Abstract

Simple Summary

   Chronic ammonia-N exposure always exists in realistic cultivation, and
   it is important to know about the mechanisms of shrimp in response to
   it and to shorten the breeding cycle. The aim of this study was to
   investigate the influence of chronic ammonia-N exposure on the growth
   performance, morphological and physiological alterations, and
   transcriptome changes of shrimp and to evaluate their ammonia-N
   resistance after chronic ammonia-N stress. Results showed that chronic
   ammonia-N exposure affected growth, caused significant structural
   damage and vacuolation in hepatopancreas and gill tissues, and changed
   related antioxidant enzyme activities. The gills maintained cellular
   homeostasis mainly through the high expression of cytoskeleton and
   transcription genes, whereas the hepatopancreas down-regulated related
   genes in the ribosome, proteasome, and spliceosome pathways. These
   genes and pathways are important in the biosynthesis and transformation
   of living organisms. In addition, both tissues maintained organismal
   growth primarily through lipid metabolism, which may serve as an
   effective strategy for ammonia-N resistance in L. vannamei.

Abstract

   Ammonia nitrogen is one of the main toxic substances in aquatic
   cultivation environments. Chronic exposure to excessive amounts of
   ammonia-N creates toxic consequences, retarding the growth of aquatic
   organisms. This study investigated the growth performance,
   morphological and physiological alterations, and transcriptome changes
   in the hepatopancreas and gills of white shrimp Litopenaeus vannamei.
   The results showed that there was no significant difference in the
   survival rate (p > 0.05), whereas growth performance was reduced
   significantly in the treated groups compared to the control groups (p <
   0.05). Significant structural damage and vacuolation occurred in
   hepatopancreas and gill tissues in the treated groups. Superoxide
   dismutase (SOD) activity and Na^+/K^+-ATPase content were significantly
   increased by chronic ammonia-N exposure in the two tissue groups. In
   addition, catalase (CAT) activity and malondialdehyde (MDA) levels were
   significantly altered in the hepatopancreas groups (p < 0.05), whereas
   no differences were observed in the gill groups (p > 0.05). There were
   890 and 1572 differentially expressed genes identified in the
   hepatopancreas (treated versus control groups) and gills (treated
   versus control groups), respectively, of L. vannamei under chronic
   ammonia-N exposure. Functional enrichment analysis revealed
   associations with oxidative stress, protein synthesis, lipid
   metabolism, and different serine proteases. The gills maintained
   cellular homeostasis mainly through high expression of cytoskeleton and
   transcription genes, whereas the hepatopancreas down-regulated related
   genes in the ribosome, proteasome, and spliceosome pathways. These
   genes and pathways are important in the biosynthesis and transformation
   of living organisms. In addition, both tissues maintained organismal
   growth primarily through lipid metabolism, which may serve as an
   effective strategy for ammonia-N resistance in L. vannamei. These
   results provided a new perspective in understanding the mechanisms of
   ammonia-N resistance in crustaceans.

   Keywords: ammonia-N exposure, Litopenaeus vannamei, tissue,
   differentially expressed genes, mechanisms

1. Introduction

   The Pacific white shrimp, Litopenaeus vannamei, accounts for ~80% of
   the total shrimp market because of its rapid growth rate and tolerance
   to a wide range of salinities and temperatures [[44]1]. However, the
   rapid expansion in shrimp farming has created some unavoidable
   problems, such as disease outbreaks, inadequate culture technologies,
   and variable and deteriorated aquaculture environments, which accompany
   them on account of the rapid expansion in shrimp farming [[45]2]. The
   yield and quality of shrimp are affected by the degradation of culture
   water environmental factors [[46]3].

   The most common dissolved inorganic nitrogen ions include ammonia-N,
   nitrite-N, and nitrate-N, which are interrelated via the nitrification
   cycle [[47]4]. A previous study reported that ammonia-N was usually
   converted to nitrite-N and then to nitrate-N by aerobic
   chemoautotrophic bacteria, such as Nitrosomonas and Nitrobacter
   [[48]5], and finally to N[2] via aerobic denitrification [[49]6].
   Ammonia is an essential and low-life-sustaining nutrient for marine
   phytoplankton [[50]7]. In some fish species, low concentrations of
   exogenous ammonia induce protein synthesis and body growth
   [[51]8,[52]9]. However, excessive ammonia cannot be fully absorbed and
   is excreted by the shrimp. Ammonia accumulates in the water because of
   the ammonification originating from organic detritus, such as excess
   feed and feces in the intensive culture systems [[53]10]. High
   concentrations of ammonia lead to the pollution of aquatic water,
   reducing growth rates [[54]11], affecting osmoregulation [[55]2],
   causing cellular toxicity [[56]12], increasing the susceptibility of
   aquatic organisms to infection [[57]13], and even resulting in
   mortality. Additionally, ammonia affects the profusion and
   physiological conditions of different aquatic animals; among them, the
   decapod crustaceans are particularly vulnerable to it due to the toxic
   effect of ammonia, which can influence survival, metabolism, immune
   function, and oxidative stress [[58]5,[59]14,[60]15].

   Oxidative stress in the hepatopancreas of shrimp at a total ammonia
   concentration of 20 mg/L can lead to endoplasmic reticulum stress and
   apoptosis [[61]16]. Liu et al. reported that organisms are able to
   regulate and resolve the excess reactive oxygen species (ROS) that are
   induced by chronic exposure to Pb (during a 28-day indoor exposure
   study) at low concentrations in the hepatopancreas of Macrobrachium
   nipponensis [[62]17]. In aquatic organisms, excessive ROS generated by
   accumulated ammonia in cultivation water induce oxidative stress and
   lead to toxic effects, such as the loss of cell function and even
   apoptosis [[63]14]. Enzymatic and nonenzymatic mechanisms belong to
   self-protection processes, which are established by organisms to
   eliminate ROS-mediated damage [[64]18]. A previous study suggested that
   high ammonia-N exposure induced not only oxidative stress but also ER
   stress [[65]19]. Chronic exposure to ammonia-N reduces the shrimp’s
   food intake and disrupts energy metabolism by lowering body lipids and
   increasing carbohydrate content, leading to the slower growth of the
   shrimp [[66]20]. Additionally, osmoregulation and ammonia excretion are
   also affected, and this changes Na^+/K^+-ATPase activity [[67]21].

