Integrated analysis of physiological, transcriptomic and metabolomic responses and tolerance mechanism of nitrite exposure in Litopenaeus vannamei

Journal Pre-proof Integrated analysis of physiological, transcriptomic and metabolomic responses and tolerance mechanism of nitrite exposure in Litopenaeus vannamei

Jie Xiao, Qing-Yun Liu, Jing-Hao Du, Wei-Lin Zhu, Qiang-Yong Li, Xiu-Li Chen, Xiao-Han Chen, Hong Liu, Xiao-Yun Zhou, Yong-Zhen Zhao, Huan-Ling Wang PII:

S0048-9697(19)34407-9

DOI:

https://doi.org/10.1016/j.scitotenv.2019.134416

Reference:

STOTEN 134416

To appear in:

Science of the Total Environment

Received date:

11 June 2019

Revised date:

4 August 2019

Accepted date:

11 September 2019

Please cite this article as: J. Xiao, Q.-Y. Liu, J.-H. Du, et al., Integrated analysis of physiological, transcriptomic and metabolomic responses and tolerance mechanism of nitrite exposure in Litopenaeus vannamei, Science of the Total Environment (2019), https://doi.org/10.1016/j.scitotenv.2019.134416

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

Integrated analysis of physiological, transcriptomic and metabolomic responses and tolerance mechanism of nitrite exposure in Litopenaeus vannamei

Jie Xiao a, Qing-Yun Liu b, Jing-Hao Du a, Wei-Lin Zhu b, Qiang-Yong Li b, Xiu-Li Chen, Xiao-Han Chen b, Hong Liu a, Xiao-Yun Zhou a, Yong-Zhen Zhao

b, *

,

Key Lab of Freshwater Animal Breeding, Key Laboratory of Agricultural Animal

ro

a

of

Huan-Ling Wang a, *

-p

Genetics, Breeding and Reproduction, Ministry of Education, College of Fishery

Guangxi Academy of Fishery Sciences, Guangxi Key Laboratory of Aquatic Genetic

lP

b

re

Huazhong Agricultural University, Wuhan, P. R. China

Jo ur

na

Breeding and Healthy Aquaculture, Guangxi Nanning 530021, China, P.R. China

* Co-corresponding author: Yong-Zhen Zhao Tel: +86 0771-5333857; Fax: +86 0771-5316364 E-mail address: [email protected] (Yong-Zhen Zhao).

* Co-corresponding author: Huan-Ling Wang Tel: +86 027 87282113; Fax: +86 027 87282114. E-mail address: [email protected] (Huan-Ling Wang).

1

Journal Pre-proof

Abstract Nitrite accumulation in aquatic environments is a potential risk factor that disrupts multiple physiological functions in aquatic animals. In this study, the physiology, transcriptome and metabolome of the control group (LV-C), nitrite-tolerance group (LV-NT) and nitrite-sensitive group (LV-NS) were investigated to identify the stress

of

responses and mechanisms underlying the nitrite tolerance of Litopenaeus vannamei.

ro

After LV-NT and LV-NS were subjected to nitrite stress, the hemocyanin contents

-p

were significantly decreased, and hepatopancreas showed severe histological damage

re

compared with LV-C. Likewise, the antioxidant enzymes were also significantly

lP

changed after nitrite exposure. The transcriptome data revealed differentially expressed genes associated with immune system, cytoskeleton remodeling and

na

apoptosis in LV-NT and LV-NS. The combination of transcriptomic and metabolomic

Jo ur

analysis revealed nitrite exposure disturbed metabolism processes in L. vannamei, including amino acid metabolism, nucleotide metabolism and lipid metabolism. The multiple comparative analysis implicated that higher nitrite tolerance of LV-NT than LV-NS may be attributed to enhanced hypoxia inducible factor-1α expression to regulate energy supply and gaseous exchange. Moreover, LV-NT showed higher antioxidative ability, detoxification gene expression and enhanced fatty acids contents after nitrite exposure in relative to LV-NS. Collectively, all these results will greatly provide new insights into the molecular mechanisms underlying the stress responses and tolerance of nitrite exposure in L. vannamei.

2

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

Keywords: Nitrite; Shrimp; Antioxidant System; Histopathology; Omics Analysis.

3

Journal Pre-proof

1. Introduction Nitrite stress is a major environmental factor that limits shrimp survival and growth in aquaculture. Nitrite is mainly formed from incomplete oxidation of ammonia to nitrate during denitrification or nitrification process in intensified aquaculture or recirculated water system. The increasing accumulation level of nitrite

of

in water disrupts multiple physiological functions of aquatic animals, such as retard

ro

growth and molting, high mortality, nitrogen excretion, endocrine disruption, ionic

-p

balance and oxygen transportation (Chen and Chen 1992; Chen and Cheng 1995a;

re

Jensen 2003). In crustaceans, the studies about the nitrite toxicity have also been

lP

reported that nitrite exposure could decrease hemocyanin level, delay larval development, depress the immune ability and increase susceptibility to infection

na

(Chen and Cheng 1995b; Mallasen and Valenti 2006; Tseng and Chen 2004). However,

Jo ur

the information related to the molecular mechanisms of nitrite stress response still remain very limited.

Few studies have currently addressed the biological effects of nitrite stress on shrimp. Guo et al. (2013) reported that nitrite exposure could induce hemocyte apoptosis and the changes of the antioxidant enzymes in L. vannamei. Nitrite exposure also induces overproduction of reactive oxygen species (ROS), DNA damage and reduces the total hemocyte count (THC) in Penaeus monodon (Xian et al. 2011; Xian et al. 2012). In addition, it is reported that nitrite exposure influences the immune-related gene expression both in dose- and time-dependent changes in

4

Journal Pre-proof

hepatopancreas of Marsupenaeus japonicus (Zheng et al. 2016). It has been reported that ammonia and nitrite exert synergistic effects on oxidative stress and apoptosis in Macrobrachium rosenbergii (Zhang et al. 2015), and significant changes are also observed in respiratory parameters, acid-base balance and osmoregulation in M. japonicus under ammonia and nitrite stress (Cheng et al. 2013). Moreover, ammonia

of

and nitrite exposure can constitute a serious damage and destruction to gill structure

ro

of M. amazonicum juveniles (Dutra et al. 2017). However, although high

-p

concentration of nitrite in aquatic environments causes negative effects on shrimp,

re

some families of shrimps still can withstand somewhat nitrite in our previous study,

understood.

lP

the potential mechanisms underlying this tolerance to nitrite are still poorly

na

L. vannamei as an important resource for fisheries and aquaculture represents

Jo ur

more than 50% of the total penaeid shrimp yield in the world (FAO, 2018). However, shrimp aquaculture is suffering economic losses caused by environmental stress, such as nitrite, whereas it can be accumulated to very high concentrations during the culture of shrimp especially in intensive culture systems. Our previous studies found that one family of shrimps showed the highest survival rate after 96 h of nitrite stress among 20 families, however, another family of shrimps showed the lowest survival rate (data unpublished). The hypothesis of this study was that these shrimps, which exhibited strong tolerance to excess nitrite concentration, may have specific adaptive strategies to high concentration nitrite. To

