Transcriptome analysis and histopathology of the mud crab (Scylla paramamosain) after air exposure

Journal Pre-proof Transcriptome analysis and histopathology of the mud crab (Scylla paramamosain) after air exposure

Chang-Hong Cheng, Hong-Ling Ma, Yi-Qin Deng, Juan Feng, Xiao-Long Chen, Zhi-Xun Guo PII:

S1532-0456(19)30368-0

DOI:

https://doi.org/10.1016/j.cbpc.2019.108652

Reference:

CBC 108652

To appear in:

Comparative Biochemistry and Physiology, Part C

Received date:

26 July 2019

Revised date:

29 October 2019

Accepted date:

29 October 2019

Please cite this article as: C.-H. Cheng, H.-L. Ma, Y.-Q. Deng, et al., Transcriptome analysis and histopathology of the mud crab (Scylla paramamosain) after air exposure, Comparative Biochemistry and Physiology, Part C(2019), https://doi.org/10.1016/ j.cbpc.2019.108652

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier.

Journal Pre-proof

Transcriptome analysis and histopathology of the mud crab (Scylla paramamosain) after air exposure Chang-Hong Chenga, Hong-Ling Maa, Yi-Qin Denga, Juan Fenga, Xiao-Long Chena, Zhi-Xun Guo a,b* a

Key Laboratory of South China Sea Fishery Resources Exploitation &

f

Utilization, Ministry of Agriculture, South China Sea Fisheries Research

oo

Institute, Chinese Academy of Fishery Sciences, Guangzhou, Guangdong

Jo u

rn

al

Pr

e-

pr

510300, China, PR China

* Corresponding authors. Tel./fax: +86 2089108331

E-mail addresses: [email protected] (Z.-X. Guo)

1

Journal Pre-proof

Abstract:

The

mud

crab,

Scylla

paramamosain,

is

an

economically-important crab in China. Air exposure is an important environmental stressor during mud crab culture and transportation. Adaptive mechanisms responding to air exposure in mud crabs are still poorly understood. In this study, mud crabs were exposed to air for 120 h.

f

Air exposure decreased total hemocyte counts, led to cytological damage,

oo

and caused high mortality. Transcriptomic analysis was conducted at 0, 6

pr

and 96 h after air exposure. A total of 3,530 differentially expressed genes

e-

(DEGs) were identified. DEGs were mainly involved in the oxidative

Pr

stress response, metabolism, cellular processes, signal transduction, and immune functions. Transcriptomic analysis also revealed that genes of

al

glycolysis and of the tricarboxylic acid cycle were key factors in

rn

regulating the mud crab adaptation to air exposure.

Jo u

Keywords: Scylla paramamosain; air exposure; transcriptomic analysis; oxidative stress

2

Journal Pre-proof

1. Introduction The mud crab (Scylla paramamosain) is widely distributed on the coast in the south of China. In recent years, mud crab cultivation has become very popular in China, with a production that exceeds 140 thousand tons. Mud crab aquaculture suffers from the various physical

f

and chemical challenges, such as air exposure, ammonia and nitrite stress,

oo

high temperature, and pathogen infection (Guo et al., 2013). Air exposure

pr

is one of the most severe stressors for mud crabs during culture and

e-

transportation (Dong et al., 2019). In order to reduce transportation costs,

Pr

mud crabs are transported in a semi-dry environment. Thus, mud crabs may spend several days exposed to air during transport. High mortality

al

may occur after air exposure.

rn

To survive air exposure, mud crabs have various physiological and

Jo u

behavioral adaptations. They can directly breathe air, or rely on the residual moisture on the lid to obtain dissolved oxygen (Lardies et al., 2011; Lu et al., 2016). However, crabs exposed to air for a long time may have alterations of the metabolism and may die (Whiteley et al., 2015). Therefore, understanding of the adaptation mechanisms to air exposure will be beneficial for mud crab culture and transportation. Air exposure can lead to imbalance in water and blood pH (Regnault et al., 2011). Previous studies showed that air exposure caused adverse effects on respiration, metabolism, growth, locomotion, and reproduction 3

Journal Pre-proof

of aquatic animals (Duan et al., 2016; Dong et al., 2019). Air exposure can lead to the formation of reactive oxygen species (ROS), resulting in altered cellular structures and biological functions (Defur et al., 1988). Air exposure can also reduce immunity, thus lowering the resistance to diseases (Lu et al., 2016). However, crustaceans have some physiological

f

adaptations to tolerate air exposure.

oo

Many studies have suggested that the ability of crustaceans to survive

pr

air exposure is related to set of adaptations: reducing activity and heart

e-

rate, decreasing energy costs, activation of the anaerobic metabolism, and

Pr

increasing antioxidant defenses (Romero et al., 2007; De Lima et al., 2015). Some genes related to the cellular response to air exposure have

al

been identified. Duan et al. (2016) reported that the transcript levels of

rn

HSP70 and ferritin gene rapidly changed following desiccation in the

Jo u

black tiger shrimp. Bao et al. (2019) identified immune-related genes in the gill related to the response to air exposure in Chinese mitten crabs using transcriptome analysis; however, in crustaceans the hepatopancreas is a key target organ for environmental stressors (Li et al., 2013; Sun et al., 2016). Elucidation of the molecular mechanisms of adaptation to air exposure in mud crab has been poorly studied. Recently, transcriptome sequencing has become an efficient way to evaluate the responses of aquatic organisms; this technique has provided valuable data on key genes involved in cellular development, cancer, 4