   In aquatic animals, the hepatopancreas and gills are vital tissues
   during ammonia-N exposure [[68]22]. The hepatopancreas is associated
   with accumulation and detoxification [[69]23], and the gill tissue is
   the first tissue to respond to negative environmental factors [[70]24],
   acting as a biomarker for aquatic toxicology. A previous study revealed
   that specific enzymes and proteins in the gills coordinate with each
   other to realize different functions, such as ion transport, acid–base
   balance, and osmotic balance [[71]25]. At the same time, Zhang et al.
   also pointed out that the hepatopancreas could reserve lipids and
   synthesize and secrete digestive enzymes [[72]25]. Li et al. suggested
   that chronic exposure to ammonia-N damaged the structure and disturbed
   the lipid metabolism of the hepatopancreas in shrimp [[73]5]. Zhu et
   al. reported that the hepatopancreas contained superoxide dismutase
   (SOD) and catalase (CAT), which are related to physiological metabolism
   and immune reactions in the antioxidant defense system [[74]26]. In
   addition, Zhang et al. reported a comparative transcriptomic analysis
   of three tissues to unveil a network of energy reallocation, which
   suggested that the hepatopancreas and gills cooperate with each other
   rather than muscles in response to heat stress [[75]27]. These findings
   indicated that the analysis of the histological structure,
   physiological indicators, and molecular response of the hepatopancreas
   and gills may explain various stress mechanisms of crustaceans.
   However, limited data exist on the response of shrimp tissues to
   ammonia-N exposure.

   In this study, the effects of exposure to 0 mg/L (control) and 8 mg/L
   (NH[4]CL solution) ammonia-N for 28 days were analyzed through growth
   performance, morphological, physiological, and transcriptional changes.
   We also analyzed changes in the physiological indices and the
   histological structure and characteristics of gene expression in the
   hepatopancreas and gills. Exploring the mechanism of ammonia-N
   tolerance in crustaceans will further enable shortening of the breeding
   cycle of new varieties suitable for this environment. Furthermore, the
   current study will supply us with a theoretical basis for decreasing
   the adverse effects and will increase our knowledge about the risk of
   ammonia-N stress in aquaculture.

2. Materials and Methods

2.1. Ethics Statement

   All animal experiments were carried out following the guidelines and
   approvals of the Institutional Animal Care and Use Committee of
   Guangdong Ocean University (Zhanjiang, China).

2.2. Experimental Animals

   This research used L. vannamei shrimp obtained from 36 full-sib
   families, which were produced at the Zhanjiang Guoxing Aquatic
   Technology Co., Ltd. (Zhanjiang, China), following the same methods as
   described previously [[76]28]. To determine ammonia-N tolerance, an
   acute ammonia-N stress test following a logarithmic spacing of
   concentration (20, 26.3, 34.7, 45.7, 60.3 mg/L) was conducted with
   juveniles from 36 families. The pre-experiment was adjusted and carried
   out to detect the half-lethal time under the experimental conditions
   according to the ammonia doses reported previously [[77]29]. The
   results showed that the survival time changed among these families
   under acute ammonia-N exposure compared to the control groups. We
   selected ammonia-tolerant (LV-T) and sensitive (LV-S) groups according
   to the duration of survival time. We investigated the difference in
   ammonia-N response and growth performance between groups. Shrimp from
   the same LV-T families were selected based on survival time for the
   control and chronic cultivation ([78]Figure 1). The ammonia solution
   used to modify the ammonia-N content was prepared using NH[4]Cl (Xilong
   Scientific Co., Ltd., Shantou, China). All shrimp were not fed during
   the experiment. Shrimp were acclimated for one week before the
   experiment in a cement pool (2 × 2 × 1.5 m), which contained
   sand-filtered seawater (25.0–28.0 °C, 28–30 ppt, pH 7.9–8.0), had a
   photoperiod of 12 h light and 12 h darkness, and was fitted with an air
   pump. They were fed four times each day (7:00, 11:30, 16:00, and 23:00)
   with standard feed (Quanxing Aquatic Feed Co., Ltd., Foshan, China) and
   vitamin-C soluble powder (Hebei Hao Hai Biologicag Co., Ltd.,
   Qinhuangdao, China) mixed evenly, and waste was removed from the bottom
   of the plastic frame. One third of the total aquaculture water was
   replaced daily. The hepatopancreas and gills of these individuals were
   obtained for experiments.

Figure 1.

   [79]Figure 1
   [80]Open in a new tab

   Schematic presentation of experimental shrimp from 36 families.

2.3. Experimental Treatments

   After acclimation, the shrimp were classified at random into test (with
   ammonia solution concentration of 8 mg/L) and control groups (normal
   seawater without extra ammonia solution addition) in a cement pool (15
   m^3), respectively, with three baskets of 40 shrimp in the two groups.
   Chronic exposure concentrations 8 mg/L for 28 days were based on the
   simple pre-experiment, reported environmental concentrations, and
   related studies. Lu et al. reported that the treated group was cultured
   under an ammonia-N concentration of 10 mg/L [[81]30]. Dong et al.
   suggested that the survival rate of the highest concentration group (10
   mg/L) was significantly lower than that of the control group (p <
   0.05), whereas no significant difference occurred between other
   experimental groups and the control group (p > 0.05) [[82]31]. The
   seawater temperature was maintained at 25.0–28.0 °C during the
   experiment, and a multiparameter water quality detector (Xiamen PanTian
   BioTech Co., Ltd., Xiamen, China) was used to measure dissolved oxygen
   (DO), pH, and total ammonia each day. In addition, 10–15% of the volume
   of seawater according to water quality and color was exchanged every
   day with ammonia solution or natural seawater to maintain a stable
   ammonia-N concentration and conditions in the treated and control
   groups, respectively. Cement pools were cleaned of feces daily using a
   sump pump (Goovssy, Xiamen, China). Whenever a shrimp showed loss of
   equilibrium (LOE) and was unable to recover dorso-ventral orientation
   within 5 s of external stimulation, the corresponding time was
   recorded.

2.4. Sampling and Calculations

   During the experimental period, the water temperature, pH, salinity,
   and ammonia-N concentration of the treated and control groups were
   regularly measured four times per day ([83]Table S1). At the same time,
   the shrimp were monitored and removed, and survival time was recorded
   throughout the experiment ([84]Table S2). The shrimp abstained from
   food for 24 h before sampling after 28 days of exposure. Body length
   (BL) was the distance from the base of the eye stalk to the tip of the
   telson. BL was measured using an electronic vernier caliper, accurate
   to 0.01 mm. We used an electronic balance to measure body weight (BW),
   accurate to 0.01 g. Water on the shells of freshly collected shrimp was
   absorbed using gauze prior to taking BL/BW measurements.

   The hepatopancreas and gills of three individuals were taken from each
   group, cut into pieces immediately after dissection, put into RNAhold
   (Trans, Beijing, China) reagent overnight, and then stored at −80 °C
   for experiments. These samples were delivered to Novogene Biotech Co.,
   Ltd. (Tianjin, China) for transcriptome analysis and qPCR validation.
   The two tissues from the same number of shrimp (N = 18) were used to
   assess enzyme activities and were quickly frozen with liquid nitrogen;
   they were then stored at −80 °C. To observe histological change,
   tissues from shrimp (N = 18) were fixed in 4% paraformaldehyde fixing
   solution (Biosharp, Beijing, China) and examined. We used the following
   mathematical equations to calculate various growth parameters:
   S (%) = (S[f]/S[i]) × 100%

   where S, S[f], and S[i] represent the survival rate, the numbers of
   surviving individuals at the end of the experiment, and the total
   number of individuals at the start of this stage, respectively.
   BL (cm) = B[1] − B[0]
   WG (%) = (WG[1] − WG[0])/WG[0] × 100%
   SGR (% d^−1) = [(WG[1] − WG[0])]/(T[1] − T[0]) × 100%

   where BL/WG is the growth of body length/weight gain; B[0]/WG[0] and
   B[1]/WG[1] are the data at the beginning/end of the experimental stage,
   respectively; and T[0] and T[1] are the times of the beginning/end of
   the experiment. SGR is the specific growth rate [[85]32] and is the
   average daily growth rate of the shrimp during the experiment.

