Short communication
Molecular characterization of a heat shock protein 21 (Hsp21) from red swamp crayfish, Procambarus clarkii in response to immune stimulation

https://doi.org/10.1016/j.dci.2020.103755Get rights and content

Highlights

  • A Heat shock protein 21 from Procambarus clarkii (Pc-Hsp21) was identified and characterized for the first time.

  • Transcripts and expression levels of Pc-Hsp21 in the hepatopancreas were detected after PGN, LPS and Poly I:C challenge.

  • RNA interference of Pc-Hsp21 influenced the expression of immune-related genes.

Abstract

Small heat shock proteins are a molecular chaperone and implicated in various physiological and stress processes in animals. However, the immunological functions of Hsp genes remain to elucidate in the crustaceans, particularly in red swamp crayfish, Procambarus clarkii. Here we report the cloning of heat shock protein 21 from the P. clarkii (hereafter Pc-Hsp21). The open reading frame of Pc-Hsp21 was 555 base pairs, encoding a protein of 184 amino acid residues with an alpha-crystallin family domain. Quantitative real-time PCR (qRT-PCR) analysis revealed a constitutive transcript expression of Pc-Hsp21 in the tested tissue, with the highest in hepatopancreas. The transcript abundance for this gene enhanced in hepatopancreas following immune challenge with the lipopolysaccharide, peptidoglycan, and poly I:C compared to the control group. The depletion of Pc-Hsp21 by double-stranded RNA altered transcript expression profiles of several genes in hepatopancreas, genes involved in the crucial immunological pathways of P. clarkii. These results suggest that Pc-Hsp21 plays an essential biological role in the microbial stress response by modulating the expression of immune-related genes in P. clarkii.

Introduction

The Red swamp crayfish, Procambarus clarkii, is one of the major farmed crustacean species, and is widely cultured and exported across the world (Söderhäll et al., 2005; Liu et al., 2019; Sirikharin et al., 2020). The crayfish is highly delicious that contains all the essential nutrients required for the human body. However, crayfish farming and production have severely reduced due to microbial infection, such as viruses and bacteria (Wu et al., 2017; Dai et al., 2018; Hernández-Pérez et al., 2020). Aeromonas species (vibriosis) infection can profoundly reduce the production of crayfish and is a severe threat to the aquaculture industry. Besides, the outbreak of white spot syndrome and other diseases also affected the farm production of crayfish. Although considerable progress has been made to characterize crayfish immune response against microbial pathogens (Dai et al., 2017; Cerenius and Soderhall, 2018;Sirikharin et al., 2018), there is still no therapeutic strategies available to effectively control microbial diseases in the field. Therefore, more understanding of the crayfish defense system against microbial infection remains to be elucidated.

Heat shock proteins have shown to implicate in the innate immunity of animals. They are classified into different groups based on their sequence information and molecular weight: Hsp90, Hsp70, Hsp60, Hsp40, and small Hsps (Lindquist and Craig, 1988; Wu et al., 2017). In animals, Hsps are produced in response to different environmental stress factors, including microbial pathogens, heavy metals, drought, extreme temperatures, osmotic stresses, UV radiation, and oxidation (Franck et al., 2004; Haslbeck et al., 2005; Abbas et al., 2020a). Over-expression of Hsps has been generally used as an indicator of invertebrate tolerance to thermal stress (Hu et al., 2014; Kausar et al., 2017).