Journal Pre-proof

immune responses, and reproduction (Mortazavi et al., 2008). Using this sequencing technique, many immune-related genes and polymorphic microsatellite markers were identified in crabs, including Scylla paramamosain (Ma et al., 2014; Yang et al., 2018; Zhao et al., 2019), Portunus sanguinolentus (Zhang et al., 2018), and Charybdis feriatus

f

(Zhang et al., 2018). A few studies have used this method to analyze

oo

differences in transcriptional responses in mud crabs exposed to air. The

pr

aim of our study was to describe the physiological responses and

e-

transcriptional profiles of mud crabs under air exposure. This study will

Pr

provide valuable genetic resources for understanding of the molecular mechanisms activated after air exposure in mud crabs.

rn

2.1. Animals

al

2. Materials and methods

Jo u

Mud crabs (70 ± 3.5 g) were obtained from a mud crab farm in Taishan (Guangdong, China). Mud crabs were maintained in water tanks at 10% salinity. Oyster meat was fed to them twice a day, at a rate of 4%–6% of wet body weight before the experiments. During the experimental period, water temperature was 25 °C, pH was 7.8, dissolved oxygen was above 6.0 mg L−1, and ammonia nitrogen was lower than 0.05 mg L−1. 2.2. Air exposure Three hundred mud crabs were divided into two groups: a control 5

Journal Pre-proof

group and an air-exposed group. In the control group, mud crabs were maintained in filtered aerated seawater as described above. In the air exposure group, mud crabs were placed individually in tanks without seawater at 25 °C. There were three replicates for each group. After 0, 3, 6, 12, 24, 48, 72, 96, and 120 h of air exposure, mortality was recorded.

f

Six mud crabs were randomly sampled from each group. The hemolymph

oo

of individual mud crab was withdrawn from the base of the third

pr

pereiopod using a 1 ml sterile syringe (25 gauge). Hemolymph samples

e-

were collected total hemocyte counts. Hepatopancreas samples were

2.3. Experimental settings

Pr

removed for histopathological examination.

al

According to the results obtained from the air exposure experiment, the

rn

first death occurred at 24 h. Total hemocyte counts significantly

Jo u

decreased at 12 h. Thus, 6 h was selected as the earliest stage of air exposure time. After air exposure for 96 h, crabs showed serious symptoms, such as low activity, reduced breathing and high mortality (47%). Thus, 96 h was selected as the latest stage of air exposure time. After 0, 6, and 96 h of air exposure, nine mud crabs from each group were randomly selected. Hepatopancreas samples were removed, immediately frozen in liquid nitrogen, and stored at -80 °C until they were needed for RNA extraction. 2.4. Total hemocyte count 6

Journal Pre-proof

Hemolymph samples were placed on a hemocytometer to count cells with a light microscope (Olympus). 2.5. Histopathologic analysis Hepatopancreas

samples

were

collected

for

histopathologic

examination. Tissues were fixed in formalin (10% formaldehyde), cleared

f

in xylene, and embedded in paraffin. Sections of 5 μm were cut and

oo

stained with hematoxylin and eosin. Stained samples were observed

pr

under a light microscope.

e-

2.6. Total RNA extraction, cDNA library construction, and sequencing Total RNA was isolated from the hepatopancreas using TRIzol reagent

Pr

(Invitrogen, USA) according to the manufacturer’s instructions. The

al

quantity and integrity of RNA were assessed using an Agilent 2100

rn

bioanalyzer. RNA integrity numbers were over 8.5. RNA from three

Jo u

individuals (one per biological replicate) of each group was pooled for constructing a sequencing library. In total, nine sequencing libraries were constructed. The mRNA was purified and fragmented using the TruSeq RNA Sample Prep Kit (Illumina, USA) following the manufacturer’s instructions. The cDNA libraries were prepared with SuperScript II reverse transcriptase kit (Invitrogen, USA) and amplified by PCR using a TruSeq PE Cluster kit (Illumina, USA). The final quality of the fragments was checked in an Agilent 2100 bioanalyzer. The transcriptome sequencing was performed by Hengchuan Company (Shengzhen, China). 7

Journal Pre-proof

The sequenced raw reads were submitted to NCBI with the following accession

numbers:

SAMN12748591,

SAMN12748592,

SAMN12748593,SAMN12748594, SAMN12748595, SAMN12748596, SAMN12748597, SAMN12748598, and SAMN12748599. 2.7. Raw data cleaning, de novo assembly and gene annotation

f

Before de novo assembly, adaptors and low-quality sequences were

oo

removed using Cutadapt version 1.2.1 and FastQC. De novo assembly of

pr

the clean reads was conducted using Trinity software. All unigenes were

e-

subjected to BLASTx similarity search with a cut-off E-value of 1e-5

Pr

based on different databases, including non-redundant protein sequence (Nr), non-redundant nucleotide (Nt), Swiss-Prot, Clusters of Orthologous

al

Groups (COG), Gene Ontology database (GO), and Kyoto Encyclopedia

rn

of Genes and Genomes (KEGG).