2.5. Histological Examination of the Two Tissues

   The hepatopancreas and gills of a shrimp from each plastic frame in the
   experimental and control groups of the two tissues were dissected from
   the cephalothorax. The two tissues were fixed with 4% paraformaldehyde
   for fixation and stored at 4 °C. Paraffin sections a thickness of about
   5 μm were prepared following the standard H&E staining processes using
   Servicebio (Wuhan, China) and examined under a light microscope (SDPTOP
   RX50, Zhejiang, China) to observe the histopathological changes.

2.6. Assessment of Enzyme Activities

   The deionized water was used to grind the two tissues at 4 °C. The
   supernatant was gathered from the homogenate after centrifuging at 3000
   rpm for 20 min at 4 °C for use in biochemical determination. The
   activities of antioxidant enzyme, such as superoxide dismutase (SOD),
   malondialdehyde (MDA), catalase (CAT), and Na^+/K^+-ATPase in the two
   tissues of shrimp, were measured using ELISA kits from Enzyme-linked
   Biotechnology Co., Ltd. (Shanghai, China) according to the
   manufacturer’s instructions.

2.7. Transcriptome Sequencing

   Total RNA was extracted from the samples using Trizol reagent
   (Invitrogen Life Technologies, Carlsbad, CA, USA) following the
   manufacturer’s instructions. The extracted RNA was treated with
   RNase-free water, which was prepared with 75% ethanol to remove any
   potential contamination from DNA molecules. Subsequently, the RNA Nano
   6000 assay kit of the Bioanalyzer 2100 system (Agilent Technologies,
   Santa Clara, CA, USA) was used to assess the total amount and integrity
   of the RNA following the manufacturer’s protocol.

   Briefly, the poly-T oligo-attached magnetic beads were used to purify
   total RNA to mRNA, and divalent cations were used to fragmentate under
   elevated temperature in First Strand Synthesis Reaction Buffer (5×).
   The first strand cDNA was synthesized by M-MuLV reverse transcriptase
   and random hexamer primer, and the RNA was degraded using RNaseH. DNA
   Polymerase I and dNTP were used to synthesize the second-strand cDNA.
   The remaining overhangs were translated into blunt ends through
   exonuclease/polymerase activities. An adapter with a hairpin loop
   structure was ready for hybridization after the adenylation of the 3′
   ends of DNA fragments. The AMPure XP system (Beckman Coulter, Beverly,
   CA, USA) was used for the selection of cDNA fragments with 370–420 bp.
   Eventually, AMPure XP beads were used to purify the PCR product
   following PCR amplification so as to acquire the library.

   The library was tested to ensure the quality. After construction of the
   library, quantification was first performed using a Qubit2.0
   fluorometer; dilution to 1.5 ng/µL was then performed. The insertion
   fragment size of the library was measured using an Agilent 2100
   bioanalyzer. After the insertion fragment size was as expected, the
   effective concentration of the library was accurately quantified using
   qRT-PCR (the effective concentration of the library was higher than 2
   nM) to ensure the quality of the library. When the libraries were
   qualified, they were sequenced with an Illumina NovaSeq 6000 to produce
   end reads of 150 bp. The raw data were stored in the National Center
   Biotechnology Information (NCBI) Sequence Read Archive (SRA) with
   bioproject number PRJNA993130.

2.8. Transcriptome Analysis

   CASAVA base recognition was used to image data from high-throughput
   sequencing measurements into sequence data (readings). In-house perl
   scripts were used to process raw data/reads into the fasta format.
   Clean data/reads were obtained at this stage by removing reads
   containing adapters, low-quality reads, and N bases, and then the Q20,
   Q30, and GC contents were calculated. Q20 means that every 100 bp of
   sequencing reading may contain an error, with a call accuracy of 99%.
   Similarly, a sequencing quality reaches Q30 is perfect and represents
   an accuracy of 99.90%, with no errors or ambiguities. HISAT2 (version
   2.0.5) software was used to map the filtered reads with high-quality
   clean data from the L. vannamei reference genome (NCBI accession No.
   PRJNA927338). FeatureCounts v1.5.0-p3, using the default parameters,
   was used to count the readings mapped to every gene. In addition, the
   number of transcript fragments per kilobase (FPKM) per million mapping
   readings of every gene was computed according to the gene length and
   readings mapped to it. To determine the effects of long-term ammonia-N
   exposure (8 mg/L) and normal aquatic concentrations (less than 0.25
   mg/L) on the hepatopancreas and gill of shrimp, differentially
   expressed genes (DEGs) were analyzed in groups using the DESeq2 R
   package (1.20.0). In the study, |log2 (fold change)| ≥ 1 and false
   discovery rate (FDR) < 0.05 were used to represent the DEGs of the
   transcriptomic differences between the two groups. DEGs analyzed using
   the tools including GO ([86]http://www.geneontology.org/) and KEGG
   ([87]https://www.genome.jp/kegg/) pathway enrichment analysis, which
   accessed on 1 November 2022 and 17 December 2019, respectively, to
   determine the significant pathways (p ≤ 0.05) of the regulatory genes.

2.9. Data Validation via qPCR

   To validate the gene expression profiles and an accuracy of the RNA-seq
   data analysis, samples from the hepatopancreas groups (with three
   replicates), were analyzed by qPCR. Eight gene-specific primers for
   DEGs were designed, using Primer5 software (Premier Biosoft
   International, Palo Alto, CA, USA) ([88]Table S3) for qPCR
   amplification, according to the L. vannamei genome and assembled
   transcriptome data. The qPCR testing was performed following the
   manufacturer’s instructions, using a CFX96 Real-Time PCR detection
   system (Bio-Rad, Hercules, CA, USA) and Perfect Start Green qPCR
   SuperMix (TransGen). The qPCR was performed as described in a previous
   study by Liang et al. [[89]33]. The 2^−ΔΔCt method was used for
   evaluating the relative genes expression levels through the cycle
   thresholds (Cq), which were transformed from fluorescence information,
   with β-actin (GenBank accession No. [90]XM_027364953.1) and served as
   an internal reference gene for normalization.