5

Journal Pre-proof

investigate

this

hypothesis,

comparative

physiological,

transcriptomic

and

metabolomic analysis were performed between these two families under nitrite stress. Then the study aimed to identify molecular mechanisms of nitrite responses based on multi-omics analysis compared with non-challenged group.

of

2. Materials and methods

ro

2.1 Acute nitrite exposure in shrimps

-p

L. vannamei obtained from Guangxi Academy of Fishery Sciences (Nanning,

re

Guangxi) were used to nitrite exposure experiment. To evaluate the nitrite tolerance of

lP

these shrimps, in brief, 20 families (n=40 per family) of healthy L. vannamei were collected for a pre-experiment to detect the performance in response to nitrite stress

na

for 96 h. Consequently, two extreme families with the lowest and highest survival rate

Jo ur

were found and defined as the nitrite-sensitive family (LV-NS) and the nitrite-tolerant family (LV-NT), respectively (data unpublished). Before acute nitrite exposure, the shrimps (n=40 per family) from these two families were acclimated for one week in the aquaculture environment condition (salinity at 30‰, pH at 7.9 ± 0.1, temperature at 27 ± 0.5 °C, and dissolved oxygen higher than 6 mg/L). After temporary acclimation, the shrimps were subjected to nitrite (112.5 mg/L, NaNO2) for further analysis. For the transcriptomic and metabolomic analysis, each family of shrimps (n=40) were exposed to nitrite for 24 h. Likewise, for the physiological analysis, each family of shrimps (n=40) were exposed

6

Journal Pre-proof

to nitrite and randomly collected from each group at each time point (4, 12, 24, 48, 72 and 96 h). The shrimps (n=40) without nitrite exposure were treated as control group (LV-C). 2.2 Determination of hemocyanin contents, histopathology observation and antioxidant parameter analysis

of

Hemolymph was extracted from each shrimp at each time point (4, 12, 24, 48, 72

ro

and 96 h, n=3), then added an equal volume of anticoagulant solution (0.115 M

-p

glucose, 0.34 M sodium chloride, 10 mM EDTA, and 30 mM trisodium citrate, pH

re

7.55) and stored at liquid nitrogen for the hemocyanin content determination. The

lP

hemolymph was centrifuged (800 g, 4 °C for 10 min), then the supernatant was used for absorbance value measurement at 335 nm after 1:99 dilution with Tris-Ca buffer

na

(50 mM Tris, 10 mM CaCl2, and pH=8.0) using UV spectrophotometer (1 cm path

Jo ur

length). The hemocyanin concentration was calculated using the following method: E335 nm (mg/mL) =2.3 × OD335 nm (E stands for hemocyanin; 2.3 is the extinction coefficient of hemocyanin for mg/mL) (Nickerson and Van Holde 1971; Wei et al. 2016). Likewise, the hemocyanin contents from LV-C were also examined. All experiments were conducted in three biological replicates and three technical replicates. Hepatopancreas from control and nitrite-exposed groups (exposure for 96 h) were collected and fixed in 4% paraformaldehyde solution. Then these samples were embedded in paraffin after a series of dehydrations in a gradient of alcohol and

7

Journal Pre-proof

hyalinization in xylene. Paraffin blocks of hepatopancreas were cut into continuous 5 μm sections, and observed under light microscopic after haematoxylin and eosin (HE) staining. Hepatopancreas were collected from each group at each time point (4, 12, 24, 48, 72 and 96 h, n=3) and homogenized (1:9, w/v) in cold 0.9% sodium chloride solution,

of

respectively. Then homogenates were centrifuged (3000 g, 4 °C for 10 min), and the

ro

supernatant was used to detect antioxidant indices including total superoxide

-p

dismutase (T-SOD), catalase (CAT) and total antioxidant capacity (T-AOC) by

re

commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

lP

Protein contents of hepatopancreas homogenates were measured by Biosharp Protein Assay Kit (Beijing, China). All experiments were conducted in three biological

na

replicates and three technical replicates.

Jo ur

2.3 Quantitative real-time PCR (qPCR) Muscle, gill and hepatopancreas (n=3) were collected from each group at 48 and 96 h of nitrite exposure, and total RNA was isolated by TRIzol Reagent (TaKaRa, Dalian, China) following the manufacturer’s procedure, respectively. The cDNA was synthesized and the qPCR was performed as described previously (Xiao et al. 2019). The expression levels of HIF-1α were determined by qPCR using following primers: HIF-1α-F (5’-GACTTGACCCACTTGGCTCC-3’) and HIF-1α-R (5’-CCTGCTGC TAAGACGCTT CTC-3’) (Wei et al. 2016) and 18S rRNA was used as the internal control gene (Zhang et al. 2008). The relative expression levels of the target gene

8

Journal Pre-proof were calculated based on the 2–ΔΔCt method (Livak and Schmittgen 2001). 2.4 Metabolomics analysis To investigate the changes in metabolites of LV-NT and LV-NS in response to nitrite stress, hepatopancreas (n=10, nitrite exposure for 24 h) were collected, and the metabolites were extracted for a gas chromatography-mass spectrometry (GC-MS)

of

platform analysis. Briefly, a volume of 0.4 mL extraction liquid (VMethanol:

ro

VChlorofrom=3:1) and 20 µL of L-2-Chlorophenylalanine was added into 50 mg

-p

hepatopancreas as the internal standard, and metabolites were extracted as previously

re

described (Li et al. 2017). After metabolites extraction, Agilent 7890 gas

lP

chromatograph system coupled with a Pegasus 4D time-of-flight mass spectrometer (GO-TOF-MS) was used for metabolite compounds determination (Li et al. 2017).

na

The multivariate statistical analysis of metabolome data was performed as described

Jo ur

previously (Xiao et al. 2019).

2.5 Transcriptomics analysis

To investigate the changes at mRNA levels of LV-NT and LV-NS in response to nitrite, hepatopancreas (n=6, 3 samples per pool, exposure for 24 h) from the two nitrite exposure groups (LV-NT, LV-NS) were collected and used for RNA extraction and RNA-seq analysis. Total RNA was isolated by TRIzon Reagent (TaKaRa, Dalian, China) following the manufacturer’s procedure. The cDNA library construction and sequencing were performed on an Illumina Hiseq 2000 platform as described previously (Xiao et al. 2019). The obtained raw reads were cleaned by removing reads

9

Journal Pre-proof

adapter sequences, containing ploy-N and low-quality reads. Quality score≥Q30, GC content and sequence duplication levels were used for calculating the quality of reads. High quality of clean reads was used for transcriptome assembly using Trinity software (Grabherr et al. 2011). 2.5.1 Differentially expressed gene (DEG) analysis

of

Gene expression levels were estimated by Fragments Per Kilobase of transcript

ro

per Million mapped reads (FPKM) (Trapnell et al. 2010). Differential expression

-p

analysis was performed using the DESeq (Anders and Huber 2010), and the False

re

Discovery Rate (FDR) < 0.05 and the absolute value of Fold Change (FC) > 2 were

lP

used to determine the expression changed significantly in this study. To determine the reliability of the transcriptome data, 7 DEGs were randomly selected to perform

na

qPCR to determine their expression as described in part 2.3. Primer Premier 5.0

Jo ur

software was used for primer design and all the primers were listed in Table S1. 2.5.2 Gene annotation, GO enrichment and KEGG analysis To obtain more information from DEGs, gene functional annotation was carried out based on following database: NR, Pfam, KOG/COG/eggNOG, Swiss-Prot, KEGG and GO (Xiao et al. 2019). GO and KEGG pathway enrichment analysis was conducted by topGO R packages and KOBAS software. 2.6 Statistical analysis Data analysis was performed by one-way ANOVA or student’s t-test using SPSS software (SPSS version 20.0, Chicago, IL). The difference at p <0.05 was considered

10

Journal Pre-proof

as significant.

