4. Discussion
Using the PacBio RS II sequencing platform, a 159.1 GB subread base with 73,146,314 subreads and 2,175 average reads were generated. 84,627 unigenes were detected in M. rosenbergii . All unigene sequences were assessed using BUSCO and 93.2% unigenes showed high homology and complete matches, suggesting that our full-length cDNA sequences are a rich resource for further functional genomics analysis in M. rosenbergii .
All unigene sequences were functionally annotated using the NR database. 37.6% of the unigenes were aligned with Hyalella azteca which is a widespread species of amphipod crustacean, followed by decapod crustaceans with lower match rates (4.67% for Procambarus clarkii, and 3.02% for M. rosenbergii ). These results suggest that our full-length transcripts provide additional novelty and complexity to functionally important proteins previously unannotated in the M. rosenbergii transcriptome. Transcripts annotated using the GO database were enriched in categories associated with biological process, cellular components, and molecular function, and subcategories including metabolic processes, biological regulation, catalytic activity, and transporter activity. According to KEGG annotations, genetic information processing was the dominant enriched transcript group, especially in pathways for spliceosomes, ribosomes, protein processing in the endoplasmic reticulum, and RNA transport. According to the gene function interpretation of SMRT transcripts using the KOG database, a large number of genes were functionally involved in neuron cell commitment, neuropeptide signal transduction, and neurotransmitter biosynthesis. This might provide clues for further study of brain- and nerve ganglia-regulation in sexual dimorphism pertaining to growth, development, and morphologies of M. rosenbergii . Neurosecretory cells in the brain produce various neuropeptides with regulatory effects on biological functions, and allatostatin, crustacean female sex hormone, crustacean hyperglycemic hormone, and eclosion hormone were identified from transcriptome of the brain of the Chinese mitten crab (Liu et al. 2019).
Transcription factors are proteins that modulate the transcription of specific genes by binding to DNA which regulates the proliferation, migration and differentiation of neural tissue (Feng et al. 2021; Qian et al. 2022). 6,367 putative transcription factors were identified in M. rosenbergii , and their function requires further investigation. LncRNAs constitute an important layer of regulation in gene expression at either the transcriptional or post-transcriptional levels, during fundamental processes (Necsulea et al. 2014). Transcriptome profiles of LncRNAs in the brain of zebrafish showed significant sex differentiation between male and female individuals (Yuan et al. 2019). A total of 6,287 LncRNAs have been obtained by four analytical methods fromM. rosenbergii , and the number are similar to that seen in the Platypus, but significantly lower than in other mammals (Necsulea et al. 2014). LncRNA evolution and biological function are driven by transposable elements, particularly transposable element inserts at transcription starting sites, governing LncRNA transcription (Kelley & Rinn 2012). It is interesting that the number of transposable elements identified from the transcription of M. rosenbergii is lower than what is seen in vertebrates (Liu et al. 2020).