2.10. Data Analysis

   DESeq2 R package (1.20.0), |log2 (fold change)| ≥ 1 and false discovery
   rate (FDR) < 0.05 were used to determine the DEGs. Growth and qPCR data
   of the study were analyzed with a T test in SPSS 23.0 (SPSS Inc.,
   Chicago, IL, USA). The T test was used to determine the significant
   differences between genes after the normality of distribution and
   homogeneity of variance. Excel 2016 (Microsoft, Redmond, WA, USA),
   GraphPad Prism 8.0.2 (La Jolla, CA, USA), and Origin 18 (OriginLab,
   Northampton County, MA, USA) were used to analyze all statistical data
   and generate images. The results were expressed as mean ± standard
   error (SD). The differences were statistically significant at p < 0.05.

3. Results

3.1. Survival Rate and Growth Performance

   We observed abnormal behavior in the exposed shrimp, such as reduction
   in motility, decrease in feed intake, and erratic swimming. There were
   no behavioral alterations during the experiment in the control groups,
   were no significant differences in survival rate (p > 0.05), whereas
   BL, WG, and SGR were significantly reduced (p < 0.05) between the
   control and experimental groups ([91]Table 1). In addition, the BL, WG,
   and SGR decreased by 27.96%, 43.87%, and 37.02%, respectively, compared
   to the control groups.

Table 1.

   Survival rate (SR), body length (BL), weight g.ain (WG), and specific
   growth rate (SGR) of L. vannamei exposed to ammonia nitrogen for 28
   days.
   Group Survival Rate (%) Body Length (mm) Weight Gain (%) Specific
   Growth Rate (% d^−1)
   Experimental 85.00 ± 1.00 ^a 2.86 ± 0.28 ^a 234.04 ± 0.50 ^a 15.94 ±
   0.01 ^a
   Control 89.17 ± 0.58 ^a 3.97 ± 0.15 ^b 416.99 ± 0.47 ^b 25.31 ± 0.01 ^b
   [92]Open in a new tab

   Note: The corresponding data above are the mean ± standard deviation of
   two groups; the different superscript letters within the same column
   indicate significant differences (p < 0.05).

3.2. Histological Analysis of the Two Tissues

   The hepatopancreas and gill tissues showed significant histological
   changes after 28 days of ammonia-N stress ([93]Figure 2). The damage
   reduced storage cells (R cells), increased secretory cells (B cells),
   and distorted or disappeared the star-shaped polygonal structures of
   the lumen ([94]Figure 2a). At this time, the boundaries of the
   hepatopancreatic tubules were vague, with significantly expanded
   volume, and some cells were disassociated ([95]Figure 2a). The
   boundaries of hepatopancreatic tubules were distinct, with complete
   basement membranes and arranged hepatocytes in the control group
   ([96]Figure 2b). The shrimps exposed to 0 mg/L ammonia-N had normal
   gill epithelium arrangement and cell contour without cell vacuolation
   or congestion ([97]Figure 2d). The gills of L. vannamei subjected to 8
   mg/L ammonia-N exposure displayed damaged histological structure
   compared to the control groups. The gill filaments began to deform and
   swell, quantities of hemocytes increased, and a large number of
   epithelial cells shed compared to the control groups ([98]Figure 2c).

Figure 2.

   [99]Figure 2
   [100]Open in a new tab

   Histological examination of hepatopancreas and gills of L. vannamei
   subjected to ammonia-N for 28 days with the treated group (a,c) and
   with the control group (b,d). Note: R: storage cells (R-cell); B:
   secretory cells (B-cell); T: the star-shaped polygonal structure of the
   hepatopancreatic lumen. BM: basement membrane; Ep: epithelial cell; N:
   epithelial nucleus; Hc: hemocytes. Stained using hematoxylin and eosin
   (H&E), 400×.

3.3. Enzyme Activities of the Two Tissues

   The changes in the antioxidant statuses of the two tissues of L.
   vannamei between chronic-ammonia-exposed and control groups are showed
   in [101]Figure 3. The results found that SOD activity significantly
   increased (p < 0.05) between the treated and control groups in the two
   tissues ([102]Figure 3a). CAT activity had no significant differences
   in the gills (p > 0.05), whereas it was significantly decreased (p <
   0.05) in the hepatopancreas in the 28-day exposure study ([103]Figure
   3b). The activity of Na^+/K^+-ATPase was significantly increased (p <
   0.05) in the two tissues ([104]Figure 3c). Moreover, MDA content, which
   represents the degree of oxidative stress in the tissues, was
   significantly increased in the hepatopancreas compared to the control
   groups (p < 0.05), without significance in the gill tissue ([105]Figure
   3d).

Figure 3.

   [106]Figure 3
   [107]Open in a new tab

   Effects of chronic ammonia-N on the nonspecific immune indices in the
   hepatopancreas and gill of L. vannamei. (a) SOD activity, (b) CAT
   activity, (c) Na^+/K^+-ATPase activity, (d) MDA content. Note:
   different letters indicate significant differences between treated (T)
   and control (C) groups (p < 0.05).

3.4. Assembly, Read Mapping, and Sequence Alignment

   The low-quality sequences were removed following RNA-seq analysis of
   hepatopancreas and gill samples from hepatopancreas and gill groups,
   respectively. The sequencing data with Q20, Q30, and GC contents of
   about 98%, 93%, and 49%, respectively, in the hepatopancreas in treated
   vs. control groups were considered reliable. Similarly, in the gill in
   treated vs. control groups, the Q20, Q30, and GC contents were about
   97%, 92%, and 43%, respectively ([108]Table S4). Compared to the
   reference genome, there were 266, 212, and 458 valid reads,
   respectively, and 85.79–89.94% of them were mapped to the genome in the
   hepatopancreas in treated vs. control groups. In the gill in treated
   vs. control groups, there were 127, 371, and 814 valid reads,
   respectively, and 85.79–89.94% of them were mapped to the genome
   ([109]Table S5).

3.5. DEG Analysis in the Two Tissues under Ammonia-N Exposure

   The transcriptome data of the two tissues in the two groups of L.
   vannamei were analyzed to identify the DEGs. Altogether, 890 (326
   up-regulated and 564 down-regulated) and 1572 (956 up-regulated and 616
   down-regulated) candidate genes were significantly differentially
   expressed between the experimental and control groups of the two
   tissues, respectively ([110]Figure 4a). Sixty-two overlapping DEGs were
   screened with Venn diagrams in order to explore logical association
   between the two tissues ([111]Figure 4b). The top 10 up-regulated,
   down-regulated, and overlapping DEGs are displayed in [112]Table 2 with
   descriptions. A total of 62 DEGs in both tissues—6.97% and 3.94%,
   respectively—confirmed that the response of different tissues to
   ammonia-N stress was tissue-specific. Notably, the top terms of DEGs
   that were differentially expressed after chronic ammonia-N stress
   included serine-type endopeptidase activity, oxidoreductase activity,
   and serine hydrolase activity. The cytoskeleton-associated genes were
   listed in [113]Table 3.

