Transcriptome Analysis of The Whole Body Provides Novel Insights Into The Molecular Mechanisms of Growth, Immunity, and Metabolism During The Molting Cycle of The Juvenile Mud Crab Scylla Paramamosain

Background: Molting is an inherent cyclic process that occurs in all crustaceans and is essential for growth, metamorphosis, and reproduction. Recent studies have shown that many organs and systems are involved in molting regulation, including the hepatopancreas, the immune system, and production of ecdysteroids by Y-organs. However, the molecular mechanisms underlying the dynamic process of crab molting remain poorly understood at the whole-individual level. Therefore, we investigated global expression changes in the transcriptomes of the mud crab Scylla paramamosain, the most cultured crab species worldwide. We revealed a cascade of sequential expression events of molting-related genes involved in various aspects of the molting process by using whole-body sequencing of juvenile crabs. Results: RNA-sequencing (RNA-seq) produced 139.49 Gb of clean reads and 20,436 differentially expressed genes (DEGs) among different molting stages. The expression patterns for genes involved in several molecular events critical for molting, such as cuticle reconstruction, cytoskeletal structure remodeling, hormone regulation, immune responses, and metabolism, were characterized and considered as mechanisms underlying molting in S. paramamosain. Among these genes, we identied 10,695 DEGs in adjacent molting stages. In further GO and KEGG analyses, the signicantly enriched pathways included structural constituents of cuticle, binding and chitin metabolic processes, steroid hormone biosynthesis, insulin resistance, and amino sugar metabolic processes. The expression proles of 12 functional genes detected via RNA-Seq were corroborated via real-time RT-PCR assays. The results indicated gene expression proles across the molting cycle and identied possible activation pathways for future investigation of the underlying molecular mechanisms. Conclusions: This study establishes a comprehensive transcriptomic repertoire for the molting process in S. paramamosain. The data generated in this study will serve as an important transcriptomic resource for the crab research community to facilitate gene and genome annotation and provide fundamental support for future research on molecular mechanisms in this species. Furthermore, the comprehensive analysis results will improve our understanding of morphological variation in the molting cycle and serve as a potential blueprint for future research on molting in crustaceans and other animals.


Introduction
Molting is an important and ongoing process of physiological change in the life history of all crustaceans, with periodic shedding of the old cuticle and subsequent reconstruction of a new rigid exoskeleton [1,2]. Several factors are common to the molt cycle in all crustaceans, including transcription of cuticular genes, chitin synthesis, exoskeleton reconstruction, and hormone regulation [3,4]. Previous studies have made signi cant advances in our understanding of the molting cycle concerning growth characteristics, material changes, energy metabolism, and hormone regulation at the ecological and physiological levels [5][6][7][8][9]. Moreover, recent studies have explored molting-related genes and their effects on protein synthesis [10], chitin metabolism [11], and muscle hydration control [12] during the molting recently, a transcriptome study on the hepatopancreas immune response during the molting cycle in S. paramamosain has provided a better understanding of the hepatopancreas immune system [17], but our knowledge of the molecular mechanisms remains limited. Therefore, it is important to understand the mechanisms underlying crab molting and to characterize the cascade of sequential expression events of molt-related genes involved in various aspects of the molting process. This will facilitate further investigation of functional genomics for S. paramamosain and other closely related species.
The present study attempted to construct a comprehensive transcriptional landscape for further studies of the molecular mechanisms governing the molting cycle process in S. paramamosain using RNAsequencing. The assembled unigenes were annotated against public protein databases followed by NR-NCBI, NT, PFAM, Swiss-Prot, GO, KOG, and KEGG classi cation to identify DEGs among all molting stages. To investigate expression pro les, we identi ed genes that were differentially expressed during separate molting stages by sequencing whole juvenile crabs. This resource and the associated ndings will provide insights into biological processes of the molting cycle in this species and will help to improve its performance in aquaculture.