Jo u

2.8. Differential gene expression analysis Relative expression levels of the unigenes were assessed using fragments per kilobase of exon per million fragments. Statistical comparison between different groups was conducted using DEGseq2. Unigenes with changes in gene expression over 2-fold and p-values < 0.05 were considered differentially expressed genes (DEGs). For pathway enrichment analysis, all DEGs were annotated using GO and KEGG databases. 2.9. Real-time quantitative PCR validation 8

Journal Pre-proof

To examine the reliability of the RNA-Seq results, genes related to oxidative stress and immune responses were selected for validation by qRT-PCR. Primers were designed using the Primer Premier 5. The mud crab 18S ribosomal RNA gene was selected as the internal control. Total RNA extraction from hepatopancreas in control and air exposure groups

f

was performed. Reverse transcription of the RNA was performed using

oo

the PrimeScript RT reagent Kit With gDNA Eraser (Takara, Dalian, China)

pr

following the manufacturer’s instructions. The qRT-PCR was amplified

e-

in a Bio-Rad RealTime PCR system (Bio-Rad, US) using SYBR Green. The reaction mixtures were 20 μL, containing 2 μL diluted cDNA sample

Pr

(50 ng/μL), 10 μL 2×SYBR Premix Ex Taq, 0.4 μL of each primer

al

(10μM), and 7.2 μL dH2O. The qRT-PCR conditions were as follows:

rn

94 °C for 10 min, then 45 cycles at 95 °C for 30 s, 60 °C for 30 s, and

Jo u

72 °C for 30 s, followed by 10 min at 72 °C. The standard equation and correlation coefficient were determined after constructing a standard curve of serial cDNA dilutions. The cDNA from six mud crabs in each treatment was analyzed by qRT-PCR. Each sample was amplified by triplicate. Relative gene expression levels were evaluated using the 2−ΔΔCT method (Livak and Schmittgen, 2001). Then, the data were analyzed by Wilcoxon test using the SPSS 18.0 software (SPSS, Chicago, IL, USA). A p value<0.05 was considered statistically significant. 3. Results 9

Journal Pre-proof

3.1. Effects of air exposure on the survival of mud crabs There were no deaths in the control group. Mud crabs in the air exposure group began to die at 24 h (Fig.1). After air exposure for 96 h, crabs showed serious symptoms, such as low activity, reduced breathing and high mortality (47%).

f

3.2. Effects of air exposure on total hemocyte counts

oo

Total hemocyte counts did not change at 3 h and 6 h after air exposure.

pr

However, total hemocyte counts significantly decreased at 12, 24, 48, 72,

e-

96, and 120 h after air exposure (Fig.2).

Pr

3.3. Histological changes

Compared with the control group, the hepatopancreas of mud crabs in

al

the air exposure group showed damage symptoms, such as increased

rn

secretion, fuzzy cell outline, and cell lysis (Fig.3).

Jo u

3.4. Sequencing and assembly The hepatopancreas of the control group (0 h) and the air exposure group (6 and 96 h) were used for transcriptome analysis. A total of nine libraries were constructed (Table 1). The Q30 was higher than 92.71%. The GC content ranged from 52.25%–54.10%. De novo assembly generated 138,333 contigs with a mean length of 593 bp (Table S1). Lengths of N50 and N90 were 841 bp and 253 bp, respectively. Among these unigenes, 56,744 contigs (41.01%) were between 200–300 bp, 38,388 contigs (27.75%) were within 300–500 bp, and 43,201 contigs 10

Journal Pre-proof

(31.24%) were longer than 500 bp (Fig.S1). In order to obtain the translation frame and the conserved protein domains of distinct unigenes, all unigenes were subjected to BLASTx search against six public databases. A total of 56,878 genes were annotated in COG, GO, KEGG, Nr, Nt, and Swiss-Prot databases. The numbers of unigenes were 19,092

f

in COG (33.56%), 11,489 in GO (20.20%), 35,441 in KEGG (62.31%),

oo

45,730 in Nr (80.40%), 30,283 in Nt (53.23%), and 34,335 (60.36 %) in

pr

Swiss-Prot (61.04%) (Fig.S2).

e-

3.5. Identification and analysis of differentially expressed genes DEGs were analyzed according to log2 ratios≥1 and false discovery

Pr

rates ≤ 0.01. At 6 h after air exposure, 158 genes were differentially

al

expressed (63 up-regulated and 95 down-regulated), and 3,413 genes

rn

were differentially expressed at 96 h (1953 up-regulated and 2,186

Jo u

down-regulated) (Fig.S3a). Many DEGs had a similar expression tendency at 6 h and 96 h after air exposure (Fig.S3b). Some genes involved in response to environmental stress were identified through the analysis of DEGs (Table 2). 3.6. GO enrichment and pathway analysis GO annotation was performed on DEGs using GOseq. After air exposure for 6 h, 23 GO annotations were assigned to DEGs, representing three main GO categories molecular function (13), cellular component (5), and biological process (5). After air exposure at 96 h, 23 GO annotations 11

