Short communicationMolecular characterization of a heat shock protein 21 (Hsp21) from red swamp crayfish, Procambarus clarkii in response to immune stimulation
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).
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2021, GeneCitation 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).
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These authors contributed equally to this work.