Experimental design and animal sampling
Healthy juvenile mud crabs from a full-sib family with an average carapace width of 1 cm at growth stage II (Fig. 1) were sampled from our research facility at Ningbo University. These individuals were cultured in tanks with adequate aeration, temperature (25 °C), and food (given twice daily). All procedures involving animals throughout the experiments were conducted in strict accordance with the the National Institutes of Health guide for the care and use of Laboratory animals.
All crabs were molting staged by examination of pleopod paddles for epidermal retraction, pigmentation, setae development, and the presence of matrix or internal cones in the setal lumen. Molting stages were classi ed as follows: pre-molt (PrM, Fig. 1A), post-molt (PoM, Fig. 1B), and intermolt (InM-I, InM-II, InM-III, InM-IV, Fig. 1C-F) [3,4,18]. According to the descriptions and de nitions of molting stages in previous studies [19,20], PoM should correspond to stage B; InM-I, InM-II, InM-III, and InM-IV correspond to stage C, and PrM corresponds to stage D. We collected three whole crabs from each of these six molting stages and combined them to generate a single sample for sequencing. Three samples were taken as biological replicates for each molting stage. Nine individual crabs were sampled at each molting stage. All samples were immediately frozen and stored in liquid nitrogen until RNA isolation.
A total of 1.5 µg RNA per sample was used as input material for the RNA sample preparation. Sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer's recommendations, and index codes were added to attribute sequences to each sample. First-strand cDNA was synthesized using random hexamer primers, and M-MuLV Reverse Transcriptase (RNase H-) Second-strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. After adenylation of the 3' ends of DNA fragments, NEBNext Adaptor with a hairpin loop structure was ligated to prepare for hybridization. In order to preferentially select cDNA fragments of 250-300 bp in length, the library fragments were puri ed with an AMPure XP system (Beckman Coulter, Beverly, USA). Then, 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. The PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. Finally, PCR products were puri ed (AMPure XP system), and library quality was assessed on the Agilent Bioanalyzer 2100 system [21].

Differential gene identi cation, enrichment, and pathway analysis
The gene expression levels were calculated by using FPKM (expected number of Fragments Per Kilobase of transcript sequence per Million base pairs sequences) [24]. Differential expression analysis of two samples was performed using the DEGseq (2010) R package. DESeq provides statistical routines for determining differential expression in digital gene expression data. Genes with an adjusted P-value < 0.05 found by DESeq were assigned as differentially expressed using a model based on the negative binomial distribution. The P-value was adjusted using the q value [25]. The q value < 0.005 & |log2 (foldchange)| >1 were set as the threshold criteria for signi cantly differential expression between adjacent samples (InM-I  vs PoM, InM-II vs InM-I, InM-III vs InM-II, InM-IV vs InM-III, PrM vs InM-IV, PoM vs PrM). Gene Ontology (GO) enrichment analysis of the differentially expressed genes (DEGs) was implemented by the GO seq R packages-based Wallenius non-central hyper-geometric distribution that can adjust for gene length bias in DEGs [26]. We used KOBAS software [27] to test the statistical signi cance of enrichment of differentially expressed genes in KEGG pathways.

Quantitative reverse-transcription PCR (qRT-PCR) analysis
To validate the results from RNA-Seq differential gene expression analysis, qRT-PCR was carried out among all six molting stages. Twelve genes from different gene expression clusters were randomly selected for qRT-PCR assays. PCR primers were designed based on the assembled transcriptome sequences (Table S2). The β-actin gene was ampli ed in parallel as an internal control.
Total RNA was extracted, and the rst-strand cDNA was synthesized by using a PrimeScript RT Reagent Kit with gDNA Eraser (Takara). All samples were run in triplicate in separate tubes; each cDNA sample was run in duplicate. The qRT-PCR was conducted by using SYBR Green Premix Ex Taq (Takara, Japan) in an ABI 7500 Sequence Detection System (Applied Biosystems). A standard curve was initially generated to assess accuracy, and primers with e ciency of ampli cation between 95% and 105% were chosen for the following qRT-PCR. The comparative Ct method (with the formula 2 −ΔΔCt ) was used to analyze the expression levels of different genes [28]. Student's t-test was conducted using SPSS 20.0 (http://www-01.ibm.com/software/analytics/spss/), and P-values less than 0.05 were considered statistically signi cant.

