Effects of ammonia stress on the hemocytes of the Pacific white shrimp Litopenaeus vannamei
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).
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These authors contributed equally to this paper.