Small Hsps have an extreme difference in structure and function among molecular chaperones, with molecular size ranging from 12 to 43 kDa and a conserved α-crystallin domain (Franck et al., 2004; Korkut et al., 2018). They involve in the precise folding of newly produced proteins and prevent functional proteins from aggregation and denaturation stimulated by different stresses (Basha et al., 2012; King and Macrae, 2015; Zhou et al., 2017). Furthermore, sHsps play multiple biological roles, as they are involved in the developmental processes, heat tolerance, and also regulate innate immune responses (Gehring and Wehner, 1995; Abbas et al., 2020b). For example, sHsp proteins, in many species of invertebrates (e.g., Chilo suppressalis, Choristoneura fumiferana, Frankliniella occidentalis, and Bactrocera dorsalis) have shown to upregulate under thermal stress (Wang et al., 2014; Quan et al., 2017; Dou et al., 2017). sHSPs are also developmentally regulated in many insects (Rinehart et al., 2007). sHsps in Drosophila is highly produced during gametogenesis and embryonic development (Joanisse et al., 1998). Some sHsps in B. dorsalis, Bombyx mori, and Spodoptera litura is induced by 20-hydroxyecdysone exposure, implying their involvement in metamorphosis (Shen et al., 2011; Gu et al., 2012). Additionally, some sHsps from various invertebrates (insects, crustaceans) have shown to involve in the innate immune responses (Liu et al., 2018; Junprung et al., 2019; Li et al., 2020).

In the present study, gene expression patterns and immune functions were analyzed to understand the effect of microbial infection on P. clarkii. Firstly, the cDNA sequence of heat shock protein 21 in P. clarkii (named as Pc-Hsp21) was cloned and characterized. The tissue expression profiles of the Pc-Hsp21 was clarified. The effects of immune elicitors (e.g., LPS, PGN, and poly I:C) on the expression profiles of Pc-Hsp21 was also elaborated. Besides, spatial and temporal expression patterns, the immunoregulatory role was determined by silencing Pc-Hsp21. This study provided a relatively comprehensive understanding of the microbial stress response of P. clarkii at molecular levels and assessed the potential involvement of Pc-Hsp21 in the immune functions.

Section snippets

Studied animal

In the present study, healthy red swamp crayfish (P. clarkii) was purchased from a local market in Zhejiang, China. They were cultured, as described in our previous reports (Chu et al., 2019; Zhu et al., 2019), and used as a subject to perform experiments.

Cloning of Pc-Hsp21

From the hepatopancreas of P. clarkii, total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and then reverse-transcribed using the TransScript Synthesis SuperMix (TransGen, Beijing, China). The cDNA was diluted to

Isolation of Pc-Hsp21 from P. clarkii

Based on the genomic sequence library that is constructed in our laboratory, Pc-Hsp21 cDNA was cloned by polymerase chain reaction (PCR), and the amplified PCR product was confirmed by gel electrophoresis and nucleotide sequencing. The open reading frame (ORF) of Pc-Hsp21 (Supplementary Fig. 1), comprising 555 bp nucleotides encoding 184 amino acid residues. The molecular weights and isoelectric points of Pc-Hsp21 protein were predicted to be 20.99 kDa, and 5.35. The deduced amino acid sequence

Discussion

In animals, the enhancement in the production of Hsps is one of the protective strategies that protect the cells upon rapid variations histological, physiological changes, and biochemical events. The Hsps are also implicated in immune functions, including apoptotic pathways, inflammatory processes, etc. (Yuan et al., 2017; Junkunlo et al., 2018). The Hsp proteins were initially described in the early 1960s by the pioneering work of Ferruccio Ritossa on the Drosophila melanogaster (Ritossa, 1964

Declaration of competing interest

The authors have declared that no competing interests exist.

Acknowledgement

This work was supported by the Natural Science Foundation of Zhejiang Province, China (No. LQ20C190009), the scientific and technological innovation special project for seed and seedling of Wenzhou Science & Technology Bureau (Z20170014).

References (57)

  • Y. Hong et al.

    Effects of deltamethrin on the antioxidant defense and heat shock protein expression in Chinese mitten crab, Eriocheir sinensis

    Environ. Toxicol. Pharmacol.

    (2019)
  • J.T. Hu et al.

    Thermal plasticity is related to the hardening response of heat shock protein expression in two Bactrocera fruit flies

    J. Insect Physiol.

    (2014)
  • P. Jiravanichpaisal et al.

    A highly virulent pathogen, Aeromonas hydrophila, from the freshwater crayfish Pacifastacus leniusculus

    J. Invertebr. Pathol.

    (2009)
  • K. Junkunlo et al.