3. Results 3.1 Hemocyanin contents and the expression levels of HIF-1α after nitrite exposure Hemocyanin contents were calculated at different nitrite exposure time in

of

different groups (Fig 1). Compared to 0 h, nitrite stress caused a significant decrease

ro

of hemocyanin contents both in LV-NT and LV-NS after 96 h of exposure. In LV-NT,

-p

the hemocyanin contents decreased significantly after 12 h of exposure and increased

re

to the control level after 24 h and 48 h of exposure. However, after 72 h and 96 h of

lP

exposure, the hemocyanin contents showed a significant decrease. In LV-NS, the hemocyanin contents showed a downtrend during nitrite exposure. However, no

na

significant difference (P >0.05) was found between LV-NT and LV-NS.

Jo ur

Nitrite stress had a significant effect on HIF-1α expression in the hepatopancreas, gill and muscle of LV-NT and LV-NS (Fig 2). Compared to 0 h, HIF-1α expression was significantly increased in the hepatopancreas, gill and muscle of LV-NT under nitrite stress for 48 h and 96 h except in muscle at 48 h. Interestingly, significant changes of HIF-1α expression were observed in hepatopancreas, gill and muscle under nitrite stress for 48 h and 96 h between LV-NT and LV-NS. Additionally, the HIF-1α expression all was significantly higher in LV-NT than LV-NS, except in muscle for 48 h exposure. 3.2 Histological alterations in shrimps after nitrite exposure

11

Journal Pre-proof

The hepatopancreas histological structure was observed after 96 h of nitrite exposure in L. vannamei. The hepatopancreas of the control group exhibited a well-organized glandular tubular structure, and tubules were closely arranged (Fig 3A). After nitrite exposure, the hepatopancreas exhibited obvious histological damages both in LV-NT (Fig 3B) and LV-NS (Fig 3C). Compared to the control group,

of

there was an evident separation among tubules. Furthermore, the storage cells (R-cell),

ro

secretory cells (B-cell) and star-shaped polygonal structures of the tubules were

-p

disappeared and necrotic. It was worth mentioning that LV-NS showed a more severe

re

damage of the hepatopancreas structure than LV-NT after nitrite stress.

lP

3.3 Antioxidant system alteration after nitrite exposure The CAT activity in LV-NT decreased significantly after 12 and 24 h of nitrite

na

exposure, and increased significantly to the control level at 48 h (Fig 2D). However,

Jo ur

CAT activity decreased and remained at a stable level at 72 h and 96 h of exposure. In LV-NS, the CAT activity decreased significantly within 12 h of exposure, and increased significantly to the maximum level after 24 h of exposure. Afterwards, the CAT activity decreased gradually to the minimum level at 96 h exposure. There were significant changes in CAT activity at 4, 24 and 48 h of exposure between LV-NT and LV-NS. There was a no-significant change in T-SOD activity in LV-NT during 96 h of exposure (Fig 4B). But, the T-SOD activity in LV-NS decreased gradually, reached the minimum level at 12 h of exposure, and then increased to the control level at 24 h, 48

12

Journal Pre-proof

h, 72 h and 96 h of exposure. Although no significant difference was found between LV-NT and LV-NS, T-SOD activity in LV-NT appeared to be more stable and higher than that in LV-NS at most time points. The T-AOC activity in LV-NT showed a no-significant change during nitrite exposure (Fig 4C). However, in LV-NS its activity decreased significantly after nitrite

of

exposure (except for 24 and 96 h of exposure). After 24 h of exposure, significant

ro

difference was also observed between LV-NT and LV-NS in T-AOC activity.

-p

3.4 Metabolomics alteration affected by nitrite stress

re

The hepatopancreas from the LV-NT and LV-NS were taken for GC-MS analysis.

lP

The metabolome data of hepatopancreas from untreated shrimps obtained in previous study (Xiao et al. 2019) were used as the control group in this study. In total, 238, 232

na

and 232 metabolites were identified in LV-C, LV-NT and LV-NS, respectively (Table

Jo ur

S2). Among them, 43 and 54 metabolites were significantly affected by nitrite stress in LV-NT and LV-NS, respectively (Table S3, Fig 5A). However, 13 metabolites, with 7 fatty acids, were significantly changed between LV-NT and LV-NS, indicating that these specially changed metabolites were suggestive to be related to the nitrite tolerance of LV-NT (Fig 5B, Table S3). Principal component analysis (PCA) was conducted to assess the intrinsic pattern of overall GC-MS dataset and the variation among these groups preliminarily (Fig S1-A). The score plots revealed that the significant separation was observed between the control and the nitrite exposure groups by PC1, which explained 67.7% of the

13

Journal Pre-proof

total variation of metabolomes among groups. Orthogonal projections to latent structures- discriminant analysis (OPLS-DA) were performed among these groups (LV-C, LV-NT and LV-NS). The results showed that the R2Y and Q2Y intercept values in different groups (LV- NT vs LV- C, LV- NS vs LV- C and LV-NS vs LV-NT) were 0.957 and 0.925, 0.46 and 0.69, and 0.8 and 0.231, respectively. OPLS-DA (Fig

of

S1-B and S1-C) illustrated a distinct separation between the control and nitrite

ro

exposure groups. However, Fig S1-D showed no statistically significant separation

-p

between LV-NT and LV-NS. Therefore, we filtered out the orthogonal variables that

re

were not related to the classification variables in the metabolites and obtained the

lP

reliable correlation among the groups. All these findings confirmed that the LV-NT and LV-NS responses to nitrite stress were represented by different metabolomic

na

patterns in hepatopancreas of shrimps.

Jo ur

After differential metabolites screening among these groups, the identified and qualified metabolites based on Human Metabolome Database (HMDB) were listed in Table S3. Most of differential metabolites were classed into "amino acids, peptides, and analogues", followed by "fatty acids and conjugates". Some amino acids, such as alanine, asparagine, valine, phenylalanine, tyrosine, etc showed obvious decrease in LV-NT and LV-NS after nitrite exposure. Some fatty acids, such as myristic acid, arachidic acid, heptadecanoic acid, arachidonic acid, stearic acid, palmitic acid, oleic acid and so on, were significantly decreased after nitrite exposure, with LV-NS showing greater changes than LV-NT. Nucleic acids including guanosine, inosine,

14

Journal Pre-proof

thymine and thymidine also showed significant decrease after nitrite exposure. In addition, the comparative metabolome analysis revealed that stearic acid, palmitoleic acid, palmitic acid, myristic acid, heptadecanoic acid, behenic acid and arachidic acid of these fatty acids were significantly decreased in LV-NS compared with LV-NT (Fig 5B). The major metabolites were associated with lipids metabolism, nucleotide

of

metabolism and amino acid metabolism, represented by the heatmap (Fig 5C).

