Elsevier

Fish & Shellfish Immunology

Volume 87, April 2019, Pages 534-545
Fish & Shellfish Immunology

Full length article
Proteomic and metabolomic responses in hepatopancreas of whiteleg shrimp Litopenaeus vannamei infected by microsporidian Enterocytozoon hepatopenaei

https://doi.org/10.1016/j.fsi.2019.01.051Get rights and content

Highlights

  • 266 differently expressed proteins and 49 differential metabolites were identified.

  • 7 immunologic proteins were verified for their immune role by Real-time PCR.

  • The change of growth associated proteins might prevent the shrimp from molting.

  • The energy metabolism pathway was down-regulated in the shrimp hepatopancreas.

Abstract

Enterocytozoon hepatopenaei (EHP) causes hepatopancreatic microsporidiosis (HPM) in shrimp. HPM is not normally associated with shrimp mortality, but is associated with significant growth retardation. In this study, the responses induced by EHP were investigated in hepatopancreas of shrimp Litopenaeus vannamei using proteomics and metabolomics. Among differential proteins identified, several (e.g., peritrophin-44-like protein, alpha2 macroglobulin isoform 2, prophenoloxidase-activating enzymes, ferritin, Rab11A and cathepsin C) were related to pathogen infection and host immunity. Other proteomic biomarkers (i.e., farnesoic acid o-methyltransferase, juvenile hormone esterase-like carboxylesterase 1 and ecdysteroid-regulated protein) resulted in a growth hormone disorder that prevented the shrimp from molting. Both proteomic KEGG pathway (e.g., “Glycolysis/gluconeogenesis” and “Glyoxylate and dicarboxylate metabolism”) and metabolomic KEGG pathway (e.g., “Galactose metabolism” and “Biosynthesis of unsaturated fatty acids”) data indicated that energy metabolism pathway was down-regulated in the hepatopancreas when infected by EHP. More importantly, the changes of hormone regulation and energy metabolism could provide much-needed insight into the underlying mechanisms of stunted growth in shrimp after EHP infection. Altogether, this study demonstrated that proteomics and metabolomics could provide an insightful view into the effects of microsporidial infection in the shrimp L. vannamei.

Introduction

Microsporidia are small obligate intracellular parasites and recently reclassified with the fungi and not to be protists [1,2]. Almost half of the reported genera of microsporidia infect aquatic hosts, and usually has chronic and sublethal effects on hosts [3,4]. However, microsporidia pathogenesis are vastly under-reported in aquatic systems. Hepatopancreatic microsporidiosis (HPM), one of the serious epidemics in Litopenaeus vannamei, is caused by Enterocytozoon hepatopenaei (EHP) and is currently dramatically increasing the economic losses in the shrimp harvest in Southeast Asia [[5], [6], [7]]. EHP was reported for the first time in pond-reared Penaeus monodon in Thailand [8] and later in Vietnam, China, Indonesia, Malaysia and India [5,9]. As other microsporidia, the sign of stunted growth was observed in EHP-infected shrimp. Further research revealed that EHP mainly infect epithelial cells of the hepatopancreas of wild and farmed decapod crustaceans [8]. In addition, the research of microsporidian intensively focused on the pathogen aspects including the molecular phylogenetic analysis, development of novel detection assays, histopathology and comparative genome research [6,7,10]. However, limited information was available about the host responses, which could provide important information to elucidate the diverse aspects of the host-pathogen interactions.

It is well known that host's immune response can be activated when pathogenic microorganism invading host. Invertebrate innate immune response plays a dominant role in protecting hosts from invading pathogens because of lack of adaptive immunity [11]. The innate defense system of invertebrates is composed of cellular and humoral immunity response mechanisms that include enzymes and proteins in the prophenoloxidase (proPO) activation and blood coagulation systems [12]. So EHP may cause the activation of proPO or other immune system in L. vannamei. In addition, as obligate intracellular parasites, microsporidians are highly dependent on their hosts, and have an expanded repertoire of transport proteins to exploit the rich environment in the host cell cytoplasm [10]. This feature suggests that microsporidia have a potential to change host nutritional and metabolic pathways. How EHP parasitization produces the metabolic stresses in the shrimp L. vannamei is still unknown. To study the change of host immune response and metabolic pathway, many traditional approaches have been applied [[13], [14], [15]]. However, these researches obviously presented a primary understanding of responses of L. vannamei to EHP challenges.