Illumina sequencing and transcriptome assembly
The assembly generated 953,784,150 raw reads from 18 samples taken at the six molting stages. A draft assembly of the S. paramamosain transcriptome was constructed with 139.49 Gb of quality ltered shortread Illumina sequence data (Table S1). All transcriptome data are available in the NCBI Short Read Archive (SRA) database under accession PRJNA687923. Finally, we obtained an average of 51,664,527 clean reads for our 18 samples, with Q20 of all samples greater than 97% (Table S1). The numbers of unigenes with FPKM > 0.3 ranged from 70,319 to 78,247 among the eight molting stages ( Fig. 2A). The number of reads mapped to the stage-speci c transcriptome was more than 100 million clean reads ( Fig. 2B). More than 70% of the reads were mapped to the ltered reference transcriptome and the stagespeci c corresponding transcriptome (Fig. 2B, Table S1).
In our assembly, 350,531 transcripts (each with more than 200 bp) with a total length of 339,582,267 bp were obtained, and these were assembled into 248,744 unigenes totaling 312,762,368 bp. A large portion of the transcripts were between 200 bp and 500 bp ( Table 1). The transcripts had an N50 size of 2,224 bp, mean transcript length of 969 bp, maximum transcript length of 33,949 bp, and mean unigene length of 1,257 bp (Table 1).

Expression pro le of DEGs across the molting cycle of S. paramamosain
A total of 20,436 DEGs were identi ed across the six molting stages (Table S4). Expression levels of DEGs during the molting cycle were divided into 22 categories based on K-means clustering that revealed different expression patterns of DEGs among the six molting stages (Fig. S3) S3).
GO enrichment analysis (FDR < 0.005) of these DEGs demonstrated enrichment of multiple terms of biological processes, cellular components, and molecular functions associated with the molting cycle (e.g., structural constituents of cuticle, amino sugar metabolic processes, chitin metabolic processes, and hormone receptor binding) (Fig. 3). Pro ling gene expression and GO enrichment analysis of DEGs found that genes in clusters 3, 7, 8, 10, 11, 18, and 20 were predominantly up-regulated in the molting stages InM-I, InM-II, and PoM and were down-regulated in InM-III, InM-IV, and PrM, stages that were mainly associated with "myosin complex," "motor activity," and "actin cytoskeleton"; and "structural molecule activity," "structural constituent of cuticle," and "chitin binding"; and "amino sugar metabolic process." Conversely, DEGs in clusters 12, 16, 17, and 19 were predominantly down-regulated in the molting stages InM-I, InM-II, and PoM and were up-regulated in InM-III, InM-IV, and PrM, stages that were mainly associated with "glucosamine-containing compound metabolic process," "amino sugar metabolic process," "GTP binding," "oxidoreductase activity," "guanyl ribonucleotide binding," "3-beta-hydroxy-delta5steroid dehydrogenase activity," "C21-steroid hormone metabolic process," and "regulation of hormone levels." Interestingly, DEGs in clusters 2, 4, and 13 were only up-regulated in stage PrM, and these were strongly enriched in "structural constituent of cuticle," "structural molecule activity," "chitin metabolic process," "glucosamine-containing compound metabolic process," and "amino sugar metabolic process." Moreover, DEGs in clusters 1 and 16 showed that the expression of genes increased with the number of days following molting. Details of the top 30 GO categories of the 22 clusters are shown in Table S5. 3.4. Expression pro les of DEGs associated with growth, immunity, and metabolism among molting stages The molting of crustaceans is accompanied by structural changes and material metabolism in various tissues of the body, among which muscles and the carapace undergo the most dramatic changes during the molting cycle. In this study, dozens of DEGs associated with carapace, muscle, and hemolymph were identi ed (Table 2). Of these DEGs, we identi ed many transcripts related to the composition and The muscle-and skeleton-related genes included many aspects of muscle formation and function from structural protein to muscle contraction such as actin, muscle LIM protein, muscle and skeletal receptor tyrosine protein kinase, cardiac muscle actin, and myosin. In addition, six other genes were found to be associated with hemocyte homeostasis and composition, including cryptocyanin, hemocyte kazal-type proteinase inhibitor, hemocyte homeostasis associated protein, hemocyanin, and spz3 (Table 2). Interestingly, some DEGs showed large changes in expression. For example, the FPKM values of cuticle protein CUT2 increased more than 10,000-fold from InM-I to InM-II, but decreased from InM-III, reaching zero at stage PrM. Additionally, the FPKM values of cuticle proprotein proCP and cuticular protein 15 decreased more than 3,000-fold from InM-III to InM-IV, and the same pattern was found for gastrolith protein 30, with a more than 3,000-fold decrease from InM-II to InM-III.
