., 2004). In the study, the author divided the genomic SNPS into four categories, including Class 1 SNPs are C/T and G/A transitions that produce C::G and A::T homoduplexes and C::A and T::G heteroduplexes. In contrast, class 2 SNPs (C/A and G/T) are transversions that produce C::T and A::G heteroduplexes. Class 3 SNPs (C/G) produce C::G homoduplexes with C::C and G::G heteroduplexes. Class 4 SNPs (A/T) produce A::T homoduplexes with A::A and T::T heteroduplexes. We have supplemented the reference in the revised manuscript (L255).

Round 2

Reviewer 1 Report

Please see attachment

Comments for author File: Comments.pdf

Minor editing of English language required

Author Response

Please see attachement

Author Response File: Author Response.pdf

Reviewer 2 Report

After the corrections have been made, it is noticeable that the manuscript has improved. At the same time there is a comment in the form of an appeal to the authors.
 
Remember that you are not responding to a reviewer, but to a potential reader who wishes to disclose some issues not added to the introduction or discussion. Accordingly, your response to points 2,3 and 4 goes nowhere. Why not provide a summary of these ideas in the relevant places in the article?

Author Response

Dear Editor and reviewers,

Thank you for your letter and the reviewers’ comments concerning our manuscript entitled “Comparing transcriptomes reveals molecular mechanisms in superior growth performance Marsupenaeus japonicus” (fishes-2432032). Those comments are valuable and very helpful for revising and improving our manuscript as well as important for guiding the significance of our research. We have read through the comments carefully and made corrections. Based on the instructions provided in your letter, we uploaded the file of the revised manuscript. The revised portions are indicated in blue in the manuscript. The responses to the reviewer’s comments are as follows.

Point 1: After the corrections have been made, it is noticeable that the manuscript has improved. At the same time there is a comment in the form of an appeal to the authors. Remember that you are not responding to a reviewer, but to a potential reader who wishes to disclose some issues not added to the introduction or discussion. Accordingly, your response to points 2,3 and 4 goes nowhere. Why not provide a summary of these ideas in the relevant places in the article?

Response 1: We sincerely appreciate the valuable comments. We have made changes in the revised manuscript (L299-310, 337-339, 342-349, 354-355, 360-361).

 

Thank you for your consideration of our work.

Sincerely yours

Corresponding author: Chaofan Xing

Address: Jiangsu Key Laboratory of Marine Bioresources and Environment, Jiangsu Key Laboratory of Marine Biotechnology, Jiangsu Ocean University, Lianyungang 222005, China.

E-mail: [email protected] (Chaofan Xing)

Tel.: +86 15805911240 (C.f. Xing)