Figure 4.

   [114]Figure 4
   [115]Open in a new tab

   The expression profiles of differentially expressed genes (DEGs). Note:
   The number of DEGs in each comparison group (a). Venn diagram of DEGs
   between adjacent and nonadjacent pairwise comparison groups (b). Orange
   and blue circles represent genes expressed between hepatopancreas and
   gill groups, respectively (|log2(FC)| ≥ 1 and FDR < 0.05).

Table 2.

   Top 10 up- and down-regulated DEGs in hepatopancreas and gill groups.
   Gene Names log2FC Description (KEGG) Description (GO)
   Hepatopancreas in treated vs. control groups
   LOC113820612 9.50639 chloroplastic-like /
   LOC113807369 6.40349 transmembrane protease serine 13-like /
   LOC113803831 5.52520 techylectin-5A-like /
   ctrb1 5.52161 chymotrypsin BII-like serine-type endopeptidase activity
   (MF-up)
   LOC113802089 5.46694 alcohol dehydrogenase [NADP(+)]-like
   alpi.1 5.36745 alkaline phosphatase-like hydrolase activity, acting on
   ester bonds (MF-UP)
   tubb6 5.24083 tubulin beta chain-like hydrolase activity, acting on
   acid anhydrides (MF-Down)
   LOC113812240 5.18027 methylenetetrahydrofolate reductase-like /
   LOC113820103 5.00419 mucin-12-like /
   kcp 4.96579 basic proline-rich protein-like /
   LOC113802550 −13.28640 triosephosphate isomerase B-like /
   LOC113827511 −12.04027 C-type lectin domain family 17, member A-like /
   LOC113824334 −8.99337 arylsulfatase B-like /
   mmp3 −8.66135 inactive pancreatic lipase-related protein 1-like /
   LOC113807283 −8.26976 ovochymase-2-like /
   LOC113816677 −7.90349 small integral membrane protein 8-like /
   LOC113802551 −7.60858 triosephosphate isomerase-like /
   LOC113810108 −7.00134 anti-lipopolysaccharide factor-like /
   slc46a1 −6.93143 solute carrier family 46-member 1 /
   mrpl2 −6.76469 39S ribosomal protein L2, mitochondrial-like
   ribonucleoprotein complex (CC-Down)
   Gill in treated vs. control groups
   wdr54 8.28549 trypsin-1-like serine hydrolase activity (MF-Down)
   LOC113819943 8.23980 lactosylceramide
   4-alpha-galactosyltransferase-like /
   LOC113817714 7.89112 glutamate-gated chloride channel subunit beta-like
   /
   LOC113822681 7.45779 large subunit ribosomal RNA /
   hacd2 7.41957 1-acyl-sn-glycerol-3-phosphate acyltransferase
   epsilon-like transferase activity, transferring acyl groups (MF-UP)
   LOC113800217 7.39103 CAP-Gly domain-containing linker protein 4-like /
   LOC113819942 7.27277 lactosylceramide
   4-alpha-galactosyltransferase-like /
   cyp27c1 7.24919 probable cytochrome P450 49a1 oxidoreductase activity,
   acting on paired donors, with incorporation or reduction of molecular
   oxygen (MF-UP)
   LOC113823234 7.09559 facilitated trehalose transporter Tret1-like /
   fbxo32 6.99950 F-box only protein 32-like /
   LOC113820879 −7.57815 galanin receptor type 1-like /
   LOC113820814 −6.84140 ETS-related transcription factor Elf-1-like /
   znf143 −6.46198 protein SpAN-like endopeptidase activity (MF-Down)
   LOC113819193 −6.27849 myosin heavy chain, muscle-like /
   cs −6.15781 gastric triacylglycerol lipase-like /
   LOC113830294 −6.01273 cuticle protein 7-like /
   LOC113818578 −6.00421 histone-lysine N-methyltransferase, H3 lysine-79
   specific-like /
   LOC113830353 −5.97509 extensin-like /
   ct55 −5.95853 trypsin-1-like serine-type endopeptidase activity
   (MF-Down)
   LOC113830353 −5.95598 carbohydrate sulfotransferase 10-like /
   [116]Open in a new tab

Table 3.

   List of differentially expressed genes related to the cytoskeleton.
   Genes Hepatopancreas in Treated vs. Control Groups Gills in Treated vs.
   Control Groups
   paramyosin (prm) / up
   tropomyosin (tpm) down /
   flotillin (flot) / up
   titin (ttn) up up
   beta tubulin (tub) down up
   [117]Open in a new tab

3.6. GO and KEGG Enrichment Analysis

   The distribution of DEGs’ enriched functions via GO analysis between
   the ammonia-treated and control groups was generally consistent
   ([118]Figure 5). The DEGs from GO enrichment analysis were assigned to
   3 major functional classes—biological process (BP), cellular component
   (CC), and molecular function (MF)—and 107 subcategories between treated
   vs. control groups in hepatopancreas ([119]Figure 5a). BP-enriched DEGs
   were associated with 47 significantly terms, including translation,
   peptide biosynthetic process, amide biosynthetic process, peptide
   metabolic process, cellular amide metabolic process, and so on. Another
   31 highly enriched terms were classified as CC, including
   ribonucleoprotein complex, ribosome, protein-containing complex,
   cytoplasmic part, cytoplasm, proteasome complex, and endopeptidase
   complex. The remaining 29 significantly enriched terms belonged to MF.
   Among the nine pathways, ribosome and ribosome biogenesis were
   significantly affected by ammonia-N exposure, with most genes being
   mostly down-regulated, except the first gene of the ribosome pathway in
   our study ([120]Table 4).

Figure 5.

   [121]Figure 5
   [122]Open in a new tab

   Functional annotation analysis of DEGs under long-term ammonia-N
   stress. GO enrichment analysis of DEGs between the control and treated
   groups in hepatopancreas (a) and gills (b). KEGG pathway enrichment
   analysis of DEGs between the control and treated groups in
   hepatopancreas (c) and gills (d).

Table 4.