Journal Pre-proof

were assigned to DEGs; the three main GO categories were molecular function (93), cellular component (74), and biological process (28). Most DEGs were enriched in environmental information processing and metabolism including the categories “signal transtiation”, global and overview maps, and energy generation. Some other DEGs categories

f

were related to oxidative stress response, heat shock proteins, immune

oo

alteration, and apoptosis.

pr

To understand the pathways affected by air exposure, DEGs were

e-

analyzed against the KEGG pathway. At 6 h and 96 h after air exposure

Pr

124 and 290 pathways were enriched, respectively. The top 30 KEGG pathways are shown in Fig.4. Pathways were allocated into six categories

al

according to their biological function: environmental information

rn

processing, human diseases, organismal systems, cellular processes,

Jo u

metabolism, and genetic information processing (Fig.S4). The maximum number of DEGs corresponded to metabolic pathways, (e.g., amino acid metabolism, energy metabolism, carbohydrate metabolism, and lipid metabolism) (Fig.5). 3.7. Validation of DEGs from RNA-Seq To validate the DEGs identified by Illumina sequencing, some DEGs were selected for RT-qPCR analysis. The results showed that the expression patterns of the selected DEGs obtained by RT-qPCR analysis were consistent with the sequencing results, indicating that results were 12

Journal Pre-proof

reliable (Fig.S5). 4. Discussion Air exposure is one of the most important ambient stressors influencing the survival rate of crustaceans during cultivation and transportation process. During air exposure, crustaceans suffer oxygen deficiency, high

f

temperatures, irradiation, and sudden changes of pH, which could cause

oo

serious tissue damage. Some crustacean species develop several

pr

mechanisms to survive and recover under air exposure. However, the

e-

ability of desiccation tolerance is different among crustacean species. Bao

Pr

et al. (2019) reported that the mortality rate of Chinese mitten crabs was 50 % after air exposure 26.5 h. Dong et al. (2019) suggested that all

al

swimming crabs died after air exposure 24 h at 25°C. In the present study,

rn

the mortality of mud crab occurred as early as 96 h post the air exposure,

exposure.

Jo u

suggesting that mud crab have the poor ability to survive in the air

Haemocytes play an important role in the physiology and immunity of crustaceans. Total haemocyte counts are considered to be a good indicator for immune function. It could be easily affected by environmental stressors, including sulfide, copper, ammonia and salinity extremes. Ridgway et al. (2006) reported that a significant reduction in the total haemocyte counts was observe in Norway lobsters after air exposure. In our study, total hemocyte counts began to decrease at 12 h after air 13

Journal Pre-proof

exposure. The decrease of the total haemocyte counts may lead to impaired immunity(Cheng, et al., 2017). In this study, after air exposure, a total of 138,333 unigenes were assembled from the hepatopancreas of mud crabs. The number of unigenes generated in this study was much higher than that by Liu et al.

f

(2017) and Cheng et al. (2019). Compared with three databases of mud

oo

crabs, the mean length was higher than the length reported by Yang et al.

pr

(2018) and Cheng et al. (2019), and lower than that reported by Lin et al.

e-

(2019). In our study N50 was significantly higher than in previously

Pr

assembled transcriptomes (Ma et al., 2014; Liu et al., 2018; Yang et al., 2018). After air exposure, a total of 3,413 genes were identified as DEGs;

al

they were annotated in categories and pathways related to metabolism,

rn

detoxification, and immune response.

Jo u

Many studies suggested that exposure to air could cause to an increase of endogenous ROS (Ridgway et al., 2006; Duan et al., 2016). When the production of ROS is beyond the organism’s capacity to deal with these reactive species, there is an oxidative stress. Oxidative stress could lead to DNA strand breaks, protein oxidation, and metabolite peroxidation, finally resulteding in significant damage to cell structure (Wang et al., 2009). Ridgway et al. (2016) reported that air exposure led to a progressive degradation of the structure of the hepatopancreas tissue in Norway lobsters. Duan et al. (2016) reported that in black tiger shrimp 14

Journal Pre-proof

air exposure caused the inflation of the liver tubules, scattered cell nucleus in the gap of tubules, and disappearance of connective tissue. Our results also showed that air exposure damaged tissue structure. These results suggested that air exposure could lead to cell injury, thereby disrupting the normal functioning of cells. On the other hand,

f

organismstissues. Our results also showed that air exposure damaged

oo

tissues. These results suggested that air exposure could lead to cell injury,

pr

thereby disrupting the normal functioning of cells. Organisms have

e-

developed the antioxidant mechanisms to prevent the oxidative damages.

Pr

According to our DEGs analysis, some genes related to antioxidant enzymes, such as glutathione peroxidase (Gpx), thioredoxin and

al

thioredoxin reductase were induced after air exposure. GPx3 is a member

rn

of the antioxidant enzyme family that protects effectively the organism

Jo u

cells from the oxidative damage (Ursin et al., 2015). Thioredoxin can scavenge ROS, and reactivate damaged proteins by oxidative stress (Pacitti et al., 2014). Thioredoxin reductase belongs to the the pyridine nucleotide-disulfide oxidoreductase family, which includes several enzymes involved in cellular oxidation and reduction (Mustacich and Powis, 2000). Thus, up-regulation of these genes after air exposure may relieve the oxidative stress damage induced by air exposure in mud crabs. HSPs are a group of molecular chaperones that play a critical role in protein folding, intracellular transport, and protein (Pelham, 1986; 15

Journal Pre-proof

Geething and Sambrook, 1992). HSPs are induced by stressors, such as heat or cold shock, toxins, bacterial infection and a variety of other stresses. Up-regulated HSPs are considered as key indicator of protein damage due to stressors. Increased production of HSPs in organism could enhance cell resistance to environmental stress (Feder and Hofmann,

f

1999). In our study, HSP90 and HSP70 were induced after air exposure,

oo

suggesting that HSPs played an important role in protecting organisms

pr

against air exposure.

e-

The immune system is generally sensitive to environmental stress.