ro

3.5 Transcriptomic alteration affected by nitrite stress

-p

The hepatopancreas were taken respectively from LV-NT and LV-NS for RNA

re

sequencing to determine DEGs responsible for nitrite stress and tolerance. The

lP

transcriptome data in the previous study (Xiao et al. 2019) of hepatopancreas from untreated shrimps were used as control group (LV-C) in the present study. After

na

sequencing, each sample contained paired-end reads ranging from 21.6 to 40.6 million

Jo ur

and the quality score≥Q30 and GC content of each sample was more than 86% and 51%, indicating the high quality of reads (Table S4). Here, 65.00% - 77.23% of the clean reads were mapped to the unigenes or transcripts assembled by Trinity software (Fig S2). To determine the reliability of the transcriptome data, expression of 7 randomly selected genes was detected by qPCR and all 7 genes showed a similar expression pattern, which indicated that the transcriptome data were highly reliable for further analysis (Fig 46). Among all the expressed genes, 1256 (LV-NT) and 1490 (LV-NS) DEGs in hepatopancreas were found in comparison with LV-C (Fig 7A). It was also apparent

15

Journal Pre-proof

that more DEGs were down-regulated in LV-NS than LV-NT (Fig 7B). Likewise, 170 DEGs were observed between LV-NT and LV-NS (Fig 7B). Therefore, these results indicated a significant difference between these two groups in response to nitrite stress at the level of transcription. To further explore genes and pathways related with the nitrite responses and

of

tolerance, DEGs from each group were assigned to annotation (Table S5). In the GO

ro

analysis (Fig S3), DEGs were assigned gene ontology terms and classified into the

-p

biological process, molecular function and cellular component of three categories. Of

re

the categorized as biological process, the dominant subcategories were metabolic

lP

process, cellular process and single-organism process. In the cellular component category, most of DEGs were categorized into cell, cell part, organelle,

na

macromolecular complex and membrane. Within the molecular function, the

Jo ur

categories of catalytic activity, binding, structural molecule activity and transporter activity were most enriched. In the KEGG analysis, most of DEGs were enriched in ribosome (ko03010), lysosome (ko04142), protein processing in endoplasmic reticulum (ko04141) and phagosome (ko04145) (Table S6). Based on gene function annotation, many of DEGs were implicated in immune system, apoptosis and cytoskeleton, such as alpha-2-macroglobulin (A2M), serine proteinase inhibitor B3 (SERPINB3), serine proteinase 1 (SP1), C-type lectin (CTL), caspase-2 (CASP2), apoptosis inhibitor 2-like (API2), apoptosis inhibitor 1 (API1), alpha-I tubulin (AITUB), actin 1 (ACT1), flotillin-1 (FLOT1) and so on (Table 1). The

16

Journal Pre-proof

DEGs associated with metabolism process, including "amino acid metabolism", "nucleotide metabolism" and "lipid metabolism", were both overrepresented after nitrite exposure (Table S7). In addition, some DEGs involved in detoxification were observed, such as metallothioneins (MTs), carboxylesterase A4 (CA4), JHE-like carboxylesterase 1 and 2 (CXE1, CXE2), glutathione S-transferase (GST), with higher

of

expression in LV-NT than LV-NS (Fig 7C). Besides, hemocyanin-related genes were

ro

also significantly changed after nitrite exposure (Fig 7C).

re

4.1 Stress responses of nitrite exposure

-p

4. Discussion

lP

In crustacean, the nitrite stress severely restricts artificial breeding and large-scale cultivation. To elucidate the nitrite stress responses, the physiological,

na

transcriptome and metabolome responses of hepatopancreas of L. vannamei in LV-NT

Jo ur

and LV-NS compared with LV-C were investigated. In fish, the nitrite is toxic due to oxidation of functional hemoglobin (Fe2+) to methemoglobin (Fe3+), which cannot bind oxygen (Jensen 2003). Hemocyanin, as a major component of the hemolymph in crustacean, is a respiratory protein and responsible for O2 binding and CO2 transporting in shrimps (Cheng et al. 2003; Coates and Nairn 2014). It has been reported that nitrite stress decreases significantly the fraction of oxyhemocyanin and increases that of deoxyhemocyanin in Penaeus monodon, which decreases the hemocyanin oxygen binding (Cheng and Chen, 1999). After 96 h of nitrite exposure, the hemocyanin contents were all significantly decreased, which indicated that nitrite

17

Journal Pre-proof

stress may result in hypoxia of organisms (Fig 1). Hypoxia inducible factor-1α (HIF-1α) is one of the main transcription factors for the responses to hypoxia stress (Kodama et al. 2012; Semenza 2001). The results found that the expression of HIF-1α was all significantly changed in hepatopancreas, gill and muscle after 48 h and 96 h of exposure. Probably nitrite exposure could influence the hemocyanin contents, and thus

of

lead to hypoxia, which is one of the toxicity mechanisms of acute nitrite stress in

ro

shrimps and may threaten their survival.

-p

Oxidative stress is one of the toxicity mechanisms of environmental stress in

re

aquatic organisms (Guo et al. 2013b; Liang et al. 2016). Previous studies indicated

lP

that nitrite stress could induce the ROS production and cause oxidative stress in aquatic animals (Xian et al. 2011). CAT, T-SOD and T-AOC are important antioxidant

na

enzymes, which are crucial in eliminating the ROS production and protecting cells

Jo ur

from oxidative stress. In this study, the activities of CAT, T-SOD and T-AOC showed time-dependent changes during 96 h of nitrite exposure. T-SOD plays important roles in the antioxidative system by metabolizing O2- to H2O2 and molecular oxygen, which is the first step of ROS elimination. CAT has the ability to reduce H2O2 to oxygen and water, which could counteract the influence of oxidative stress (Ha et al. 2005). In the present study, the CAT activity was inhibited from 48 h to 96 h of exposure, suggesting that the prolonged exposure to nitrite could lead to excessive ROS or H2O2, which further suppressed the activity of CAT. However, there were no significant changes in T-SOD activity as a whole, which indicated that the T-SOD activity may

18

Journal Pre-proof

be less sensitivity to nitrite stress. T-AOC could reflect the overall activity of antioxidants in organisms, including enzymatic antioxidants and non-enzymatic antioxidants. The changes of T-AOC activity during nitrite exposure reflected that the overall antioxidant ability of shrimp was suffering from nitrite stress (Xu et al. 2018). However, after 96 h of exposure, the T-AOC activity reached to the control level,

of

which may be attributed to a series of changes in antioxidants to maintain the balance

ro

of reactive oxygen free radicals. Moreover, analysis of KEGG identified significant

-p

changes to protein processing in endoplasmic reticulum and ribosome after nitrite

re

stress (Table S6), which further indicated that acute nitrite exposure could impact

lP

protein biosynthesis and metabolism (Pelava et al. 2016). Likewise, histological analysis showed that nitrite stress caused severe damages of the hepatopancreas

na

structure in L. vannamei (Fig 3). These results confirmed that nitrite exposure could