   List of functional groups and related liver DEGs from hepatopancreas
   and gill groups in response to long-term ammonia-N exposure.
   KEGGID Funnation Description Gene Names
   Hepatopancreas in treated vs. control groups
   pvm03010 Ribosome LOC113826494, mrpl2, LOC113826493, LOC113822410,
   rsl24d11, rpl37, rpl7, rps27a, LOC113803764, rpl32, LOC113817813,
   LOC113822082, mrps6, LOC113812878, rpl29, rplp2, LOC113826804,
   LOC113817249, LOC113830328, LOC113827080, mrpl12, rpl36, rpl19, rps26
   pvm03050 Proteasome LOC113803698, psma4, psma1, psma2, psma3, psmd3,
   LOC113807430, psmb3, LOC113830026
   pvm03040 Spliceosome lsm8, snrpa1, LOC113820651, sf3b5, alyref,
   LOC113802307, snrpb, ppih, LOC113803628, snrpg, sf3a3, tra2b,
   LOC113823506, u2af1, sf3b6, snrpc, LOC113820949, lsm3
   pvm00670 One carbon pool by folate LOC113812240, mtr, shmt1, atic, dhfr
   pvm00531 Glycosaminoglycan degradation LOC113824334, xpnpep2, shisa7,
   LOC113804791, sgsh
   pvm01240 Biosynthesis of cofactors psat1, alpi.1, akr1b1.1,
   LOC113802089, ugdh, LOC113826969, shmt1, dhfr, si:ch73-334d15.1, ak6,
   LOC113828810, LOC113802542, LOC113823260, LOC113807674, LOC113807126,
   coq7
   pvm00040 Pentose and glucuronate interconversions akr1b1.1,
   LOC113802089, ugdh, si:ch73-334d15.1, sord, asrgl1
   pvm00600 Sphingolipid metabolism cpxm1a, xpnpep2, LOC113829078,
   LOC113806775, LOC113810646, LOC113824094, LOC113804791, smpd1
   pvm03008 Ribosome biogenesis in eukaryotes LOC113826178, ak6,
   LOC113828897, LOC113816393, LOC113801028, LOC113826254, dkc1,
   LOC113816679, rrp7a
   Gill in treated vs. control groups
   pvm00512 Mucin type O-glycan biosynthesis galnt3, plbd1, LOC113806687,
   LOC113806752, LOC113801114, LOC113826876, LOC113814026
   pvm03013 Nucleocytoplasmic transport LOC113821023, kpnb3, kidins220a,
   nup58N, ran, LOC113814257, LOC113811339, LOC113822864, ipo7, nup153,
   ipo9, LOC113807607
   pvm00100 Steroid biosynthesis LOC113822972, LOC113810916, cs
   [123]Open in a new tab

   Note: the bold indicates the up-regulated genes, and non-bold indicates
   down-regulated genes in these pathways.

   In the hepatopancreas in treated vs. control groups, there were nine
   significantly enriched terms classified as BP, including the cellular
   carbohydrate metabolic process, protein de-ubiquitination, protein
   modification via small protein removal, and the nucleobase-containing
   compound biosynthetic process ([124]Figure 5b). The cell junction
   (GO:0030054) was the only one highly ranked in the CC category. The MF
   category included the remaining 23 significantly enriched terms.
   However, there were no significantly enriched overlap terms classified
   in the three functional classes between the transcriptome analysis of
   the two tissues. It is important to explore gene interactions because
   of their value in biological functions. To understand potential gene
   interactions under chronic ammonia-N stress, all DEGs were searched and
   assigned to nine and three pathways in hepatopancreas and gills,
   respectively, and the top 20 enrichment pathways shown in [125]Figure
   5. The analysis revealed that the DEGs in the groups were significantly
   enriched in the following pathways: ribosome, proteasome, spliceosome,
   and one carbon pool by folate; glycosaminoglycan degradation,
   glycosaminoglycan degradation, biosynthesis of cofactors, pentose and
   glucuronate interconversions, sphingolipid metabolism, and ribosome
   biogenesis in eukaryotes ([126]Figure 5c). In the gills in treated vs.
   control groups, mucin-type O-glycan biosynthesis, nucleocytoplasmic
   transport, and steroid biosynthesis were the significantly enriched
   pathways of the DEGs ([127]Figure 5d). [128]Table 4 lists the major
   enriched KEGG pathways associated with the DEGs. In addition, the two
   tissues maintain shrimp growth primarily through lipid metabolism
   ([129]Table 5).

Table 5.

   The related genes in the lipid metabolism pathway.
   KEGGID Funnation Description Gene Names
   LA vs. LAC
   pvm00565 Ether lipid metabolism LOC113829078
   pvm00600 Sphingolipid metabolism cpxm1a, xpnpep2, LOC113829078,
   LOC113806775, LOC113810646, LOC113824094, LOC113804791,
   smpd1
   pvm00564 Glycerophospholipid metabolism LOC113808891, LOC113810367,
   PCYT2, LOC113802189, LOC113805268, LOC113814898
   pvm00561 Glycerolipid metabolism akr1b1.1, LOC113802089
   LB vs. LBC
   pvm00565 Ether lipid metabolism agps, LOC113821399, LOC113824844
   pvm00600 Sphingolipid metabolism gba, asah1
   pvm00564 Glycerophospholipid metabolism hacd2, LOC113821399
   pvm00561 Glycerolipid metabolism LOC113823713, hacd2, LOC113819701,
   LOC113817093
   [130]Open in a new tab

   Note: the bold indicates the genes up-regulated; non-bold indicates
   down-regulated genes in these pathways.

3.7. Validation of RNA-Seq Data by qPCR

   Eight DEGs were randomly selected from RNA-seq data for qPCR analysis
   to verify the accuracy of the Illumina sequencing results ([131]Figure
   6). The eight DEGs (six up-regulated and two down-regulated) related to
   hepatopancreas transcriptome data were lysozyme-C-like (dnajc5),
   lysosomal phospholipase A2 (lpla2), sodium- and chloride-dependent
   GABA-transporter-2-like (slc6a8), titin, ferritin-subunit-like,
   Penaeidin-2b (naa38), protein-transport-protein-sec61-subunit-beta-like
   (sec61b), and phosphoenolpyruvate carboxykinase (pck2). Overall, the
   qPCR analysis confirmed that the RNA-seq data were consistent with the
   expression patterns of the DEGs.

Figure 6.

   [132]Figure 6
   [133]Open in a new tab

   Eight validated genes expression levels in the hepatopancreas groups at
   28 days.

4. Discussion

4.1. Survival Rate and Growth Performance under Ammonia-N Exposure

   Previous studies have shown that excess ammonia increased susceptivity
   to pathogens, thereby inhibiting growth, reducing osmoregulation,
   increasing molting frequency, and even leading to high mortality in
   shrimp [[134]34]. Non-ionic ammonia exposure at 0.54 mg/L in the
   hepatopancreas of M. rosenbergii significantly reduced the survival
   rate, whereas it had no obvious effect on growth compared to other
   exposure concentrations (0, 0.108, 0.216, 0.324, or 0.54 mg/L) in a
   20-day experiment [[135]31]. Similarly, the survival rate has no
   significance in blunt snout bream after long-term ammonia-N exposure
   [[136]22]. The research revealed that the BL, WG, and SGR of shrimp
   decreased significantly with long-term ammonia-N stress, which affected
   the growth performance (p < 0.05). This result is consistent with the
   reported long-term experiment with a higher temperature in summer
   [[137]35]. Chronic ammonia-N exposure could affect the food intake and
   then disturb this growth [[138]20], which explains why the growth
   performance of the shrimp differed compared from that of the control
   groups. The sustained ammonia-N stress decreased the growth of shrimp,
   which extended the cultivation culture cycle (generally about four
   months) and increased cost. Aquatic animals may be subjected to
   potential toxic interactions with nitrate-N because wastewater
   components often contain more than one nitrogen-containing waste
   [[139]36]. Moreover, three pollutants in the culture water often exceed
   those found in nature [[140]37]. In summary, it has been reported that
   many aquatic species showed a reduction after long-term exposure to
   ammonia-N, which demonstrates the importance of the cultivation
   environment. In our study, there was no significant difference in the
   cumulative death rate after long-term stress of 28 days, indicating
   that ammonia-N with 8 mg/L concentration was in the adaptive range of
   the shrimp and could affect their growth.

