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.