4. Discussion
Litopenaeus vannamei , Penaeus monodon , M. japonicus , and Penaeus chinensis are the four most cultured prawns worldwide. The genomes of L. vannamei , P. monodon , and F. chinensis have been published (Zhang et al., 2019; Uengwetwanit et al., 2021; Wang et al., 2021b); however, so far, no genome of M. japonicus has been reported, despite the economic and ecological significance of this species. M. japonicus is a major research object due to its position as the most important cultured shrimp. Previously, this species was believed to show adaptive plasticity, and this view was supported by the observed expansion of stress response‑related gene families in the present study. Genomic information regarding this species could help to gain a better understanding of its habit of sand diving as well as its environmental adaptation. The present study reports the whole-genome sequence ofM. japonicus , which was assembled using PacBio long‑read data and Hi-C techniques. The genome was assembled based on a 10-Mb benchmark (Reference standard for genome biology, 2018), and is considered to be a high-quality reference genome because of its N50 scaffold length of 38.26 Mb. We believe that it is one of the highest quality crustacean genomes available currently.
Previously, the M. japonicus genome was estimated to be 1.94 Gb (Yuan et al., 2018). In the present study, the genome size was 1.54 Gb, which is smaller than that of two other penaeid shrimps, L. vannam ei (2.60 Gb) (Zhang et al., 2019) and P. monodon (2.59 Gb) (Uengwetwanit et al., 2017), and larger than that of F. chinensis (1.38 Gb) (Wang et al., 2021b). Compared with the genome of L. vannam ei ( Zhang et al., 2019), the M. japonicus genom e contained slightly fewer genes (24,317vs . 25,596). The M. japonicus and L. vannameigenomes contain a similar gene numbers. However, the M. japonicusgenome contains a markedly lower proportion of repetitive sequences compared with that of L. vannamei (56.07% vs . 78%, respectively) (Zhang et al., 2019), which might have contributed to genome contraction in M. japonicus.
M. japonicus is a benthic species that needs sand to survive, while L. vannamei is planktonic. The microenvironment of the sediment is very complex, comprising both water and soil. In addition, benthic bivalves have become adapted to extreme environments comprising enriched pathogens and ions, and a low oxygen content. Therefore, benthic crustaceans are likely to have specific molecular mechanisms endowing them with tolerance to extreme environments. M. japonicus ’ adaption to a lifestyle comprising burial in sediment is likely to involve specific gene families associated with complex signaling systems, ion binding systems, and the immune system.
M. japonicus is commercially valuable because of its consumer-appreciated coloration and its capability of being transported live without water (Cheng & Chen, 2002). An HO-like, gene, whose encoded protein is involved in heme oxygenase (decyclizing) activity and heme oxidation, was identified under positive selection in M. japonicus . Heme is an important cofactor for oxygen transfer, oxygen storage, and oxygen activation (Shimizu et al., 2019; Tsiftsoglou et al., 2006). It exists in the form of hemocyanin in crustacea. The main function of hemocyanin is oxygen transport (Burmester, 2004; Zhang et al., 2020). M. japonicus can tolerate an extremely hypoxic environment; therefore, we speculated that the positive selection on this heme-related gene could benefit the resistance to low oxygen of M. japonicus .
Meanwhile, the HO-like protein might participate in shell color regulation. Shell color plays a significant role in consumer acceptability of crustacean species. True green pigments in animals are mostly porphyrinoids. Endogenous porphyrins resulting from the breakdown of heme are usually known as bile pigments, especially biliverdin (Kikuchi et al., 2005; Martins et al., 2019; Schmid & McDonagh, 1975). In mammals, heme oxygenase (HO) is a universal enzyme that degrades heme to biliverdin-IX alpha (BV-IXalpha), liberating ferrous iron (Fe2+) and carbon monoxide (CO) as by-products (Mahawar & Shekhawat, 2018; Shekhawat & Verma, 2010). Porphyrins and metalloporphyrins show different colors when they coordinate with different metal ions (Bonkovsky et al., 2013). A previous study onF. chinensis reported that porphyrin metabolism participates in body color formation (Wang et al., 2019).