Pr

Compared with vertebrates, mud crabs lack acquired immunity; they must rely on innate immunity to deal with environmental stresses and pathogen

al

invasion. In our study, lysozyme was induced after air exposure.

rn

Lysozyme is an important immune protein involved in innate immunity,

Jo u

and possesses high antimicrobial activities (Hinds Vaughan and Smith, 2013). Thus, lysozyme might play an important role in the response to air exposure. In contrast, some immune-related genes (C-type lectin and toll-like receptor) were down-regulated after air exposure. C-type lectins are pattern recognition receptors that play important roles in non-self-recognition and pathogen elimination (Wang et al., 2011). Inhibiting the expression level of C-type lectins could reduce the phagocytosis activity. Toll-like receptors are a large family of pattern recognition receptors that that recognize pathogens (Wang et al., 2018). In 16

Journal Pre-proof

our study, C-type lectin and toll-like receptor significantly decreased after air exposure, suggesting air exposure might impair immunity by inhibiting the expression of immune-related genes. Accumulation of ROS can modify DNA bases, and lead to DNA damage. DNA damage is usually accompanied by DNA repair.

f

According to our DEGs analysis, the expression of DNA repair-related

oo

genes (ATR and Chk1) were changed after air exposure. The ataxia

pr

telangiectasia-mutated and Rad3-related (ATR) plays a central role in the

e-

repair of replication-associated DNA damage. After DNA damage or

Pr

oxidative stress, ATR can be activated through phosphorylation (Falck et al., 2005). ATR phosphorylate checkpoint kinase 1 (Chk1), resulting in

al

cell-cycle check point activation and maintaining genomic stability

rn

(Bucher and Britten, 2008). Our result indicated that ATR and Chk1 may

Jo u

be involved in the process of air exposure damage-induced repair. Irreversible cell damage in cells can lead to apoptosis. We identified some DEGs involved in apoptosis. Inhibitor of apoptosis-proteins(IAP) maintain a balance between cell proliferation and cell death (You et al., 1997). In this study, IAPs were up-regulated after air exposure for 96 h. Activated IAPs could inhibit caspase activity, acting as negative regulators of apoptosis (Eckelmane et al., 2006). Caspase-3 is the major executioner of caspases, playing a vital role in the cascade apoptosis reaction (Elmore, 2007). After caspase-3 is stimulated, it activates the 17

Journal Pre-proof

downstream components, resulting in apoptosis (Fan et al., 2005). This study showed that caspase-3 was significantly decreased at 96 h after air exposure, suggesting that long time air exposure induced too much ROS production which causing damage to the apoptosis. The finding is consistent with our histopathologic results.

f

In crustaceans, metabolism is one of the major functions of the

oo

hepatopancreas (Sun et al., 2016). In response to environmental changes,

pr

aquatic animals adjust their metabolic process to adapt to the new energy

e-

requirements. In this study, many genes and pathways involved in

Pr

glycolysis, tricarboxylic acid (TCA) cycle, lipid metabolism, and amino metabolism were altered after air exposure (Fig.5). In both vertebrates

al

and invertebrates, glycolysis is the main pathway of energy generation.

rn

Previous studies suggested that hypoxia stress was associated with the

Jo u

activation of glycolysis (Sun et al., 2018; Lia et al., 2018). In this study, the key enzymes of the glycolysis, such as hexokinase, aldolase, triose phosphate isomerase, and pyruvate kinase were significantly up-regulated after air exposure, suggesting that glycolysis plays an important role in energy supply when mud crab are coping with air exposure. LDH catalyzes the transformation of pyruvate into lactate. The present study showed that air exposure induced the expression of LDH, which is consistent with the findings of another study on Oriental river prawn (Sun et al., 2018). The TCA cycle is a central pathway for ATP production and 18

Journal Pre-proof

for providing precursors for many biosynthetic pathways. We observed that many genes involved in TCA cycle, were down-regulated after air exposure, which indicates a negative of air exposure impact on the TCA pathway. 5. Conclusions

f

In this study, we demonstrated the molecular mechanism of mud crab

oo

adaptation to air exposure (Fig.6). Our results indicated that long time air

pr

exposure decreased total haemocyte count, and led to cell death.

e-

Transcriptome analysis revealed that mud crab adapted to to air exposure

detoxification-related

and

Pr

by increasing expression levels of genes related to metabolism-associated, anti-oxidative

defense-related

genes.

rn

and disrupt metabolism.

al

Additionally, long time air exposure might impair the immune functions,

Jo u

Acknowledgements

This research was supported by Science and Technology Program Project of Guangzhou (201904010327), China Agricultural Research System (CARS-48), Basic and applied basic research fund of Guangdong Province (2019A1515011548), Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture (GDKLHSA0805), Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology (2019011007).

