Elsevier

Chemosphere

Volume 239, January 2020, 124759
Chemosphere

Effects of ammonia stress on the hemocytes of the Pacific white shrimp Litopenaeus vannamei

https://doi.org/10.1016/j.chemosphere.2019.124759Get rights and content

Highlights

  • Transcriptomics and metabolomics approaches were used to analyze the effects of ammonia stress on shrimp hemocytes.

  • The characteristics of differentially expressed genes in hemocytes under ammonia stress were analyzed.

  • Ammonia stress led to disorder of amino acid metabolism, nucleotide metabolism and lipid metabolites.

  • High concentrations of hemolymph ammonia could induce apoptosis of hemocytes and decrease the hemocytes count.

Abstract

Ammonia is an important environmental stress factor in aquaculture. Long-term ammonia stress could affect the normal growth, and also increase the risk for the occurrence of various diseases. In order to learn the mechanism that ammonia caused the outbreak of the shrimp disease, transcriptomics and metabolomics approaches were used to analyze the differential expressions of the genes in hemocytes and different metabolites in the serum of the Pacific white shrimp Litopenaeus vannamei under ammonia exposure. Transcriptional analysis showed that 17 cell apoptosis related genes, seven phagocytosis related genes, 10 immunity related genes and seven cell cycle and lipid metabolism related genes showed differential expressions after ammonia exposure. Metabolomics analysis on the serum showed that 25 differential metabolites were identified in positive and negative ion patterns. They are involved in purine metabolism, amino acids metabolism and lipid metabolism. Injection of two up-regulated metabolites triethanolamine and oxypurinol to normal shrimp could induce apoptosis in normal shrimp. The total hemocytes counts in shrimp showed a significant decrease and the apoptotic cell ratio increased significantly under ammonia exposure. These results suggested that ammonia exposure increased the apoptosis of hemocytes, which affected the immunity of shrimp, and thus caused susceptibility to pathogenic infection. These data will help us understand the mechanism of ammonia stress leading to the immunity decline of shrimp.

Introduction

The Pacific white shrimp Litopenaeus vannamei is an important shrimp species in aquaculture. It is most widely cultured throughout the world due to its rapid growth, disease tolerance, adaptability to high-density culture (Cuzon et al., 2004; Kim et al., 2014). However, with the expansion of cultivation scale and the promotion of ultrahigh-density culture mode, aquaculture environment begins to deteriorate, and the diseases occur more frequently, which seriously affect the production and quality of L. vannamei (Alagappan et al., 2010; Cuzon et al., 2004). Disease outbreak of shrimp is usually the result of interplay among pathogen, host and environment (Kautsky et al., 2000). Ammonia is a primary environmental factor in the aquatic culturing system that poses a great threat to the survival of shrimp (Cobo et al., 2014).

In aquatic environments, ammonia originates from excess feed, excrement of the animals, decomposing organic solids, the surplus ammonia produced in the surrounding agricultural production which is brought into the aquaculture pool through rain or wind (Kuhn et al., 2010). Ammonia is usually present in ionized (NH4+) and un-ionized (NH3) states in water (Emerson et al., 1975; Fromm and Gillette, 1968), while NH3 diffuses easily across cell membrane and into the hemolymph. The ratio of NH4+/NH3 can be affected by pH, temperature and salinity (Chen and Chin, 1989; Kir et al., 2004; Lin and Chen, 2001). Studies showed that a high concentration of ammonia could reduce the growth of shrimp, increase the frequency of molting, and lead to high mortality (Chen and Lin, 1992; Wickins, 1976). In addition, ammonia can also affect osmoregulatory capacity, physiological state of the gills and hepatopancreas (Lin et al., 1993; Lu et al., 2016). Excessive ammonia concentration in aquaculture water could inhibit the immune system of shrimp and increase the susceptibility of shrimp to pathogens. L. vannamei is more susceptible to Vibrio infection under ammonia stress (Liu and Chen, 2004; Qiu et al., 2008). Therefore, it is valuable to systematically understand the molecular mechanism of L. vannamei responsive to ammonia stress. In the present study, metabolomics was used to investigate the changes in serum metabolites of L. vannamei under ammonia stress, and transcriptome were used to analyze the characteristics of the gene expression of hemocytes under ammonia stress.

Section snippets

Animals

Healthy Pacific white shrimp L. vannamei cultured in the aquarium of Institute of Oceanology, Chinese Academy of Sciences, Qingdao, with an average weight of 16.7 g ± 5.8 g, were used for the ammonia exposure experiments. They were acclimated at 25 ± 1 °C with the salinity of 30‰ and pH 7.5 ± 0.1 for 3 days, prior to the experiments. All shrimp were fed twice daily at 8:00 a.m. and 17:00 p.m. with commercial food pellets (Dale Feed Company, Yantai, China).