Jo ur

cause oxidative stress in L. vannamei, and further damage hepatopancreas structure. A variety of genes showing the largest magnitude of expression changes including immune system, apoptosis and cytoskeleton-associated genes were detected after nitrite exposure according to RNA-seq (Table 1). The innate immune system is the only defense mechanisms to protect organism against microbial infection and environmental stress for invertebrates (Bachere et al. 2004; Cheng and Chen 2000). Previous studies indicated that nitrite stress would impair the immune defense system of aquatic animals (Chand and Sahoo 2006; Zheng et al. 2016). In the present study, some immune-related genes, such as CTL, A2M, SERPINB3, P44L, SERPIN, LAC1,

19

Journal Pre-proof

FLP1, GPCR, SOCS-2, CBP, etc were significantly changed. Moreover, analysis of KEGG pathway enrichment also showed that most of DEGs were significantly enriched in immune response pathway, such as lysosome and phagosome (Luzio et al. 2007). Possibly the nitrite stress could influence the immune system in L. vannamei and further cause an increase of susceptibility of shrimps to pathogens. Among these

of

DEGs, genes involved in cytoskeleton remodeling, such as IFRD1, ACT1, FLOT1,

ro

TNT, MYH1, etc were also overrepresented after nitrite exposure. Sinha et al. (2014)

-p

reported that the changes of cytoskeleton remodeling-related genes were mostly likely

re

to protect against stress. These results indicated that the changes of these genes were

lP

involved in nitrite stress responses and, however, it should be explored to how these genes participate in the remodeling of cytoskeleton structure in the further studies.

na

Notably, nitrite stress caused significant changes of apoptosis-related genes, including

Jo ur

CASP2, API2, API1, BIRCP7 and p53-like protein. It has been suggested that elevated concentrations of nitrite in pond water can induce apoptosis (Xian et al. 2012; Zheng et al. 2016). All these results revealed involvement of immune system, cytoskeleton remodeling and apoptosis in responses to nitrite stress of L. vannamei. Overall, metabolomics data showed that nitrite stress reduced the levels of all significantly changed metabolites in L. vannamei. Remarkably, most of amino acids, nucleotides, lipids and other metabolism-related metabolites were significantly changed after nitrite exposure (Table S3). Interestingly, a series of genes associated with "amino acid transport and metabolism", "lipid transport and metabolism" and

20

Journal Pre-proof

"nucleotide transport and metabolism" were also significantly changed after nitrite exposure (Table S7). These findings implied that nitrite exposure could disrupt metabolism processes in L. vannamei, especially in amino acid metabolism, nucleotide metabolism and lipid metabolism. 4.2 Nitrite tolerance mechanisms of L. vannamei

of

In order to investigate the nitrite tolerance mechanisms of shrimps, the

ro

comparative analysis was also performed between LV-NT and LV-NS. Oxidative

-p

stress is one of the toxicity mechanisms in responses to nitrite in shrimps based on

re

present study. However, antioxidant systems are vital to detoxify harmful ROS and

lP

respond to oxidative stress. Based on analysis of T-AOC, CAT and T-SOD activities after nitrite exposure, LV-NT also showed greater ability of antioxidant relative to

na

LV-NS. The T-SOD and T-AOC activities in LV-NT showed no significant changes

Jo ur

after 96 h of nitrite exposure, but the antioxidant enzymes in LV-NS were much more sensitive to nitrite exposure.

Nitrite could influence the contents of hemocyanin in shrimps, which may further result in hypoxia after nitrite exposure. Although no significant difference was observed in hemocyanin contents between LV-NT and LV-NS, these two groups showed different expression patterns of HIF-1α. Previous study indicated that the up-regulation of HIF-α mRNA is an important component in the adaptation to hypoxia (Liu et al. 2014). Hepatopancreas is an important metabolic organ and a glucose source that may release glucose to the hemolymph via glucose transporters

21

Journal Pre-proof

for energy supply under hypoxia stress in L. vannamei (Martínez-Quintana et al. 2015). Muscle is the storage center of glycogen and an important fuel substrate for animals under hypoxic condition. The expression of HIF-1α was higher in LV-NT than LV-NS, which meant that LV-NT had greater ability to utilize and regulate energy supply in nitrite mediated hypoxia stress. Gill is the main organ involved in gaseous

of

exchange in aquatic organisms and plays a key role in sensing and adapting hypoxia

ro

stress (Piontkivska et al. 2011). The higher expression of HIF-1α in LV-NT may be

-p

related to enhanced gaseous exchange to obtain more oxygen to adapt to nitrite stress.

re

Likewise, higher expression of hemocyanin-related genes (hemocyanin subunit L3,

lP

hemocyanin subunit Y) was also observed in LV-NT than LV-NS, which may influence the hemocyanin contents of shrimps.

na

Detoxification is an important adaptive strategy in responses to nitrite stress.

Jo ur

Metallothioneins (MTs) are highly conserved cysteine-rich proteins that are involved in detoxification of heavy metals, including cadmium and cooper, and protecting against oxidative stress and apoptosis (Felix-Portillo et al. 2016; Felix-Portillo et al. 2014; Mahmood et al. 2009; Ruttkay-Nedecky et al. 2013; Zhang et al. 2014). Previous study has mentioned that MTs are relevant to protect against ROS generated during hypoxia stimuli and ROS-detoxifying processes (Felix-Portillo et al. 2014). In this study, the increased expression of MTs was also observed after nitrite exposure, with much higher expression in LV-NT than LV-NS. This may be related to detoxification process against nitrite stress. Carboxylesterases constitute a metabolic

22

Journal Pre-proof

enzyme superfamily, which are important detoxifying enzymes and play important roles in protecting organisms against oxidative damage. Previous studies have indicated that the members of carboxylesterases are involved in the insecticide detoxification in Locusta migratoria (Zhang et al. 2013; Zhang et al. 2011). Likewise, carboxylesterases

are

reported

to

involvement

in

the

detoxification

of

of

organophosphorus insecticides and the oxidative resistance responses during adverse

ro

stress (Jackson et al. 2013; Zhang et al. 2011). In the present study, the mRNA

-p

expression of carboxylesterase A4 (CA4) (p >0.05), JHE-like carboxylesterase 1 and 2

re

(CXE1, CXE2) in LV-NT was much higher than LV-NS. Besides, glutathione

lP

S-transferase (GST) was involved in detoxification process (Ren et al. 2015) and also showed higher mRNA expression in LV-NT. Therefore, we speculated that all these

na

detoxification-related genes may play important roles in detoxification process to

Jo ur

cope with nitrite stress, and LV-NT probably may have higher ability of nitrite detoxification than LV-NS.

Fatty acids are the critical component of lipid and basic components of cells involved in multiple biological processes. Fatty acids were reported to play important roles in immune system in response to stress (Gao et al. 2018; Zuo et al. 2017). In this study, comparative metabolome analysis revealed that most of fatty acids, including monounsaturated fatty acids (MUFAs) and saturated fatty acids (SFAs), were significantly increased in LV-NT relative to LV-NS (Fig 5B). Thus, we presumed that increased concentration of these fatty acids may activate the defense system to

23

Journal Pre-proof

strengthen its tolerance to nitrite. However, the mechanisms how fatty acids deal with nitrite are unknown, which is still worthy of further investigations.