    Clotting protein–An extracellular matrix (ECM) protein involved in crustacean hematopoiesis

    Dev. Comp. Immunol.

    (2018)
  • W. Junprung et al.

    Litopenaeus vannamei heat shock protein 70 (LvHSP70) enhances resistance to a strain of Vibrio parahaemolyticus, which can cause acute hepatopancreatic necrosis disease (AHPND), by activating shrimp immunity

    Dev. Comp. Immunol.

    (2019)
  • S. Kausar et al.

    Biotic and abiotic stress induces the expression of Hsp70/90 organizing protein gene in silkworm, Bombyx mori

    Int. J. Biol. Macromol.

    (2020)
  • G.G. Korkut et al.

    The effect of temperature on bacteria-host interactions in the freshwater crayfish, Pacifastacus leniusculus

    J. Invertebr. Pathol.

    (2018)
  • C.S. Li et al.

    Heat shock protein 20 from Procambarus clarkii is involved in the innate immune responses against microbial infection

    Dev. Comp. Immunol.

    (2020)
  • M. Liu et al.

    Involvement of gamma interferon inducible lysosomal thiol reductase in the innate immune responses of red swamp crayfish, Procambarus clarkii

    Dev. Comp. Immunol.

    (2019)
  • Q.N. Liu et al.

    A small heat shock protein 21 (sHSP21) mediates immune responses in Chinese oak silkworm Antheraea pernyi

    Int. J. Biol. Macromol.

    (2018)
  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method

    Methods

    (2001)
  • B. Oidtmann et al.

    Detection of genomic DNA of the crayfish plague fungus Aphanomyces astaci (Oomycete) in clinical samples by PCR

    Vet. Microbiol.

    (2004)
  • F. Ritossa

    Experimental activation of specific loci in polytene chromosomes of Drosophila

    Exp. Cell Res.

    (1964)
  • Y. Shen et al.

    Cloning and expression analysis of six small heat shock protein genes in the common cutworm, Spodoptera litura

    J. Insect Physiol.

    (2011)
  • R. Sirikharin et al.

    Characterization of a cold-active transglutaminase from a crayfish, Pacifastacus leniusculus

    Fish Shellfish Immunol.

    (2018)
  • J.M. van Noort et al.

    The link between small heat shock proteins and the immune system

    Int. J. Biochem. Cell Biol.

    (2012)
  • H.H. Wang et al.

    The mRNA expression profiles of five heat shock protein genes from Frankliniella occidentalis at different stages and their responses to temperatures and insecticides

    J. Integr. Agric.

    (2014)
  • P.H. Wang et al.

    Molecular cloning, characterization and expression analysis of two novel Tolls (LvToll2 and LvToll3) and three putative Spatzle-like Toll ligands (LvSpz1-3) from Litopenaeus vannamei

    Dev. Comp. Immunol.

    (2012)
  • Cited by (9)

    • A review on the DNA methyltransferase family of insects: Aspect and prospects

      2021, International Journal of Biological Macromolecules
      Citation Excerpt :

      Therefore, studies related to microbial infections in the economically important insect species mainly focusing on host-pathogen interaction [92,93]. Microbial pathogens hijack host metabolic machinery and utilize it for their proliferation, finally paralyzing the host cellular and biochemical events via secreting proteases or by generating PGs [46,94,95]. On the other hand, insects activate innate immune systems in response to pathogen infection as a defense strategy, which begins by detecting microbial pathogen and synthesizing immune effectors to eliminate them [96].

    • Analysis of transcriptome difference between rapid-growing and slow-growing in Penaeus vannamei

      2021, Gene
      Citation Excerpt :

      In a study of Calanus finmarchicus, HSP21 plays a role in short-term stress responses, stress tolerance, and protecting proteins from degradation (Aruda et al., 2011). A novel study suggests that HSP21 is both an inducible and a multifunctional protein involved in the immune defense reaction of Procambarus clarkii (Dai et al., 2020). Under salinity stress, HSP21 expression levels increased significantly in the muscle and gut tissues of P. monodon (Shekhar et al., 2013).

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text