In our study, 21 important hormone-related genes that may be directly or indirectly involved in controlling the crustacean molting cycle were identi ed ( Table 2). Of these genes, the expression levels of ecdysis triggering hormone receptor subtype-B and ecdysone induced protein showed characteristic patterns of down-regulation during the entire molting cycle from PoM to PrM. The expression of ecdysteroidregulated protein was up-regulated from PoM to InM-II and then declined from InM-III to PrM. Estrogen sulfotransferase was up-regulated from PoM to InM-IV but increased suddenly at stage PrM, and the same pattern was found for estradiol 17-beta-dehydrogenase 8 and juvenile hormone epoxide hydrolase (Table 3). To identify the possible factors involved in the molting process, we also investigated the expression pro les of members of the immune system and metabolism during molting. There were 21 and 28 genes found to be related to the immune system and metabolism, respectively. Of the immune-related genes, the C-type lectin receptor was down-regulated in PoM but up-regulated in PrM, and the same pattern was found for crustin 2, crustin 3, beta-thymosin 4, and beta-thymosin 5. Interestingly, most immune system genes reached their maximum expression levels in InM-II; these included C-type lectin domain family 4member, C-type lectin-2, C-type lectin receptor, C-type lectin, crustin-2, crustin 4, and ALF1. Twenty-eight genes related to substance metabolism, storage, and transportation were detected, including four sugar transporters, one sterol regulatory element-binding protein, six serine proteinases, three serine proteinase inhibitor accases, six cytochrome P450s, three lipoprotein receptors, one lipid storage droplet protein, and one lipase (Table S6).

GO and KEGG enrichment analysis of DEGs in adjacent molting stages
Our analysis speci cally investigated the interactions among DEGs between adjacent molting stages and their functioning in biological processes by GO and KEGG enrichment. A total of 10,695 DEGs between any two adjacent molting stages (i.e., InM-I vs PoM, InM-II vs InM-I, InM-III vs InM-II, InM-IV vs InM-III, PrM vs InM-IV, and PoM vs PrM) were identi ed (Table S7, Fig. 2D). The 10,695 DEGs were further subjected to GO enrichment to predict their potential functions and to KEGG enrichment to obtain relevant metabolic pathways. To associate different expression patterns of these DEGs with morphological and physiological changes during the molting cycle, we performed GO enrichment analysis and sorted DEGs into different sub-categories that belonged to three main GO categories: biological processes, cellular components, and molecular functions. GO enrichment analysis revealed that structural constituents of the cuticle were the most signi cantly enriched pathway for 10,695 DEGs accounting for 451 unigenes, followed by chitin-binding and chitin metabolic process (Table S8, Fig. S4A). Notably, these top three enrichment pathways were all related to the molecular function and biological process of shell formation regulation during crab molting.