19

Journal Pre-proof

References Bao, J., Xing, Y.N., Jiang, H.B., Li, X.D., 2019. Identification of immune-related genes in gills of Chinese mitten crabs (Eriocheir sinensis) during adaptation to air exposure stress. Fish Shellfish Immunol. 84,885-893. Bucher, N., Britten C.D., 2008. G2 checkpoint abrogation and checkpoint kinase-1 targeting in the treatment of cancer. Br. J. Cancer, 98,523-528. Cheng, C.H., Ye, C.X., Guo, Z.X., Wang, A.L., 2017. Immune and physiological

f

responses of pufferfish (Takifugu obscurus) under cold stress. Fish Shellfish

oo

Immunol. 64:137-145.

De Lima, T.M., Geihs, M.A., Ner, L.E., Maciel, F.E., 2015. Air exposure behavior of

pr

the semiterrestrial crab Neohelice granulata allows tolerance to severe hypoxia but

e-

not prevent oxidative damage due to hypoxia-reoxygenation cycle. Physiol Behav, 151, 97-101.

Pr

Defur, P.L., Pease, A., Siebelink, A., Elfers, S., 1988. Respiratory responses of blue crabs, Callinectes sapidus, to emersion, Comp. Biochem. Physiol. A, 89A, 97-101.

al

Dong, Z.G., Mao, S., Chen, Y.H., Ge, H.G., Li, X.Y., Wu, X.G., Liu, D.W., Zhang, K.,

rn

Bai, CW., Zhang, Q.Q., 2019. Effects of air-exposure stress on the survival rate and

429-434.

Jo u

physiology of the swimming crab Portunus trituberculatus. Aquaculture, 500 (20

Duan, Y.F., Zhang, J.S., Dong H.B., Wang Y., Liu Q.S., Li, H., 2016. Effect of desiccation and resubmersion on the oxidative stress response of the kuruma shrimp Marsupenaeus japonicus. Fish Shellfish Immunol. 49, 91-99. Duan, Y.F., Zhang, Y., Dong H.B., Zhang, J.S., 2016. Effect of desiccation on oxidative stress and antioxidant response of the black tiger shrimp Penaeus monodon. Fish Shellfish Immunol. 58, 10-17. Eckelman B.P., Salvesen G.S., Scott F.L., 2006. Human inhibitor of apoptosis proteins: why XIAP is the black sheep of the family. EMBO 7,988-994 Elmore, S., 2007. Apoptosis: a review of programmed cell death. Toxicol. Pathol. 35,495-516. 20

Journal Pre-proof Falck, J. Coates, J. Jackson S.P., 2005. Conserved modes of recruitment of ATM, ATR and DNA–PKcs to sites of DNA damage. Nature, 434,608-611. Fan, T.J., Han, L.H., Cong, R.S., Liang J., 2005. Caspase family proteases and apoptosis Acta. Biochim. Biophys. Sin.37, 719-727. Feder, M.E., Hofmann, G.E., 1999. Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annu. Rev. Physiol. 61,243-282. Geething, M.J., Sambrook, J., 1992. Protein folding in the cell. Nature 355,33-45.

oo

f

Guo, Z.X., He, J.G., Xu, H.D., Weng, S.P., 2013. Pathogenicity and complete genome sequence analysis of the mud crab dicistrovirus-1.Virus Research, 171,8-14.

pr

Hinds Vaughan, N., Smith, S.L., 2013. Isolation and characterization of a c-type

e-

lysozyme from the nurse shark. Fish Shellfish Immunol. 35, 1824-1828. Lardies, M.A., Muñoz, J.L., Paschke, K., Bozinovic, F., 2011. Latitudinal variation in

Pr

the aerial⁄aquatic ratio of oxygen consumption of a supratidal high rocky-shore crab. Mar. Ecol. 32, 42-51.

al

Li, X., Cui, Z., Liu, Y., Song, C., Shi, G., 2013. Transcriptome analysis and discovery

rn

of genes involved in immune pathways from hepatopancreas of microbial challenged mitten crab Eriocheir sinensis. PLoS One 8, e68233.

Jo u

Lia, M.G., Wang, X.D., Qi, C.L., Li, E., Du, Z.Y., Qin, J.G., Chen, L.Q., 2018. Metabolic response of Nile tilapia (Oreochromis niloticus) to acute and chronic hypoxia stress. Aquaculture, 495,187-195. Liu, S., Chen, G., Xu, H., Zou, W., Yan, W., Wang, Q., Deng, H., Zhang, H., Yu, G., He, J., Weng, S., 2017. Transcriptome analysis of mud crab (Scylla paramamosain) gills

in

response

to

Mud

crab

reovirus

(MCRV).Fish

Shellfish

Immunol.60,545-553. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real time quantitative PCR and the 2−ΔΔCT method. Methods 25,402-408. Lu, Y.l., Zhang, D., Wang, F., Dong, S.G., 2016 Morbidity and mortality in Norway lobsters, Nephrops norvegicus: physiological, immunological and pathological effects of aerial exposure. J Therm Biol., 60, 33-40. 21