4.2. Tissue Damage and Physiological Responses

   In this study, the ammonia-treated groups of the two tissues showed
   obvious histopathological changes in the irregular appearance of
   tubular structure after four weeks of rearing compared to the control
   groups. R cells have many small lipid-filled vacuoles for the synthesis
   of lipoproteins and their export to other organs, heavy metal
   detoxification, and uric acid excretion. B cells are regarded as an
   important lipid reserve in tissues because of their functions of
   producing, secreting, and recycling fat emulsifiers [[141]38]. In
   addition, they take charge of the accumulation and transport nutrients,
   which are also sensitive to toxic substances and other stresses
   [[142]39]. They are important components belonging to the
   hepatopancreas. Compared to the control groups, a common feature in the
   hepatopancreas was moderate-to-high vacuolation in R cells. Similar
   results have been found with Marsupenaeus japonicus [[143]40] and M.
   nipponense [[144]38]. This suggests that ammonia-N caused damage to the
   hepatopancreas structure, leading to a reduction in R cells, and then
   secretion and exretion of toxic substances through B cells, resulting
   in an increase in B cells. The previous study reported that the
   infestation by Probopyrus bithynis affected the structure of gills in
   M. amazonicum, including swelling, elevation of epithelial cell
   hyperplasia, and hemocyte accumulation [[145]41]. Guo et al. observed
   that the structure change in gills was different at various
   points—cellular tumefaction and laminar epithelial thickening in
   particular [[146]42]. Similarly, the symptomatology of the study was
   consistent with this when the shrimp were exposed to 15 mg/L ammonia-N
   for 28 days [[147]43]. These studies suggest that the histopathological
   changes may reflect an adaptation to the stress caused by chronic
   ammonia-N and lead to thickening of the epithelium, which increases the
   distance between the internal and external media.

   Ammonia in aquaculture water will accumulate and produce excess ROS in
   tissues, which induces oxidative stress, creates toxic effects on
   aquatic organisms, and leads to functional defects and even apoptosis
   [[148]14]. Antioxidant enzymes are produced by organisms fighting
   against ROS and balancing the homeostasis between oxidants and
   antioxidants in living organisms. In the study, levels of SOD were
   increased between the experimental and control groups (p < 0.05), and
   CAT was increased just in the hepatopancreas in treated vs. control
   groups. The antioxidant defense system can eliminate the ROS under
   non-stress conditions. However, excessive ROS are produced by chronic
   ammonia-N exposure, which inhibited the detoxification function, and
   other antioxidant enzymes may be needed to solve the problem. MDA is
   one of the major indicators reflecting the extent of oxidative damage
   [[149]44]. The study showed that MDA content significantly increased
   with long-term ammonia-N exposure (p < 0.05), suggesting that the
   shrimp tissues were damaged by oxidative stress, while the results of
   the gill in treated vs. control groups indicated that there was no
   significant difference (p > 0.05). The increase in antioxidant enzyme
   activities, such as SOD, CAT, and MDA contents, suggested that the
   increased SOD and CAT activities were not sufficient for protection
   against stress-induced damage over a long period of time. It is
   commonly observed in many aquatic animals, such as rainbow trout
   (Oncorhynchus mykiss) [[150]45], mangrove crab [[151]46], and Pinctada
   fucata [[152]47], that Na^+/K^+-ATPase (NKA) plays a significant role
   in the process of bronchial ammonia excretion. In the study, NKA
   activity in the two tissues significantly increased between the treated
   and control groups (p < 0.05). The results indicated that NKA
   participates in hepatopancreas- and gill-mediated NH4^+ transport and
   that the latter is more sensitive to ammonia-N stimulation than the
   former. Similarly, the effects of chronic ammonia–N stress on juvenile
   ivory shell snails, Babylonia areolata, showed the same result
   [[153]9]. Moreover, the histopathological variation in the two tissues
   showed trends of degeneration and necrosis, suggesting that chronic
   ammonia-N exposure may lead to tissue damage without recovery in L.
   vannamei.

4.3. Comparison Analysis between the Two Tissues

   Transcriptome sequencing is an important tool for studying the
   mechanisms of growth, metabolism, immunity, etc. in non-model oganisms
   [[154]48]. To better understand the molecular mechanism of chronic
   ammonia-N exposure, we carried out a comparative transcriptomics
   analysis between the hepatopancreas and gill groups. In the two groups,
   62 overlapping DEGs were identified, indicating that the response to
   chronic ammonia-N stress varies significantly across the two tissues.

   In the hepatopancreas in treated vs. control groups, DEGs were enriched
   in nine KEGG pathways, indicating that ammonia-N stress could affect
   carbohydrate metabolism, glycan biosynthesis, metabolism, and the
   immune system in the hepatopancreas of L. vannamei. Cellular stress and
   mutations in genes encode important factors and lead to reduced or
   abnormal ribosome production, which activates the tumor suppressor p53
   and then reprograms cellular transcription [[155]49]. Orsolic et al.
   reported that oxidative stress affects ribosome biogenesis [[156]50].
   The ribosome pathway was significant in this study (p < 0.05); most of
   the genes in it were down-regulated. In addition, dismutase (SOD) and
   catalase (CAT) are antioxidants that belong to the first line of
   defense antioxidants [[157]51]. However, the study found that CAT was
   significantly up-regulated in the hepatopancreas in the treated vs.
   control groups (p < 0.05), whereas there was no significant difference
   in the gill in the treated vs. control groups (p > 0.05).

   We speculate that the chronic ammonia-N stress may induce the oxidative
   stress and produce excess ROS and then restrain related genes’
   expression. Moreover, the hepatopancreas was more sensitive to
   oxidative stress than the gills were. Proteasome and spliceosome
   pathways were also down-regulated after chronic ammonia-N stress
   ([158]Table 4), which influenced the degradation of redundant and
   superfluous protein and metal RNA splices. The pentose and glucuronate
   interconversions pathway is involved in carbohydrate metabolism
   [[159]52] and significantly enriched DEGs are related to detoxification
   [[160]53]. This pathway is enriched after different types of stress,
   such as salinity stress [[161]53], Zn exposure [[162]33], and exposure
   to phoxim and prometryne residues in shrimp [[163]54]. These pathways
   were significantly changed after ammonia-N exposure, indicating that it
   impacted protein biosynthesis and metabolism.