In the comparison of InM-I vs PoM, 597 DEGs were up-regulated, and 711 DEGs were down-regulated. These were sorted into 2,585 GO sub-categories with six signi cantly enriched sub-categories (Corrected P-value < 0.05) of amino sugar metabolic processes, structural constituents of cuticle, chitin metabolic processes, glucosamine-containing compound metabolic processes, chitin binding, and viral release from host cell (Fig. 2D, Fig. S5). In the comparison of InM-II vs InM-I, 338 DEGs were up-regulated, and 410 DEGs were down-regulated; these were sorted into 2,152 GO sub-categories with four signi cantly enriched sub-categories (Corrected P-value < 0.05) of structural constituents of cuticle, histidine biosynthetic processes, histidine metabolic processes, and imidazole-containing compound metabolic processes (Fig. 2D, Fig. S5). A total of 322 DEGs were up-regulated and 627 DEGs were down-regulated in the comparison of InM-III vs InM-II, and no signi cantly enriched sub-categories (Corrected P-value < 0.05) were observed. In the comparison of InM-IV vs InM-III, 789 DEGs were up-regulated, and 780 DEGs were down-regulated; these were sorted into 2,506 GO sub-categories with 13 signi cantly enriched subcategories (Corrected P-value < 0.05) (Fig. 2D, Fig. S5)  Figure S5.
To more precisely analyze the expression patterns of DEGs in the molting cycle, we examined the expression pro les (Table S10) and listed the top 10 GO enrichment categories of DEGs in pairwise comparisons among molting stages of PrM, PoM, and InM (Table S11). Interestingly, Venn diagram analysis revealed a total of 73 and 1418 DEGs for pairwise comparisons between the four intermolt stages (InM-I, InM-II, InM-III, InM-IV) and PrM, PoM, respectively (Fig. 4). In InM vs PoM, 73 DEGs were sorted into the most enriched GO sub-categories associated with energy metabolism (e.g., adenosine kinase activity, AMP biosynthetic process, AMP metabolic process, and ribo avin synthase complex). In InM vs PrM, 1418 DEGs were sorted into the most enriched GO sub-categories associated with shell formation in the molting cycle (e.g., structural constituent of cuticle, structural molecule activity, chitin binding, and chitin metabolic process).

qRT-PCR validation
For validation purposes, we selected 12 representative genes from the 22 clusters identi ed above and examined their expression levels during the molting cycle by using qRT-PCR. The qRT-PCR results con rmed the ndings of RNA-Seq, with correlation coe cient (r) values from 0.844 to 0.979 depending on the speci c genes and reference genes (Fig. 5).

Discussion
Establishment of a comprehensive transcriptomic repertoire for the molting process in S. paramamosain In order to produce a comprehensive reference transcriptome, next-generation sequencing was used to construct a genome-wide transcriptional landscape for further studies of the developmental and molecular aspects of the molting process in S. paramamosain. An S. paramamosain molt cycle-speci c transcriptome derived from whole juvenile crabs, including individual organs such as the brain, gills, nerves, eyestalks, hepatopancreas, muscle, MO, and Y organs from all molt cycle stages, was developed for this study. There were some similarities to previous studies [3,4] involving molting cycle transcriptome analyses in crustaceans compared to the results of our work; for example, some common factors related to the molting cycle were identi ed, such as the cuticular genes, chitin synthase, exoskeleton reconstruction, and hormone regulation. However, several differences exist between our results and those of previous studies. These differences may be due to the materials and techniques used. The transcriptome database obtained using Illumina RNA-seq in our study was larger than those obtained by microarray-based analysis in other studies, and more genes were detected at each stage of the molting process in our study. Second, previous studies mainly focused on the functional analysis of single organs during the molting cycle or on a speci c stage, for example, focusing on muscle [29], the hepatopancreas [4,17], the eyestalks and heart [30], or the mandibular organ and Y-organ [31]. Moreover, our analysis revealed a cascade of sequential expression events of genes involved in various aspects of the molting process, including chitin degradation and synthesis, tissue development, hormone regulation, immune response, and energy metabolism. The present transcriptomic data are of high quality compared with previous studies and provide a comprehensive survey from each molting cycle stage of S. paramamosain.