Conclusion In this study, physiological and molecular differences of nitrite responses were

of

investigated among LV-NT, LV-NS and LV-C in L. vannamei. These results showed

ro

that nitrite exposure could decrease the hemocyanin contents and damage

-p

hepatopancreas structure. Also, most of DEGs were involved in immune system,

re

cytoskeleton remodeling and apoptosis after nitrite stress. Furthermore, the

lP

combination of transcriptomic and metabolomic analysis revealed nitrite exposure disturbed metabolism processes in L. vannamei, including amino acid metabolism,

na

nucleotide metabolism and lipid metabolism. Shrimps in LV-NT were much more

Jo ur

nitrite-tolerant than LV-NS in terms of its greater ability of antioxidant to cope with ROS under nitrite stress. Nitrite exposure could decrease hemocyanin contents, thus influence the HIF-1α expression. The higher HIF-1α expression in LV-NT may be correlated with enhanced energy supplies and gaseous exchanges, which may be one of adaptive strategies to nitrite exposure. Additionally, higher expression of detoxification genes and enhanced fatty acids contents based on transcriptomic and metabolomic analysis may be related to strengthen its tolerance to nitrite in LV-NT in relative to LV-NS. These results could provide useful insights into the mechanisms underlying nitrite stress responses and tolerance mechanisms in L. vannamei and its

24

Journal Pre-proof

utilization for shrimp breeding with nitrite tolerance.

Acknowledgments This work was supported by Guangxi Scientific Research and Technology Development project (NO. AB16380189), Science and Technology Major Project of

of

Guangxi (NO. AA17204080, AA17204088), National Modern Agriculture Industry

ro

Technology System Project (nycytxgxcxtd-14-01), Modern Agroindustry Technology

-p

Research System (CARS-47), Eight Osmanthus Scholars Special Funding (NO.

re

BGXZ-NMBDX-04).

lP

References

Jo ur

Genome Biol. 11, R106.

na

Anders, S., Huber, W., 2010. Differential expression analysis for sequence count data.

Bachere, E., Gueguen, Y., Gonzalez, M., de Lorgeril, J., Garnier, J., Romestand, B., 2004. Insights into the anti-microbial defense of marine invertebrates: the penaeid shrimps and the oyster Crassostrea gigas. Immunol Rev. 198, 149-168. Chand, R.K., Sahoo, P.K., 2006. Effect of nitrite on the immune response of freshwater prawn Macrobrachium malcolmsonii and its susceptibility to Aeromonas hydrophila. Aquaculture. 258, 150-156. Chen, J.C., Chen, S.F., 1992. Effects of nitrite on growth and molting of Penaeus monodon juveniles. Camp Biochem Physiol C. 101, 453-458.

25

Journal Pre-proof

Chen, J.C., Cheng, S.Y., 1995a. Accumulation of urea in the haemolymph and ammonia excretion of Penaeus japonicus exposed to ambient nitrite. Comp Biochem Physiol C Toxicol Pharmacol. 110, 1-6. Chen, J. C., Cheng, S. Y., 1995b. Changes of oxyhemocyanin and protein levels in the hemolymph of Penaeus japonicus exposed to ambient nitrite. Aquat Toxicol. 33,

of

215-226.

ro

Cheng, S.Y., Chen, J.C., 1999. Hemocyanin oxygen affinity, and the fractionation of

re

nitrite. Aquat Toxicol. 45, 35-46.

-p

oxyhemocyanin and deoxyhemocyanin for Penaeus monodon exposed to elevated

lP

Cheng, S.Y., Shieh, L.W., Chen, J.C., 2013. Changes in hemolymph oxyhemocyanin, acid–base balance, and electrolytes in Marsupenaeus japonicus under combined

na

ammonia and nitrite stress. Aquat Toxicol. 130-131, 132-138.

Jo ur

Cheng, W., Chen, J.C., 2000. Effects of pH, temperature and salinity on immune parameters of the freshwater prawn Macrobrachium rosenbergii. Fish Shellfish Immunol. 10, 387-391.

Cheng, W., Liu, C.H., Kuo, C.M., 2003. Effects of dissolved oxygen on hemolymph parameters of freshwater giant prawn, Macrobrachium rosenbergii. Aquaculture. 220, 843-856. Coates, C.J., Nairn, J., 2014. Diverse immune functions of hemocyanins. Dev Comp Immunol. 45, 43-55. Dutra, F.M., Rönnau, M., Sponchiado, D., Forneck, S.C., Freire, C.A., Ballester,

26

Journal Pre-proof

E.L.C., 2017. Histological alterations in gills of Macrobrachium amazonicum juveniles exposed to ammonia and nitrite. Aquat Toxicol. 187, 115-123. FAO, Fisheries and Aquaculture Information and Statistics Branch, 2018. Available at: http://www.fao.org/fishery/statistics/software/fishstatj/ Felix-Portillo, M., Martínez-Quintana, J.A., Arenas-Padilla, M., Mata-Haro, V.,

of

Gómez-Jiménez, S., Yepiz-Plascencia, G., 2016. Hypoxia drives apoptosis

ro

independently of p53 and metallothionein transcript levels in hemocytes of the

-p

whiteleg shrimp Litopenaeus vannamei. Chemosphere. 161, 454-462.

re

Felix-Portillo, M., Martinez-Quintana, J.A., Peregrino-Uriarte, A.B., Yepiz-Plascencia,

lP

G., 2014. The metallothionein gene from the white shrimp Litopenaeus vannamei:

91-100.

na

Characterization and expression in response to hypoxia. Mar Environ Res. 101,

Jo ur

Gao, Y., Zheng, S.C., Zheng, C.Q., Shi, Y.C., Xie, X.L., Wang, K.J., Liu, H.P., 2018. The immune-related fatty acids are responsive to CO2 driven seawater acidification in a crustacean brine shrimp Artemia sinica. Dev Comp Immunol. 81, 342-347. Grabherr, M.G., Haas, B.J., Yassour, M., Levin, J.Z., Thompson, D.A., Amit, I., Adiconis, X., Fan, L., Raychowdhury, R., Zeng, Q., Chen, Z., Mauceli, E., Hacohen, N., Gnirke, A., Rhind, N., di Palma, F., Birren, B.W., Nusbaum, C., Lindblad-Toh, K., Friedman, N., Regev, A., 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 29, 644-652. Guo, H., Xian, J.A., Li, B., Ye, C.X., Wang, A.L., Miao, Y.T., Liao, S.A., 2013. Gene

27

Journal Pre-proof

expression of apoptosis-related genes, stress protein and antioxidant enzymes in hemocytes of white shrimp Litopenaeus vannamei under nitrite stress. Comp Biochem Physiol. C Toxicol Pharmacol. 157, 366-371. Ha, E.M., Oh, C.T., Ryu, J.H., Bae, Y.S., Kang, S.W., Jang, I.H., Brey, P.T., Lee, W.J., 2005. An antioxidant system required for host protection against gut infection in

of

Drosophila. Dev Cell. 8, 125-132.

ro

Jackson, C.J., Liu, J.W., Carr, P.D., Younus, F., Coppin, C., Meirelles, T., Lethier, M.,

-p

Pandey, G., Ollis, D.L., Russell, R.J., Weik, M., Oakeshott, J.G., 2013. Structure and

re

function of an insect α-carboxylesterase (αEsterase7) associated with insecticide

lP

resistance. Proc Natl Acad Sci. 110, 10177-10182. Jensen, F.B., 2003. Nitrite disrupts multiple physiological functions in aquatic animals.

na

Comp Biochem Physiol A Mol Integr Physiol. 135, 9-24.