Ammonia exposure and samples collection

The stock solution of ammonia-N (10 gL-1

Reads assembly and sequence annotation

An overview of sequence information of the transcriptome was listed in Table 2. Totally 53,654,414 to 64,596,884 clean reads were obtained for each sample, 82.84%–86.82% were matched in comparison with the reference genome. All clean reads were assembled into 61,283 unigenes, GC percentage was 40.92%, N50 was 1308 bp, and the average length was 766 bp. All unigenes were BLASTX-searched against four databases. Totally 12,426 unigenes were matched to known proteins in the NR database, 9,715

Discussions

Ammonia is an important environmental stress factor in aquaculture. Hemocytes are the most important immunocytes in crustaceans (Vazquez et al., 2009). They play important roles in immune responses, such as phagocytosis, coagulation, encapsulation, nodule formation, melanization, production of antimicrobial peptides (AMPs), etc., to fight against the invasion of pathogens (Söderhäll, 2016; Tassanakajon et al., 2013). It was reported that total hemocytes count could be decreased in several

Conclusion

In conclusion, ammonia stress led to disorder of amino acid metabolism and nucleotide metabolism, which might be attributed to ammonia-induced apoptosis and functional damage of hepatopancreas. The decline of amino acid levels in serum affected the normal growth of muscles and ultimately led to slow growth of shrimp. High concentrations of hemolymph ammonia and abnormal levels of metabolites could induce apoptosis of hemocytes, decrease the hemocytes count, and ultimately reduce the immunity of

Acknowledgements

This work was financially supported by National Natural Science Foundation of China (31830100, 41776158, and 31772880) and China Agriculture Research system-48 (CARS-48).

References (60)

  • M. Kir et al.

    Effects of temperature on acute toxicity of ammonia to Penaeus semisulcatus juveniles

    Aquaculture

    (2004)
  • R.L. Konger et al.

    Oxidized glycerophosphocholines as biologically active mediators for ultraviolet radiation-mediated effects

    Prostaglandins Other Lipid Mediat.

    (2008)
  • D.D. Kuhn et al.

    Evaluation of nitrifying bacteria product to improve nitrification efficacy in recirculating aquaculture systems

    Aquacult. Eng.

    (2010)
  • Z. Liang et al.

    Ammonia exposure induces oxidative stress, endoplasmic reticulum stress and apoptosis in hepatopancreas of pacific white shrimp (Litopenaeus vannamei)

    Fish Shellfish Immunol.

    (2016)
  • Y. Lin et al.

    Acute toxicity of ammonia on Litopenaeus vannamei Boone juveniles at different salinity levels

    J. Exp. Mar. Biol. Ecol.

    (2001)
  • C.H. Liu et al.

    Effect of ammonia on the immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus

    Fish Shellfish Immunol.

    (2004)
  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2 −ΔΔ C T method

    Methods

    (2001)
  • T. Nagano et al.

    X-linked inhibitor of apoptosis protein mediates neddylation by itself but does not function as a NEDD8–E3 ligase for caspase-7

    FEBS Lett.

    (2012)
  • M.R. Pinto et al.

    Effects of ammonia stress in the Amazon river shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae)

    Aquat. Toxicol.

    (2016)
  • T. Rodríguez-Ramos et al.

    Effects of Echerichia coli lipopolysaccharides and dissolved ammonia on immune response in southern white shrimp Litopenaeus schmitti

    Aquaculture

    (2008)
  • L. Romagnoli et al.

    Early growth response-1 protein is induced by JC virus infection and binds and regulates the JC virus promoter

    Virology

    (2008)
  • I. Söderhäll

    Crustacean hematopoiesis

    Dev. Comp. Immunol.

    (2016)
  • A. Tassanakajon et al.

    Discovery of immune molecules and their crucial functions in shrimp immunity

    Fish Shellfish Immunol.

    (2013)
  • B. Verghese et al.

    Effect of environmental parameters on immune response of the Indian spiny lobster, Panulirus homarus (Linnaeus, 1758)

    Fish Shellfish Immunol.

    (2007)
  • Z. Wang et al.

    Characterization of two types of vascular endothelial growth factor from Litopenaeus vannamei and their involvements during WSSV infection

    Fish Shellfish Immunol.

    (2015)
  • D. Warnakulasuriyarachchi et al.

    Translational induction of the inhibitor of apoptosis protein HIAP2 during endoplasmic reticulum stress attenuates cell death and is mediated via an inducible internal ribosome entry site element

    J. Biol. Chem.

    (2004)
  • J.F. Wickins

    The tolerance of warm-water prawns to recirculated water

    Aquaculture

    (1976)
  • J. Xiao et al.

    Stress response and tolerance mechanisms of ammonia exposure based on transcriptomics and metabolomics in Litopenaeus vannamei

    Ecotoxicol. Environ. Saf.

    (2019)
  • K.M. Alagappan et al.

    Occurrence of Vibrio parahaemolyticus and its specific phages from shrimp ponds in east coast of India

    Curr. Microbiol.

    (2010)
  • J. Chen et al.

    Effect of ammonia at different pH levels on Penaeus monodon postlarvae

    Asian Fish Sci.

    (1989)
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