The body of crustaceans is covered by a rigid exoskeleton, the cuticle, which protects the inner organs against outer environmental factors. Immunological detection showed a diffuse distribution of hemocyanin over the cuticle of M. japonicus (Adachi et al., 2005). A red color‑related protein was purified from the shell of M. japonicus , which was identified belonging to the hemocyanin family (Pan et al., 2020). In this study, the change to the structure of the HO-like protein could result in a change in its function. The loss of the kinked helix structure and transmembrane domain possibly make it easier for the protein to dissociate in the hemolymph to exert its effects. In summary, we considered that the positive selection on the HO-like gene is at least partly responsible for colorful appearance of M. japonicus .
The reported reference genome assembly is of high quality, with an improved gene set with increased contiguity and an improved annotation rate. Indeed, 98.64% of the predicted genes could be annotated functionally. This suggested that the genome assembly could better facilitate transcriptomic studies in kuruma shrimp compared with RNA-seq mapping based on de novo transcriptome assembly. We used RNA-seq mapping based on the reference genome to identify genes associated with the shrimp’s cold-resistant performance. Profitable shrimp production relies on the low-temperature tolerance traits of this species (Jiang et al., 2019). M. japonicus is a warm water species that can thrive at temperatures between 18 and 30 °C (optimum = 28 °C), although it can survive at temperatures as low as 6 °C (Ren et al., 2020). However,M. japonicus ’s nursery temperature should not be lower than 16 °C. Thus, the breeding season and geographical distribution of M. japonicus are limited by low temperature, which also affects production and cultivation efficiency. Therefore, the identification or development of a low-temperature tolerant variety of M. japonicus remains necessary. Despite its economic and ecological importance, that lack of a high-quality reference genome has limited our knowledge of genes related to cold tolerance in this species.
In the present study, we explored the effects of acute cold stress on the eyestalk transcriptome of M. japonicus to identify candidate cold tolerance-related and cold change‑related genes. We further studied DEGs associated with the nervous and endocrine systems because of their importance in the regulation a variety of physiological functions and the maintenance of system-wide homeostasis under normal and stressful conditions (Wu et al., 2019; TsuTsui et al., 2020; Ye et al., 2021). A previous study found that increases in biogenic amines enhanced insect cold tolerance (Lubawy et al., 2013). In agreement with that study, in the endocrine system, we observed marked increases in the expression levels of genes encoding Gs-α, PC2, G-protein coupled receptors (GPCRs), and the dopamine receptor 1 (DAR1) after low temperature stress, while the DAR2 gene showed the opposite trend. As an important biogenic amine, dopamine (DA) binds to GPCRs, and depending on the target tissue and receptor type, stimulates different secondary messengers, mostly Ca2+ or cAMP (Farooqui, 2012). The regulation of many behaviors involves the participation of DA, including feeding, locomotion, and evoking systemic responses to different stressful conditions (Aparicio-Simón et al., 2018; Tong et al., 2021). G proteins, important signal transduction factors located on the cell membrane, comprise of three kinds of subunits α, β and γ, which are involved in the regulation of multiple processes (Wei et al., 2020). For example, a GPCR binds to GnRH, which activates intracellular signaling to promotes the synthesis and secretion of follicle stimulating hormone and luteinizing hormone (Ibuchi & Nagayama, 2021). It has been reported that glucose metabolism is modulated by the mature Crustacean hyperglycemic hormone (CHH) protein, produced by PC2-like protein cleavage of the CHH propeptide (Tangprasittipap et al., 2012). These results suggested that DA enhances CHH expression by promoting PC2, resulting in regulation of the hemolymph glucose concentration. Energy mobilization is one of the most important functions of the release of biogenic amines into the insect hemolymph (Lorenz & Gäde, 2009). The mammalian DAR family comprises two classes (D1-like and D2-like), classified according to their intracellular signaling pathways and pharmacological properties. D1-like receptors bind to Gs/Golf class of Gα proteins, resulting in the activation of adenylyl cyclase, which increases the intracellular cAMP level. By contrast, D2-like receptors bind to Gαi/Go proteins, resulting in inhibition of adenylyl cyclase, which decreases intracellular cAMP levels (Chen et al., 2017). Therefore, the comparison of eyestalk transcriptomes identified genes associated with cold-stress, which could lead to better management of shrimp farming, with a consequent reduction in the exploitation of natural populations.