Jo ur

Kodama, K., Rahman, M.S., Horiguchi, T., Thomas, P., 2012. Assessment of hypoxia-inducible factor-1alpha mRNA expression in mantis shrimp as a biomarker of environmental hypoxia exposure. Biol Lett. 8, 278-281. Li, T., Li, E., Suo, Y., Xu, Z., Jia, Y., Qin, J.G., Chen, L., Gu, Z., 2017. Energy metabolism and metabolomics response of Pacific white shrimp Litopenaeus vannamei to sulfide toxicity. Aquat Toxicol. 183, 28-37. Liang, Z., Liu, R., Zhao, D., Wang, L., Sun, M., Wang, M., Song, L., 2016. Ammonia exposure induces oxidative stress, endoplasmic reticulum stress and apoptosis in hepatopancreas of pacific white shrimp (Litopenaeus vannamei). Fish Shellfish

28

Journal Pre-proof

Immunol. 54, 523-528. Liu, C.C., Shin, P.K.S., Cheung, S.G., 2014. Isolation and mRNA expression of hypoxia-inducible factor α (HIF-α) in two sublittoral nassariid gastropods: Nassarius siquijorensis and Nassarius conoidalis. Mar Environ Res. 99, 44-51. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using

of

real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25,

ro

402-408.

-p

Luzio, J.P., Pryor, P.R., Bright, N.A., 2007. Lysosomes: fusion and function. Nat Rev

re

Mol Cell Biol. 8, 622-632.

lP

Mahmood, K., Yang, J.S., Chen, D., Wang, M., Yang, F., Yang, W.J., 2009. Response of metallothionein gene-1 to laboratory exposure to heavy metals and thermal stress in

na

the freshwater prawn Macrobrachium rosenbergii. J Hazard Mater. 167, 523-530.

Jo ur

Mallasen, M., Valenti, W.C., 2006. Effect of nitrite on larval development of giant river prawn Macrobrachium rosenbergii. Aquaculture. 261, 1292-1298. Martínez-Quintana, J.A., Kikuta, S., Felix-Portillo, M., Peregrino-Uriarte, A.B., Yepiz-Plascencia, G., 2015. A novel functional glucose transporter in the white shrimp Litopenaeus vannamei -LvGLUT2- is up-regulated during hypoxia in hepatopancreas. Mar Environ Res. 112, 61-67. Nickerson, K.W., Van Holde, K.E., 1971. A comparison of molluscan and arthropod hemocyanin-I. Circular dichroism and absorption spectra. Comp Biochem Physiol B Biochem Mol Biol. 39, 855-872.

29

Journal Pre-proof

Pelava, A., Schneider, C., Watkins, N.J., 2016. The importance of ribosome production, and the 5S RNP-MDM2 pathway, in health and disease. Biochem Soc Trans. 44, 1086-1090. Piontkivska, H., Chung, J.S., Ivanina, A.V., Sokolov, E.P., Techa, S., Sokolova, I.M., 2011. Molecular characterization and mRNA expression of two key enzymes of

of

hypoxia-sensing pathways in eastern oysters Crassostrea virginica (Gmelin):

ro

Hypoxia-inducible factor α (HIF-α) and HIF-prolyl hydroxylase (PHD). Comp

-p

Biochem Physiol D Genomics Proteomics. 6, 103-114.

re

Ren, X., Pan, L., Wang, L., 2015. The detoxification process, bioaccumulation and

lP

damage effect in juvenile white shrimp Litopenaeus vannamei exposed to chrysene. Ecotoxicol Environ Saf. 114, 44-51.

na

Ruttkay-Nedecky, B., Nejdl, L., Gumulec, J., Zitka, O., Masarik, M., Eckschlager, T.,

Jo ur

Stiborova, M., Adam, V., Kizek, R., 2013. The role of metallothionein in oxidative stress. Int J Mol Sci. 14, 6044-6066. Semenza, G.L., 2001. HIF-1 and mechanisms of hypoxia sensing. Curr Opin Cell Biol. 13, 167-171. Sinha, A.K., Matey, V., Giblen, T., Blust, R., De Boeck, G., 2014. Gill remodeling in three freshwater teleosts in response to high environmental ammonia. Aquat Toxicol. 155, 166-180. Trapnell, C., Williams, B.A., Pertea, G., Mortazavi, A., Kwan, G., van Baren, M.J., Salzberg, S.L., Wold, B.J., Pachter, L., 2010. Transcript assembly and quantification

30

Journal Pre-proof

by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 28, 511-515. Tseng, I.T., Chen, J.C., 2004. The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus under nitrite stress. Fish Shellfish Immunol. 17, 325-333.

of

Wei, L., Li, Y., Qiu, L., Zhou, H., Han, Q., Diao, X., 2016. Comparative studies of

ro

hemolymph physiology response and HIF-1 expression in different strains of

-p

Litopenaeus vannamei under acute hypoxia. Chemosphere. 153, 198-204.

re

Xian, J.A., Wang, A.L., Chen, X.D., Gou, N.N., Miao, Y.T., Liao, S.A., Ye, C.X., 2011.

lP

Cytotoxicity of nitrite on haemocytes of the tiger shrimp, Penaeus monodon, using flow cytometric analysis. Aquaculture. 317, 240-244.

na

Xian, J.A., Wang, A.L., Hao, X.M., Miao, Y.T., Li, B., Ye, C.X., Liao, S.A., 2012. In

Jo ur

vitro toxicity of nitrite on haemocytes of the tiger shrimp, Penaeus monodon, using flow cytometric analysis. Comp Biochem Physiol C Toxicol Pharmacol. 156, 75-79. Xiao, J., Li, Q.Y., Tu, J.P., Chen, X.L., Chen, X.H., Liu, Q.Y., Liu, H., Zhou, X.Y., Zhao, Y.Z., Wang, H.L., 2019. Stress response and tolerance mechanisms of ammonia exposure based on transcriptomics and metabolomics in Litopenaeus vannamei. Ecotoxicol Environ Saf. 180, 491-500. Xu, Z., Regenstein, J.M., Xie, D., Lu, W., Ren, X., Yuan, J., Mao, L., 2018. The oxidative stress and antioxidant responses of Litopenaeus vannamei to low temperature and air exposure. Fish Shellfish Immunol. 72, 564-571.

31

Journal Pre-proof

Zhang, J., Li, D., Ge, P., Yang, M., Guo, Y., Zhu, K.Y., Ma, E., Zhang, J., 2013. RNA interference revealed the roles of two carboxylesterase genes in insecticide detoxification in Locusta migratoria. Chemosphere. 93, 1207-1215. Zhang, J., Wang, J., Gui, T., Sun, Z., Xiang, J., 2014. A copper-induced metallothionein gene from Exopalaemon carinicauda and its response to heavy metal

of

ions. Int J Biol Macromol. 70, 246-250.

ro

Zhang, J., Zhang, J., Yang, M., Jia, Q., Guo, Y., Ma, E., Zhu, K.Y., 2011.