With the emergence and development of systems biology techniques, including genomics, transcriptomics, proteomics and metabolomics, new opportunities are offered with great potential in unraveling biological problems, and have been successfully employed in multiple areas such as environmental sciences, toxicological effects and immunology [[16], [17], [18]]. Among these approaches, proteomics is a means of comprehensive interpretation, which can be used to describe more direct molecular responses than conventional transcriptomic or genomics [19]. As a kind of highly sensitive proteomic platform, the isobaric label tandem mass tags (TMT) was used to identify differential proteome frequently [20,21]. So far, many studies have reported about using proteomic techniques to study the response of invertebrates against various pathogenic microorganisms [14,19,20]. In addition, metabolomics usually focuses on the whole set of low molecular weight (<1000 Da) metabolites. The metabolites represent the collection of all metabolites in organs, tissues, biofluids, or even whole organisms, which are the end products of various biological systems [22]. A comparative analysis of metabolomes can give significant evidence in interpreting metabolite perturbations in response to exogenous factors affecting metabolism of organisms as well as proteomics [23,24]. These perturbed metabolites and proteins are a definite set of molecular biomarkers related to biological effects of stressors [17,18]. Therefore, a combination of proteomics and metabolomics can yield a better understanding of the biological responses that an organism makes to environmental stressors [25]. To date, no attempt has been made to test the responses induced by EHP in L. vannamei using a combined proteomic and metabolomic approach.

In this study, EHP infection in L. vannamei with stunted growth was confirmed using PCR assay firstly. Secondly, the proteomic and metabolomic profiling were applied to show a global survey of differentially identified proteins and metabolites between EHP-infected and healthy shrimp. Thirdly, the patterns of some differential proteins were further assessed by qRT-PCR in EHP natural infections. It was found that not only the innate immune system of L. vannamei was activated, but also the growth hormone and energy metabolism of L. vannamei were disturbed by EHP though these studies. These results could help us to better understand the immune relationship between L. vannamei and EHP and molecular mechanisms of stunted growth of shrimps in EHP infection.

Section snippets

PCR assay for detecting EHP from shrimp samples

L. vannamei were obtained from commercial shrimp ponds in Nantong, Jiangsu Province, China. DNA extractions of hepatopancreas from healthy shrimps and slow-growing shrimps infected by EHP were prepared with the EasyPure Genomic DNA Kit (Transgen, China). Templates were tested further for concentration and purity with a spectrophotometer (Thermo, USA). The primers for detecting the EHP are EHP-510F and EHP-510R (Table S1). The PCR amplification was carried out in a 25-μL reaction mixture, which

The detection of EHP infection in shrimp

There was no specific clinical sign associated with EHP-infected shrimp, except stunted growth without distinctly increased mortality. The size of the stunted shrimp was nearly half the size of non-infected shrimp at a particular age (Fig. S1). The primers EHP-510F/R of the 18S rRNA gene [9] from the EHP genome were selected to carry out PCR using DNA extracted from stunted growth and healthy shrimps. As shown in Fig. 1A, the PCR results revealed the appearance of an obvious band at the

Discussion

HPM caused by EHP is a very serious disease of L. vannamei in aquaculture. No significant mortality was observed on all occasions of EHP outbreak, but stunted growth was observed in most EHP-positive farms [7,32]. Because the study of HPM has been very limited, little was known about the molecular mechanism of the immune responses and the stunted growth of L. vannamei infection by EHP. With advanced analytical techniques, proteomic and metabolomic analyses have become techniques used in

Acknowledgments

We thank Professor O. Roger Anderson of Columbia University in the City of New York for editing the manuscript. The current study was supported by grants from the National Natural Sciences Foundation of China (NSFC Nos. 31570176; 31602198; 31870168), Project of the fishery science and technology innovation program (Grant Nos. Y2016-28; Y2017-20; Y2017-34), the Natural Science Foundation of Jiangsu Province (Grant No. BK20151545), Opening Foundation of Jiangsu Provincial Key Construction

References (64)

  • Z. Fu et al.