According to the Illumina sequences from the brain and nerve ganglion ofM. rosenbergii , a large number of DEGs were found and annotated using the GO database. Growth function was a prevalent term in the GO database. In the giant freshwater prawn, the periodic shedding of the exoskeleton is one of the most important physiological processes essential for growth, including molting and muscle development. Molting-related genes, for instance, molt-inhibiting hormone, crustacean hyperglycemic hormone, ecdysteroid receptor, and retinoic acid X receptor (Zhang et al. 2019) were not found in DEGs in the present study. In contrast, growth- associated gene expressions were observed in DEGs. In particular, actin is one of the most abundant intracellular proteins and a preferential binding of actin-beta to myosin which contains actin binding sites, plays a key role in many essential biological processes for cell adhesion, migration, and contractility in muscle (Vedula & Kashina 2018). Increasing myofiber numbers (hyperplasia) is important in body growth of aquatic animals. For example, sea bream Sparus aurata ’s muscle hyperplasia contributes greatly to its adult size, while zebrafishBrachydanio rerio shows little hyperplasia and reaches only a small adult size (Rowlerson et al.1997). In the present study, GO enrichment results from 80 and 66 up-regulated genes involved a common actin binding process in the male brain and nerve ganglion respectively, indicating that the faster growth males compared to females in the giant freshwater prawn is under nervous system control. In the DEGs, the insulin-like androgenic gland hormone binding protein (IAGBP) gene showed up-regulated expressions in male brains. It has demonstrated that the IAGBP, as a binding partner of the insulin-like androgenic gland hormone (IAG), directly increases IAG transcripts, which play an important role in the growth and development of male M. rosenbergii (Yanget al. 2020). Meanwhile, certain growth factors, such as transforming growth factor beta (TGF-beta), and p38-MAPK binding proteins (Wyganowska-Swiatkowska et al. 2015), were found to be up-regulated in the male brain and nerve ganglion. TGF-beta is a central mediator of fibroblast activation, and, in skeletal muscle, TGF-beta signals by binding to tissue-specific combinations of receptor subtypes, triggering the activation of a muscle hypertrophy program, promoting muscle growth (Forouhan et al. 2022). In the brain/nerve ganglion, differentially expressed genes between male and female prawns lay the foundation for the further study of gene expression and manipulation in growth and development in giant freshwater prawn.
KEGG pathway analysis showed that DEGs were significantly enriched in the protein processing in the endoplasmic reticulum pathway, which not only would be a unique protein-synthesizing mechanism, but also is a crucial player in efficient neural communications (Khan 2022). Endoplasmic reticulum (ER)-to-Golgi trafficking is an essential cellular process in the secretory pathway, and selective export of proteins from the ER requires transport signals for efficient recruitment of a transmembrane intracellular lectin ER-Golgi intermediate compartment protein 53 (ERGIC-53) into budding vesicles (Nuferet al. 2003). Overexpression of exogenous ERGIC-53 has been shown to increase protein-secreted production in batch cultivation (Yutaka et al. 2022). ERGIC-53 used as an ER export determinant was only highly expressed in the male nerve ganglion of giant freshwater prawn, suggesting that it facilitates secretion of protein including neuropeptides and hormones transported to the Golgi for delivery to their final destinations. Rainbow trout that are transferred from freshwater to seawater experience growth stunting and down-regulation in growth factor IGF-I transcription has revealed endoplasmic reticulum stress to be a key mechanism underlying this growth stunting phenotype (Morro et al. 2021). The maintenance of intracellular hormones can be controlled by correctly folded models, and misfolded proteins in the endoplasmic reticulum are eliminated by a highly effective protein-degradation process known as ERAD. We found multiple genes involved in terminally misfolded protein elimination in ERAD, and members of two gene families were markedly upregulated in male giant freshwater prawns, Dailin-2 in the brain, and SVIP in the nerve ganglion. Derlin-2 is homologous with Derlin-1 and capable of recognizing the misfolded protein. It physically interacts with p97, SEL1L, and SVIP to form a retrotranslocated complex, thereby, using the Derlin-1 channel, the misfolded protein is transported into the proteasome for degradation via ERAD (Ballar et al. 2007; van de Weijer et al. 2014). Therefore, it is conceivable that male prawns are more likely than females to remove misfolded proteins, since the male grows faster than the female giant freshwater prawn.
To explain the regulation mechanism underlying DEGs in protein processing in the endoplasmic reticulum, a LncRNA and mRNA co-expression network analysis was performed to find differentially expressed LncRNAs regulating transcription of DEGs in the nervous system, such as LncRNAs DLX6-AS1, ZNFX1-AS1, and HULC which are reported to play critical roles in promoting oncogenic phenotypes of cancer cell lines (Ghafouri-Fard et al. 2022; Xian et al. 2018). LncRNAs are highly expressed in the nervous system and control gene expression in the developing and adult brain, mediating neural differentiation (Chen & Zhou 2017). However, these conclusions have some limitations due to the comparative analysis of tissue next- generation sequencing data. Further in vivo studies will help to understand these LncRNAs’ roles in the nervous system of the giant freshwater prawn.