-p

Genomics-based approaches to screening carboxylesterase-like genes potentially

re

involved in malathion resistance in oriental migratory locust (Locusta migratoria

lP

manilensis). Pest Manag Sci. 67, 183-190.

Zhang, Q., Li, F., Zhang, X., Dong, B., Zhang, J., Xie, Y., Xiang, J., 2008. cDNA

na

cloning, characterization and expression analysis of the antioxidant enzyme gene,

584-591.

Jo ur

catalase, of Chinese shrimp Fenneropenaeus chinensis. Fish Shellfish Immunol. 24,

Zhang, Y., Ye, C., Wang, A., Zhu, X., Chen, C., Xian, J., Sun, Z., 2015. Isolated and combined exposure to ammonia and nitrite in giant freshwater pawn (Macrobrachium rosenbergii): effects on the oxidative stress, antioxidant enzymatic activities and apoptosis in haemocytes. Ecotoxicology. 24, 1601-1610. Zheng, J., Mao, Y., Su, Y., Wang, J., 2016. Effects of nitrite stress on mRNA expression of antioxidant enzymes, immune-related genes and apoptosis-related proteins in Marsupenaeus japonicus. Fish Shellfish Immunol. 58, 239-252.

32

Journal Pre-proof

Zuo, H., Gao, J., Yuan, J., Deng, H., Yang, L., Weng, S., He, J., Xu, X., 2017. Fatty acid synthase plays a positive role in shrimp immune responses against Vibrio

Jo ur

na

lP

re

-p

ro

of

parahaemolyticus infection. Fish Shellfish Immunol. 60, 282-288.

33

Journal Pre-proof Fig 1. The changes of hemocyanin contents in LV-NT and LV-NS after 4, 12, 24, 48, 72 and 96 h of nitrite exposure. The column with different superscripts represents significant differences (P<0.05).

Fig 2. The expression levels of HIF-1α in hepatopancreas (A), gill (B) and muscle (C) of L. vannamei after 48 h and 96 h of nitrite exposure. *, ** and *** respectively mean P < 0.05, P < 0.01 and P<0.001 according to Tukey's test. Results are mean ± SEM

of

(n=3).

ro

Fig 3. Hepatopancreas histopathology of L. vannamei exposed to nitrite. A) Control; B)

-p

Shrimps in LV-NT after 96 h of exposure; C) Shrimps in LV-NS after 96 h of exposure.

lP

points to R-cells (storage cells).

re

One side arrow (black) points to B-cells (secretory cells), and one side arrow (red)

Fig 4. The antioxidative enzyme activities of L. vannamei exposed to nitrite after nitrite

na

exposure. A) Catalase (CAT), B) Total superoxide dismutase (T-SOD); and C) Total antioxidant capacity (T-AOC). Results are mean ± SEM (n=3). The column with

Jo ur

different superscripts manifests significant differences (P<0.05).

Fig 5. Identification of significantly differential metabolites in L. vannamei after nitrite exposure. A) Venn diagram of significantly differential metabolites in LV-NT and LV-NS compared with LV-C. B) The significantly differential metabolites in LV-NT compared with LV-NS. Saturated fatty acids (SFAs), Monounsaturated fatty acids (MUFAs). C) Heatmap of significantly differential metabolites after nitrite exposure. The value is showed by Log2 Ratio. "LV-NT/LV-C" and "LV-NS/LV-C" mean the differentially expressed genes in LV-NT and LV-NS in relative to LV-C.

Fig 6. Expression of 7 DEGs from the transcriptome validated by qPCR. Data were

34

Journal Pre-proof normalized to 18s rRNA as the reference and presented as a fold to validate the transcriptomic analysis results. CA4 (Carboxylesterase A4), CTL (C-type lectin), PK2 (Pyruvate kinase 2), TLSP1 (Trypsin-like serine proteinase 1), HSP70 (Heat shock protein 70), TGL (Triacylglycerol lipase), P44L (Peritrophin-44-like protein).

Fig 7. Identification of differentially expressed genes (DEGs) in L. vannamei after nitrite exposure. A) Venn diagram of differentially expressed genes in LV-NT and

of

LV-NS compared with LV-C. B) Number of DEGs after nitrite exposure in hepatopancreas. C) Detoxification-related DEGs in LV-NT and LV-NS compared with

ro

LV-C. FPKM means Fragments Per Kilobase of transcript sequence per Millions base

Jo ur

na

lP

re

-p

pairs sequenced. * means significant changes between LV-NT and LV-NS (P<0.05).

35

Journal Pre-proof Table 1. List of differentially expressed genes in hepatopancreas in response to nitrite stress. Nr annotation

LV-NT/LV-C Log2FC Regulate d

LV-NS/LV-C Log2FC Regulate d

Jo ur

na

lP

re

-p

ro

of

Immune System C-type lectin (CTL) 2.0 up 1.7 alpha-2-macroglobulin (A2M) 2.2 up 2.4 serine proteinase inhibitor B3 2.6 up 2.1 (SERPINB3) serine proteinase 1 1.4 up 2.0 peritrophin-44-like protein (P44L) 3.4 up / putative serine proteinase inhibitor 2.6 up 3.8 (SERPIN) laccase 1 (LAC1) 1.8 up 1.9 ficolin-like protein 1 (FLP1) 1.4 up 1.2 crustacyanin subunit C 1.9 up / G protein-coupled receptor (GPCR) 2.0 up 1.8 suppressor of cytokine signaling-2 like 2.6 up / protein (SOCS-2) chitin binding-like protein (CBP) -1.2 down -1.04 reverse transcriptase / N/A -2.0 Apoptosis caspase-2 (CASP2) 1.1 up 1.5 apoptosis inhibitor 2-like (API2) 1.4 up / apoptosis inhibitor 1 (API1) 1.6 up 1.3 baculoviral IAP repeat-containing 2.0 up 2.0 protein 7 (BIRCP7) p53-like protein / N/A 4.5 Cytoskeleton interferon-related developmental 1.3 up / regulator 1 (IFRD1) alpha-I tubulin 1.3 up / actin 1 (ACT1) 1.5 up 1.8 flotillin-1 (FLOT1) 1.02 up 1.2 filamin-A isoform X3 / N/A 1.5 troponin T (TNT) / N/A 1.04 septin-9-like isoform X2 / N/A 1.2 myosin heavy chain type 1 (MYH1) / N/A 1.04 "LV-NT/LV-C" and "LV-NS/LV-C" mean the differentially expressed genes and LV-NS in relative to LV-C; "N/A" means no significantly changed. 36

up up up up N/A up up up N/A up N/A down down up N/A up up up N/A N/A up up up up up up in LV-NT

Journal Pre-proof Highlights 1. Nitrite exposure decreased hemocyanin contents and caused severe oxidative stress. 2. Nitrite exposure influenced immune defense, cytoskeleton remodeling and apoptosis. 3. Amino acid metabolism, lipid metabolism and nucleotide metabolism were disrupted. 4. Higher antioxidative ability and enhanced fatty acids were observed in LV-NT.

Jo ur

na

lP

re

-p

ro

of

5. Increased detoxification gene and HIF-1α expression were related to tolerance.

37

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7