Gene Ontology functional annotation analysis classi ed the predicted functions of the assembled unigenes. Cell, binding, and cellular processes were the most highly represented categories, consistent with transcriptome results of the whole claw muscles in Eriocheir sinensis [29] and Penaeus monodon [30]. Our results further demonstrated that proliferation of epithelial tissues [32,33], muscle [34], and other development-related processes accompanying muscle and exoskeleton reconstruction were prominent during molting. Moreover, KEGG analysis demonstrated that the top enriched pathways were organismal systems instead of being related to metabolism as in previous studies [29,30]. These pathways included the circulatory system, environmental adaptation, the digestive system, endocrine system, the immune system, and the nervous system. Also, translation, ribosomal structure, and biogenesis were identi ed as the most present categories. Therefore, the present results suggest the biological functions and interactions of annotated unigenes involved in organ development, tissue reconstruction, and adjustment of environmental pressure during the molting cycle. These annotations are the starting point for investigating the molecular mechanisms of molting-related genes in the molting cycle of S. paramamosain, and they also provide a valuable resource for further research into speci c functions and pathways of crustacean molting.
The molting cycle is a complex process that is normally divided into four stages: intermolt, premolt, ecdysis, and postmolt [35,36]. Overall, our clustering analysis and GO enrichment of DEGs across the molting cycle of S. paramamosain revealed the variational characteristics of molecular changes responding to morphological and physiological changes. For example, previous studies have indicated that postmolt (PoM) is a critical phase in the molting cycle of crustaceans, a period in which the animal is recovering from molting and during which its exoskeleton is quickly hardened to avoid predation [4,29]. The most enriched GO terms of DEGs in PoM, InM-I, and InM-II of S. paramamosain included "myosin complex," "motor activity," "actin cytoskeleton," and "structural molecule activity," revealing a gradual upregulation of muscle-related genes and further indicating the important functions of these genes in muscle regeneration during the postmolt and intermolt periods [6,31,37]. Moreover, up-regulation of genes in the premolt stage being enriched in the GO terms "3-beta-hydroxy-delta5-steroid dehydrogenase activity," "C21-steroid hormone metabolic process," and "regulation of hormone levels" demonstrated that the premolt stage, a critical phase in preparation for ecdysis, is controlled by hormonal regulation in the molting cycle of S. paramamosain.
Expression patterns of DEGs associated with cuticle and cytoskeletal proteins among molting stages Similar to other crustaceans, growth of S. paramamosain is a stepwise process comprising periodic shedding, subsequent reconstruction of a rigid external exoskeleton and cuticle, and muscle growth to ll the new body during a molting cycle [3,38]. Some functional genes involved in the molting cycle of crabs were identi ed as associated with a series of biological processes in previous studies, for example, in cuticle reconstruction, cytoskeletal structure remodeling, protein synthesis, hormone regulation, immune response, and metabolism [4,13,15,17]. Among these processes, cuticle reconstruction and cytoskeletal structure remodeling have been considered to be essential for all four phases of the molting cycle [33]. More than 50 different cuticle-and cytoskeletal-related genes were identi ed as being differentially expressed across the molting cycle in this study. For example, chitinase digests the old exoskeleton to regenerate a new shell via chitin metabolism that involves chitin synthase genes, chitinase genes, chitin deacetylase genes, and a number of genes whose gene products contain chitin-binding or other chitin metabolism-related domains [11,39,40]. Research studies on Penaeus aztecus [41] and P. monodon [42] have identi ed chitinase 2 as a direct factor in the molting process that is up-regulated in stage PrM and down-regulated in PoM. Moreover, previous study in juvenile Penaeus chinensis [43] has shown that the expression changes of chitinase 1 and 3 can be observed at all stages of the molting cycle. Finally, we identi ed six chitinases and found that they showed differential expression patterns in stages PoM, InM, and PrM, suggesting different physiological roles and modes of action of during the molting cycle. The chitin deacetylase gene is another chitin metabolism-related gene involved in the catalysis of the acetamido group in the N-acetyl-d-glucosamine units of chitin [44,45]. To date, ve classes of chitin deacetylase have been characterized, three of which were identi ed in this study (chitin deacetylase 1, chitin deacetylase 4, and chitin deacetylase 9). In insects, it has been demonstrated that the chitin deacetylase gene is involved not only in growth but also in the immune system [45,46]. However, few studies have charactered the chitin deacetylase genes or studied their function in the molting cycle of crustaceans. Most recently, the rst study concerning the cDNA cloning of chitin deacetylase in a crab species has been carried out in Chionoecetes japonicus [47]. The authors of the latter study hypothesized that chitin deacetylase was involved in ecdysis, as it was expressed only in the epidermis. Moreover, transcriptome analysis of molting-related tissues in Cherax quadricarinatus [11] and E. sinensis [40] revealed the up-regulated expression of chitin deacetylase during PoM compared to its levels during InM. In our study, chitin Cytoskeletal reconstruction is an important process for body recovery after molting [33]. However, factors participating in enlargement of the cytoskeleton during the molting process are not yet completely clear.