    Comparative proteomic analysis of the sun- and freeze-dried earthworm Eisenia fetida with differentially thrombolytic activities

    J. Proteomics.

    (2013)
  • V. García-Hernández et al.

    A tandem mass tag (tmt) proteomic analysis during the early phase of experimental pancreatitis reveals new insights in the disease pathogenesis

    J. Proteomics.

    (2018)
  • M.S. Pedras et al.

    Metabolic responses of Thellungiella halophila/salsuginea to biotic and abiotic stresses: metabolite profiles and quantitative analyses

    Phytochemistry

    (2010)
  • H. Wu et al.

    Comparison of metabolic profiles from serum from hepatotoxin-treated rats by nuclear-magnetic-resonance-spectroscopy-based metabonomic analysis

    Anal. Biochem.

    (2005)
  • Y.P. Liu et al.

    Establishment and application of pcr assay for detection of vibrio spp

    Freshw. Fish.

    (2015)
  • M. Dashty

    A quick look at biochemistry: carbohydrate metabolism

    Clin. Biochem.

    (2013)
  • M.T. Nakamura et al.

    Regulation of energy metabolism by long-chain fatty acids

    Prog. Lipid Res.

    (2014)
  • D.P. Marancik et al.

    Proteomic characterization of the acute-phase response of yellow stingrays Urobatis jamaicensis after injection with a Vibrio anguillarum-ordalii bacterin

    Fish Shellfish Immunol.

    (2013)
  • R.L. Tellam et al.

    Peritrophic matrix proteins

    Insect. Biochem. Molec.

    (1999)
  • C.M. Elvin et al.

    Characterization of a major peritrophic membrane protein, peritrophin-44, from the larvae of Lucilia cuprina

    J. Biol. Chem.

    (1996)
  • Y. Huang et al.

    Identification and molecular characterization of a peritrophin-like gene, involved in the antibacterial response in Chinese mitten crab, Eriocheir sinensis

    Dev. Comp. Immunol.

    (2015)
  • P.B. Armstrong et al.

    Humoral immunity in long-lived arthropods

    J. Insect Physiol.

    (1996)
  • W. Liu et al.

    Proteomic analysis of differentially expressed proteins in hemolymph of Scylla serrata response to white spot syndrome virus infection

    Aquaculture

    (2011)
  • S.C. Szumowski et al.

    Microsporidia-host interactions

    Curr. Opin. Microbiol.

    (2015)
  • Y.J. Yue et al.

    Early responses of silkworm midgut to microsporidium infection - a Digital Gene Expression analysis

    J. Invertebr. Pathol.

    (2015)
  • S.C. Andrews et al.

    Structure, function, and evolution of ferritins

    J. Inorg. Biochem.

    (1992)
  • Q. Meng et al.

    iTRAQ-based proteomic study of the effects of Spiroplasma eriocheiris on Chinese mitten crab Eriocheir sinensis hemocytes

    Fish Shellfish Immunol.

    (2014)
  • L. Wang et al.

    Two Rab GTPases, EsRab-1 and EsRab-3, involved in anti-bacterial response of Chinese mitten crab Eriocheir sinensis

    Fish Shellfish Immunol.

    (2013)
  • W. Wu et al.

    Characterization of a Rab GTPase up-regulated in the shrimp Peneaus japonicus by virus infection

    Fish Shellfish Immunol.

    (2007)
  • W.W. Li et al.

    Molecular cloning, characterization and expression analysis of cathepsin A gene in Chinese mitten crab

    Eriocheir sinensis, Peptides

    (2011)
  • W.W. Li et al.

    Molecular cloning, characterization, expression and activity analysis of cathepsin L in Chinese mitten crab, Eriocheir sinensis

    Fish Shellfish Immunol.

    (2010)
  • K.C. Holford et al.

    Purification and characterization of a mandibular organ protein from the American lobster, Homarus americanus: a putative farnesoic acid O-methyltransferase

    Insect. Biochem. Molec.

    (2004)
  • Cited by (78)

    View all citing articles on Scopus
    View full text