   In the gill in treated vs. control groups, three KEGG pathways
   (mucin-type O-glycan biosynthesis, nucleocytoplasmic transport, and
   steroid biosynthesis) were significantly enriched and increased,
   indicating that ammonia-N stress could affect cellular homeostasis,
   biological processes, and protein function and structure in the gills
   of L. vannamei. The nuclear pore complex (NPC) is a kind of nuclear
   macro-complex that mediates nucleocytoplasmic transport (NCT). NPC
   plays an essential role in maintaining cellular homeostasis and
   influences transcription through interactions with chromatin. The
   depletion of NPC may lead to defects in mRNA transcription or nuclear
   output [[164]55]. Morgan et al. reported that kras (G12V)-driven
   hepatocyte proliferation was prevented when the NCT inhibitor was
   administered to heterozygous ahctf1 larvae [[165]56]. Some genes
   involved in NCT under ammonia-N stress conditions were up-regulated
   (kpnb3, kidins220a, nup58, ran, and ipo9) in this study ([166]Table 4),
   which meant that the stress could increase gene transcription and
   protect cellular homeostasis. A previous study confirmed that ipo9
   coordinates the nuclear import of functionally related factors required
   for gametogenesis [[167]57]. The ran overexpression significantly
   increased cellular tolerance to a variety of pesticides [[168]58]. In
   addition, ran relocalization and nuclear transport were affected by
   stress treatment in animal cells [[169]59]. Moreover, the expression of
   cytoskeleton-associated genes in the two tissues groups have
   significantly changed expression (p < 0.05). All genes were
   up-regulated in gill in treated vs. control groups in particular
   ([170]Table 3). A previous study reported that the changes in
   cytoskeleton-associated genes were mostly likely to protect shrimp from
   ammonia-N exposure [[171]60]. Additionally, the Na^+/K^+-ATPase,
   located on the basolateral membrane of gill cells, was associated with
   ammonia-N excretion in crustaceans, where K^+ can be replaced by NH4^+,
   which is excreted into the environment by the Na^+/NH4^+ exchanger
   [[172]61]. Combining the findings with the MDA concentration and
   structural change, we hypothesize that gills adapted to chronic
   ammonia-N stress by excreting and activating relevant
   cytoskeleton/transcription gene expression to maintain cellular
   homeostasis. These results suggested that various tissues used
   different strategies or cooperated with each other in response to
   ammonia-N stress.

4.4. DEGs Involved in Lipids Metabolism and Serine Proteases

   Cellular protection and detoxification pathways could enhance through
   environmental stress, leading to metabolic processes that require more
   energy consumption in aquatic animals [[173]15]. In general, there are
   three indicators—protein, lipid, and glycogen reserves—for assessing
   the metabolic status of animals. Among these, protein and lipid
   contents are the two important indicators of evaluating temporal
   changes in available energy reserves in aquatic animals under stress
   [[174]62]. However, three pathways involved in protein synthesis were
   down-regulated in the study, suggesting that chronic ammonia-N exposure
   affected the metabolic status of shrimp. Moreover, DEGs enriched in
   KEGG terms for ether lipid metabolism, sphingolipid metabolism,
   glycerophospholipid metabolism, and glycerolipid metabolism were found
   in both transcriptome analyses, with the expression of most genes
   showing an increasing trend ([175]Table 5). When they are subjected and
   adapted to salinity stress, lipids are the energy source for fish
   [[176]63]. Ammonia-N exposure may also cause oxidation and endoplasmic
   reticulum stress, leading to increased lipid synthesis and reduced
   lipid breakdown [[177]64]. Therefore, we speculated that lipids were
   used to offer energy via metabolic pathways in response to ammonia-N
   stress. Additionally, serine-type endopeptidase activity,
   oxidoreductase activity, hydrolase activity, and serine hydrolase
   activity were GO terms enriched in DEGs from the two groups after
   chronic ammonia-N stress ([178]Table 2). Sun et al. reported that they
   were connected to innate immunity in aquatic animals [[179]65].
   Interestingly, ct55 is a novel and effective promoter of
   colitis-associated cancer (CAC) whose down-regulation indicates reduced
   inflammatory responses [[180]66].

5. Conclusions

   In this study, a comparative analysis between the hepatopancreas and
   gills of shrimp was performed to determine differences in growth
   performance, morphological and physiological alterations, and
   transcriptional responses under long-term ammonia-N exposure to probe
   the adaptation mechanism in L. vannamei. Chronic ammonia-N exposure
   significantly reduced growth, caused histological alterations, and
   changed the enzymatic activities in the two tissues. DEGs in the two
   tissues were enriched in pathways/genes associated with oxidative
   stress, protein synthesis, lipid metabolism, and different serine
   proteases. Moreover, the shrimp may offset ammonia-N toxicity at the
   transcriptome level by regulating gene expression. These results
   provided a new perspective in understanding the mechanisms of ammonia-N
   resistance in crustaceans.

Supplementary Materials

   The following supporting information can be downloaded at:
   [181]https://www.mdpi.com/article/10.3390/ani13243799/s1, Table S1:
   Range of water quality-related indicators; Table S2: Survival time of
   the shrimp; Table S3: Primer sequences used in this study; Table S4:
   Assembly statistic of L. vannamei transcriptome; Table S5: Compare
   statistics with reference genome.
   [182]Click here for additional data file.^ (170.7KB, zip)

Author Contributions

   Y.Z.: conceptualization, formal analysis, investigation, validation,
   writing original draft, writing review, and editing. J.L. (Jianyong
   Liu): conceptualization, resources, supervision, project
   administration. H.Z.: conceptualization, supervision. L.L.: proposed
   changes. J.L. (Jinyan Li): sample collection. S.F.: proposed changes.
   H.X.: experimental record and observation. H.W.: experimental record
   and observation. X.Z.: proposed changes. C.G.: feeding. G.W.: feeding.
   All authors have read and agreed to the published version of the
   manuscript.

Institutional Review Board Statement

   The animal study protocol was approved by the Animal Care and Use
   Committee of Guangdong Ocean University (GDOU-IACUC-2022-A1457, 15
   February 2022).

Informed Consent Statement

   Not applicable.

Data Availability Statement

   Data will be made available on request.

Conflicts of Interest

   The authors declare that they have no known competing financial
   interests or personal relationships that could have appeared to
   influence the work reported in this paper.

Funding Statement

   This work was supported by the open competition program of top ten
   critical priorities of Agricultural Science and Technology Innovation
   for the 14th Five-Year Plan of Guangdong Province (No. 2022SDZG01), the
   Construction Project of Modern Seed Industry Park for Whiteleg Shrimp
   of Guangdong Province (No. GDSCYY2022-005), the Special Funds for
   Science and Technology Innovation Strategy of Guangdong Province in
   2022 (No. 2022A05023), and the Special Fund for Focus Areas of General
   Universities in Guangdong Province (No. 2021ZDZX4042).

Footnotes

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References