At the level of gene expression, we found a number of genes involved in osmotic regulation, muscle growth, and cytoskeletal structure remodeling that were up-or down-regulated at different molting stages.
For example, three aquaporin genes were identi ed with lower expression levels during the PrM stage but higher levels during PoM and early InM, possibly due to rapid water uptake that occurs after ecdysis.
Previous gene expression studies in Palaemon argentinus [12] and S. paramamosain [2] have explored the role of aquaporin with Na+/K+-ATPase and Na+/K+/2Cl− cotransporter in the uptake of water during the molting processes; these are osmoregulation-related genes with the same trends of expression levels in each of the molting stages in our study. We inferred that aquaporin and other osmoregulation-related genes act synergistically to regulate osmotic pressure and water absorption after molting. Cytoskeletal reconstruction involves not only fast water absorption but also expansion of the cytoskeleton to provide a scaffold and muscle to ll the new body [38]. We observed up-regulation of many cytoskeletal genes during early PrM and PoM that have been shown to be related to constituents of the microtubules, growth, and movement of muscle, including titin, tubulin, tropomyosin, and myosin. Among these cytoskeletal-related genes, titin binds to lamentous actin and provides elasticity to muscles in insects [51], and tubulin is another important cytoskeletal group of proteins, the major constituents of the microtubules [52]. Actin is a globular multi-functional protein that forms micro laments and affects muscle plasticity in crustaceans [53,54]. As the major contractile protein in vertebrates and invertebrates, myosin was identi ed to be associated with muscle atrophy during molting and is characterized by decreases in ber width and myo bril cross-sectional areas, an increase in inter brillar spaces, and degradation [34]. Moreover, hemocyanin is thought to play a role in cuticle formation and ecdysone transport during molting regulation and antigen non-speci c immune defense by reversibly binding oxygen [55] and displaying PO, ecdysone binding, and transportation activity [50,56], which is signi cantly increased together with other hemolymph-associated genes during the late InM and PrM stages. Additionally, energy reserves, including glycogen and lipids, are also accumulated in the hemolymph for the next molt [18].
Expression patterns of DEGs associated with hormones among molting stages The regulatory mechanism of molting is a complex process that includes a network of signals involving many hormone genes such as molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH) peptide families that control the molting process by inhibiting YO ecdysteroidogenesis and secretion [57,58]. Although it is known that CHH is released from gut endocrine cells immediately before ecdysis in crustaceans, we know little regarding the stage-to-stage expression variation in molting, although this is clearly necessary to understand the signi cance of these hormones. In this study, CHH was up-regulated more than fteenfold during the InM-IV and PrM stages. During the same period, the expression levels of ecdysteroid-regulated proteins were sharply decreased by more than tenfold, which may be attributed to the suppression effect of CHH. The expression of other ecdysteroid-regulated genes also obviously decreased during the InM-IV and PrM stages, for example, ecdysis triggering hormone receptor subtype-B, ecdysone receptors, and ecdysone-induced proteins. The similar expression results for CHH and ecdysteroid-related genes have been observed in other crustaceans just prior to ecdysis [33,59].