That cold tolerance is a complex regulatory process was demonstrated by the association of DEGs with several pathways. We further studied those DEGs related to metabolism, because they might be a strategy used byM. japonicus to resist low temperature stress. Notably, “Glycine, serine and threonine metabolism”, “Glutathione metabolism”, “Ubiquitin mediated proteolysis”, “Glyoxylate and dicarboxylate metabolism”, “Thiamine metabolism”, “Glycerophospholipid metabolism”, “Alanine, aspartate and glutamate metabolism”, and “Glycolysis/Gluconeogenesis” were enriched among the DEGs of M. japonicus exposed to low temperature for 48 h (Tab. S10). Cold stress upregulated the genes encoding glucose-6-phosphatase and ATP-generating enzymes, which suggested that heat stress induced the rapid production of ATP. This might be related to molecular chaperones requiring high levels of ATP for their functions (Chu et al., 2020). In lipid metabolism, the cold-stress upregulated DEGs were mainly associated with unsaturated fatty acids (UFA) biosynthesis. UFAs are key components of cellular membranes and affect energy metabolism. From the transcriptomic results, genes encoding fatty acid synthesis-related proteins, such as fatty aldehyde dehydrogenase, long chain fatty acid CoA ligase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase, showed high expression after acute cold stress. Fatty aldehyde dehydrogenase, which has a vital function in biomembrane structure and function, catalyzes the conversion of palmitic acid to UFAs by introducing unsaturated bonds. Accumulation of UFAs effectively restores biomembrane fluidity and membrane-related enzyme activity (Los & Murata, 1998). In the de novo synthesis of fatty acids, acetyl-CoA carboxylase is the rate-limiting enzyme, catalyzing the conversion of acetyl-CoA to malonyl-CoA (Wang et al., 2021a). As key enzymes for fatty acid synthesis, long chain fatty acid CoA ligase and stearoyl-CoA desaturase, combined with NADPH, produce palmitic acid, which is required for immune defense, biological growth, and biomembranes (Zuo et al., 2017). The observed higher expression of genes encoding fatty acid metabolism regulators in shrimp is not only important for shrimp biology, but also provides a guide for the construction of feed formulations that promote the endurance of shrimp under low temperatures. Taken together, these data indicated that in M. japonicus low temperature resistance, metabolic response pathways might have vital functions.
In addition, temperature change and temperature stress-related immune-related genes were identified among the DEGs, such as cold upregulated hsp70 , hsp90 , and hemocyanin C chain-like . Hsp70 is conserved gene that responds to various stresses, including pathogen invasion and temperature changes by promoting recovery of the cells from damaged proteins, thereby increasing the probability of survival and life span (Valenzuela-Castillo et al., 2019; Zininga et al., 2018). Hsp90 participates in various cell regulation pathways, and a remarkable proportion of its target proteins are kinases involved in signal transduction, transcriptional, cell cycle regulation, and steroid hormone receptors, as well as mediating the refolding of stress‑denatured proteins (Terasawa et al., 2005). In crustaceans, hemocyanin is a respiratory protein in the hemolymph, which is mainly responsible for oxygen binding and carbon dioxide transport (Cheng et al., 2002; Zheng et al., 2019). Overall, HSPs and hemocyanin might have important functions in low temperature adaptation. Kuruma shrimp show sand diving behavior. The sand substrates at the bottom of the sea contain an abundance of oxygen-consuming organic matter and a low oxygen content in the buried sediment (Wei et al., 2020). Resistance to low temperature is an energy consuming process. With less energy production capacity and more energy consumption, M. japonicus might not be particularly resistant to low temperature.