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-specific 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 identified, 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 specific 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 classified 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 identified 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 specific 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 up-regulation 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 fill the new body during a molting cycle [3,38]. Some functional genes involved in the molting cycle of crabs were identified 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 identified 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 identified 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 identified 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, five classes of chitin deacetylase have been characterized, three of which were identified 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 first 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 deacetylase 1 and chitin deacetylase 4 had similar expression patterns to those found in previous studies, reaching the highest expression peaks at stage PoM, decreasing in the four periods of InM, and increasing again in PrM. In addition, we also observed similar transcriptional up-regulation of chitin synthase and chitin-binding protein genes, i.e., reaching the highest expression peaks at stage PoM, decreasing in the four periods of InM, and increasing again in PrM. The functions of these chitin metabolism-related genes in the molting cycle of S. paramamosain need further exploration in the future.
Dozens of cuticle protein-related genes have also been isolated, including calcified cuticle protein, cuticle protein AMP, cuticle protein CUT, and cuticular protein, some of which have extremely high expression levels and similar expression patterns as in chitin genes during the molting cycle. These include cuticle protein CUT2, cuticle protein BD1, cuticle proprotein proCP, and cuticular protein 15. The expression levels of these genes have been shown to be associated with cuticle calcified by the role of inhibition or promotion in the molting process [48-50]. Moreover, gastrolith protein and gastrolith protein 30 showed expression profiles of up-regulation in the PoM stage and then decline during the four InM stages followed by a sharp increase in the PrM stage. Gastrolith protein 10 clearly declined from PoM to PrM. Our results suggest that the molting cycle process is complex, involving up-or down-regulation of these cuticle transcripts, indicating that formation and/or repair of the exoskeleton may need them to operate separately. The new description of DEGs and the determination of their timing in different molting stages provide temporal markers for future studies of molting progress and regulation.
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 identified 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 fill 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 filamentous 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 microfilaments and affects muscle plasticity in crustaceans [53,54]. As the major contractile protein in vertebrates and invertebrates, myosin was identified to be associated with muscle atrophy during molting and is characterized by decreases in fiber width and myofibril cross-sectional areas, an increase in interfibrillar spaces, and degradation [34]. Moreover, hemocyanin is thought to play a role in cuticle formation and ecdysone transport during molting regulation and antigen non-specific immune defense by reversibly binding oxygen [55] and displaying PO, ecdysone binding, and transportation activity [50,56], which is significantly 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 significance of these hormones. In this study, CHH was up-regulated more than fifteenfold 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 o-methyltransferase 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-specific expression profile 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 identified 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 profile of hormone genes as in other crustacean species. In summary, we infer that hormone-regulated 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 identified as differentially expressed during the molting cycle. The functions of these DEGs need to be further confirmed 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 metabolism-related 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 profile of carbohydrate metabolism-related transcripts appears to reflect 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 profiles of these transcripts indicated that molting induction creates stress that may impact on metabolic function.