The same trend of up-regulated transcript levels was found in the vitellogenin gene (VG), which has been suggested to be an ecdysteroid-responsive gene in the molting process [60]. Farnesoic acid omethyltransferase is another crustacean molting hormone-related category that may play key roles in growth regulation of crustaceans [3,61]. These enzymes were up-regulated from InM-III to PrM in the present study. In P. Chinensis, a stage-speci c expression pro le revealed that the highest expression level of farnesoic acid o-methyltransferase occurred at the intermolt stage, implying that the conversion of farnesoic acid to methyl farnesoate may be involved in the onset of molting processes [61]. Moreover, many other growth and development-related hormone genes were also identi ed as differentially expressed during the molting cycle, including estradiol 17-beta-dehydrogenase 8, estrogen sulfotransferase, hormone receptor, juvenile hormone epoxide hydrolase, and lutropin-choriogonadotropic hormone receptor. Although the functions of these genes that are potentially involved in hormone regulation are still not well characterized, it appears that S. paramamosain produces a rather complex regulation pro le of hormone genes as in other crustacean species. In summary, we infer that hormoneregulated molting signals are likely play distinctly different roles in the S. paramamosain molting cycle, and these will serve as promising candidates for future analyses.
Expression patterns of DEGs associated with immune response and metabolism among molting stages During early postmolt, S. paramamosain still has a soft cuticle and thus may be more vulnerable to bacteria, viruses, or predators [3,35,50]. As with all crustaceans, S. paramamosain does not have an adaptive immune system and instead relies upon an innate immune system to avoid exogenous stresses.
Antimicrobial peptides (AMPs) are important effectors in innate immunity, and these were up-regulated from PoM to InM in S. paramamosain. The AMPs included crustin 1, crustin 4, and ALF 1. Moreover, other transcripts homologous to a number of immune-related genes, including c-type lectin gene families, were identi ed as differentially expressed during the molting cycle. The functions of these DEGs need to be further con rmed as immune and protective tactics that allow S. paramamosain to avoid stress during the molting cycle.
It is well known that crustaceans accumulate nutrients prior to molting to provide enough energy for ecdysis. Various changes in protein, lipid, and carbohydrate content have been detected during the course of the molting cycle in crustaceans [62,63]. In our study, changes in the lipid composition were found to correspond well with the functions of these lipids during the molting cycle. For example, lipid metabolismrelated genes, including lipase 3, alcohol dehydrogenase, and fatty acid-binding protein, were accumulated by the crab before molting, reached a peak during PrM, and were depleted during PoM.
Carbohydrates such as glucose and glycogen being used primarily as a direct source of metabolic energy have been observed in several crustacean species prior to the molt [4]. The expression pro le of carbohydrate metabolism-related transcripts appears to re ect an increase in the energy requirements of S. paramamosain as the molt cycle progresses. Enzymes involved include UDP-glucuronosyltransferase and 6-phosphogluconolactonase. Most of the protein metabolism-related DEGs displayed relatively high expression during InM followed by a gradual decrease across the rest of the molting cycle. These genes included arginine kinase, glutamine synthetase 2, and glutamate dehydrogenase. In summary, the expression pro les of these transcripts indicated that molting induction creates stress that may impact on metabolic function.

Conclusion
To our knowledge, this is the rst systematic transcriptome derived from whole crab bodies, including all the individual organs, during the molting cycle in crustaceans. This transcriptome dataset signi cantly expands the available genomic information for S. paramamosain and provides fundamental support for future research on the molecular mechanisms of the molting cycle in this species. Moreover, the new description of major transcriptional events during different molting stages and the determination of their timing provide temporal markers for future studies of molting progress and regulation in crustaceans. Intermolt stage (C-F) C, setae harden, absence of setal cones; D, fully-spread epidermis; E, a clear margin of epidermal tissue at the base of the setal cones; F, a narrow zone between the setal cones and the epidermis; setal cones arise.