Journal Pre-proof Ma, H.Y., Ma, C.Y., Li S.J., Jiang W, Li X.C., Liu,Y.X., Ma, L.B., 2014. Transcriptome Analysis of the Mud Crab (Scylla paramamosain) by 454 Deep Sequencing: Assembly, Annotation, and Marker Discovery. PLoS ONE 9(7): e102668. Mortazavi, A., Williams, B.A., Mccue, K. Schaeffer, L. Wold, B., 2008. Mapping and quantifying mammalian transcriptomes by RNA-Seq, Nat. Methods 5, 621-628. Mustacich, D., Powis, G., 2000. Thioredoxin reductase. Biochem. J. 346, 1-8. Pacitti, D., Wang, T., Martin, S.A., Sweetman, J., Secombes, C.J., 2014. Insights into

oo

f

the fish thioredoxin system: expression profile of thioredoxin and thioredoxin reductase in rainbow trout (Oncorhynchus mykiss) during infection and in vitro

pr

stimulation. Dev Comp Immunol.42, 261-77.

e-

Pelham, H.R.B., 1986. Speculations on the functions of the major heat shock and glucoseregulated proteins. Cell 46,959-961.

Pr

Regnault, M., 2011. Effect of air exposure on nitrogen metabolism in the crab Cancer false king crab Paralomis granulosa, during air exposure and subsequent

al

re-submersion. Aquaculture, 319,205-210.

rn

Ridgway, I.D., Taylor, A.C., Atkinson, R.J.A., Stentiford, G.D., Chang, E.S., Chang, S.A., Neil, D.M., 2006. Morbidity and mortality in Norway lobsters, Nephrops

Jo u

norvegicus: physiological, immunological and pathological effects of aerial exposure. Journal of Experimental Marine Biology and Ecology 328, 251-264. Romero, C.M., Ansaldo, M., Lovrich, G.A., 2007. Effect of aerial exposure on the antioxidant status in the subantarctic stone crab Paralomis granulosa (Decapoda: Anomura). Comp. Biochem. Physiol. C 146, 54-59. Sun, M., Li, Y.T., Liu, Y., Lee, S.C., Wang, L., 2016. Transcriptome assembly and expression profiling of molecular responses to cadmium toxicity in hepatopancreas of the freshwater crab Sinopotamon henanense. Scientific Reports. 6, 19405. Sun. S., Guo. Z., Fu. H., Zhu. J., Ge. X., 2018. Integrated metabolomic and transcriptomic analysis of brain energy metabolism in the male Oriental river prawn (Macrobrachium nipponense) in response to hypoxia and reoxygenation. Environ Pollut. 243, 1154-1165 22

Journal Pre-proof Ursini, F., Maiorino, M., Brigelius-Flohe, R., Aumann, K.D., Roveri, A., Schomburg, D., Flohe, L., 1995. Diversity of glutathione peroxidases. Methods Enzymol. 252, 38-53 Vidya, M.K., Kumar, V.G., Sejian, V., Bagath, M., Krishnan, G., Bhatta, R., 2018. Toll-like receptors: significance, ligands, signaling pathways, and functions in mammals. Int. Rev. Immunol., 37, 20-36. Wang, L., Huang, M., Zhang, H., Song, L., 2011. The immune role of C-type lectins in molluscs. ISJ 8,241-246.

oo

f

Wang, W.N., Zhou, J., Wang, P., Tian, T.T., Zheng, Y., Liu, Y., 2009. Oxidative stress, DNA damage and antioxidant enzyme gene expression in the Pacific white shrimp,

pr

Litopenaeus vannamei when exposed to acute pH stress. Comp. Biochem. Physiol.

e-

C 150 (2009) 428-435.

Whiteley, N.M., Taylor, W., 2015. Responses to environmental stresses: oxygen,

Pr

temperature, and pH. In: Chang, E.S., Thiel, M. (Eds.), the Natural History of Crustacea. Physiology Vol. 4. Oxford University Press, New York, pp. 249-285.

al

Yang, X., Ikhwanuddin, M., Li, X., Lin, F., Wu, Q., Zhang, Y., You, C., Liu, W.,

rn

Cheng, Y., Shi, X., Wang, S., Ma, H., 2018. Comparative Transcriptome Analysis Provides Insights into Differentially Expressed Genes and Long Non-Coding RNAs

Jo u

between Ovary and Testis of the Mud Crab (Scylla paramamosain). Mar Biotechnol. 20(1),20-34.

You, M., Ku, P.T., Hrdlickova, R., Bose, H.R., 1997. ch-IAP1, a member of the inhibitor-of-apoptosis protein family, is a mediator of the antiapoptotic activity of the v-Rel oncoprotein. Mol.Cell Biologicals, 17, 7328-7341. Zhao, M., Wang, W., Chen, W., Ma, C., Zhang, F., Jiang, K., Liu, J., Diao, L., Qian, H., Zhao, J., Wang, T., Ma, L., 2019. Genome survey, high-resolution genetic linkage map construction, growth-related quantitative trait

locus (QTL)

identification and gene location in Scylla paramamosain. Sci Rep.9(1),2910. Zhang, Y., Miao, G., Wu, Q., Lin, F., You, C., Wang, S., Aweya, J.J., Ma,H., 2018. Transcriptome sequencing and molecular markers discovery in the gonads of Portunus sanguinolentus. Sci Data.5,180131. 23

Journal Pre-proof Zhang, Y., Miao, G., Fazhan, H., Waiho, K., Zheng, H., Li, S., Ikhwanuddin, M., Ma, H., 2018. Transcriptome-seq provides insights into sex-preference pattern of gene expression between testis and ovary of the crucifix crab ( Charybdis feriatus). Physiol Genomics.50(5),393-405.

Clean reads

0h_1

53,692,060

44,181,188

0h_2

50,215,216

0h_3

oo

Raw reads

GC (%)

Q20 (%)

Q30 (%)

53.68

97.49

94.00

34,960,938

54.10

96.69

92.71

51,915,364

44,630,640

52.54

96.98

93.28

6h_1

58,446,706

44,927,108

53.86

97.38

93.87

6h_2

55,069,216

43,420,026

52.25

96.63

92.69

6h_3

47,128,602

41,838,104

52.58

97.31

93.72

48,005,680

39,518,402

53.30

97.32

93.75

96 h_2

Jo u

rn

al

Pr

e-

pr

Sample

f

Table 1 Sequencing data output statistics.

53,258,462

45,476,250

52.84

97.49

93.99

96 h_1

47,901,828

38,863,696

53.81

97.59

94.17

96 h_1

Table 2 List of the DEGs involved in oxidative stress, immune responses, and metabolism after air exposure. “↑” represents the up-regulated genes. “↓” represents the down-regulated genes. “N/A”represents no change. Mechanism

Gene Title

6 h up/down

24

96 h up/down

Journal Pre-proof Apoptosis

DNA repair

Caspase-8

N/A

-2.1↓

Caspase-3

N/A

-2.60↓

Inhibitor of apoptosis protein (IAP)

N/A

1.9↑

Checkpoint kinase 1(CHK1)

N/A

2.7↑

N/A

1.5↑

HSP 90

2.1↑

2.6↑

HSP70

1.6↑

4.9↑

Cytochrome P450

3.6↑

2.8↑

Ataxia

telangiectasia-mutated

and

N/A

1.34↑

1.7↑

1.81↑

2.5↑

1.8↑

C-type lectin

-3.3

-3.54

Toll-like receptor

N/A

-1.45↓

Lysozyme

1.5↑

1.9↑

N/A

1.81↑

Aldolase

N/A

1.5↑

Triose phosphate isomerase

N/A

4.25↑

NADPH oxidase

N/A

2.48↑

Pyruvate kinase

N/A

1.6 ↑

L-lactate dehydrogenase (LDH)

N/A

1.4↑

Pyruvate dehydrogenase

1.7↑

1.8↑

Isocitrate dehydrogenase

N/A

-1.2↓

pr

Thioredoxin reductase (TrxR)

oo

Oxidative stress response

rn

Heat shock proteins

f

Rad3-related(ATR)

e-

Thioredoxin (Trx)

al

Immune response

Pr

Glutathione peroxidase 3(GPx 3)

Hexokinase

Jo u

Metabolism

25

Journal Pre-proof Glutamine synthetase

2.3↑

3.82↑

Succinate thiokinase

N/A

-2.1↓

Malate dehydrogenase

N/A

-1.9↓

Fig.1 Cumulative survival of mud crab at different time intervals after air

Jo u

rn

al

Pr

e-

pr

oo

f

exposure. Vertical bars represented the mean ± SD (N =3).

26

Journal Pre-proof

Fig.2 Total haemocyte count of mud crab at different time intervals after air exposure. Data are presented as the mean ±SD (n = 6). Data at the same time with asterisk are significantly different (P < 0.05) among

rn

al

Pr

e-

pr

oo

f

treatments.

Jo u

Fig.3 Hepatopancreas tissue of mud crab stained with HE dye after air exposure. (a) 0 h; (b) 72 h; (c) 96 h; (d) 120 h.Increased secretion(IS); Fuzzy cell outline (FO); Lysis of cell(LC).

27

Jo u

rn

al

Pr

e-

pr

oo

f

Journal Pre-proof

28

Journal Pre-proof

Fig.4 Up- and down-regulated unigenes of the top 30 KEGG pathways after air exposure in the hepatopancreas. (a) 6 h air exposure group vs

Jo u

rn

al

Pr

e-

pr

oo

f

control; (b) 96 h air exposure group vs control.

29

Journal Pre-proof

Jo u

rn

al

Pr

e-

pr

oo

f

Fig.5 The changes of metabolic pathways after air exposure.

30

Journal Pre-proof

Jo u

rn

al

Pr

e-

pr

oo

f

Fig.6 The molecular mechanism of mud crab adaptation to air exposure.

31

Journal Pre-proof

Jo u

rn

al

Pr

e-

pr

oo

f

Conflict of interest The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

32

Journal Pre-proof Highlights

 Air exposure induced oxidative stress, and led to cytological damage.  Some genes involved in response to air exposure were found.  Glycolysis and tricarboxylic acid cycle related pathways associated

Jo u

rn

al

Pr

e-

pr

oo

f

with air exposure stress were altered.

33

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6