Molecular cloning, tissue expression and regulation of nutrition and temperature on Δ6 fatty acyl desaturase-like gene in the red claw crayfish (Cherax quadricarinatus)

Abstract

Desaturase enzymes play an important role in the synthesis of unsaturated fatty acids. In this study, a complete cDNA sequence of a Δ6 desaturase-like gene was cloned from the hepatopancreas of the red claw crayfish, Cherax quadricarinatus. The full-length 1885 bp sequence comprises a 5′ UTR of 254 bp, 3′ UTR of 234 bp, and an open reading frame (ORF) of 1377 bp encoding a 458 amino acid polypeptide (GenBank accession no. MF497442). Bioinformatics analysis revealed three conserved histidine-rich regions, a cytochrome b5 domain at the N-terminus, and a haem binding site (HPGG) in the cytochrome b5 domain, all of which are typical of Δ6 desaturases. Quantitative real-time PCR demonstrated significantly higher expression in the hepatopancreas than other tissues. After feeding crayfish four formulated diets in which fish oil was replaced by 0, 33, 67, or 100% highly unsaturated soybean oil for 8 weeks, Δ6 desaturase-like mRNA expression and enzyme activity were higher than in the fish oil only group. Additionally, a 4-week low temperature treatment at 25, 20, 15, or 9 °C increased Δ6 desaturase mRNA expression and enzyme activity with decreasing water temperature. These results may help us better understand the biosynthesis of unsaturated fatty acids in C. quadricarinatus.

Introduction

Highly unsaturated fatty acids (HUFAs) are essential components of biomembranes in all cells and tissues that play important roles in many basic biological processes such as growth, reproduction, stress and immune responses, ion channel modulation, and nervous system development. (Tocher et al., 2006; Van et al., 2013; Wu et al., 2010; Xie et al., 2014). Many aquatic organisms do not possess the ability to de novo synthesise unsaturated fatty acids, but can synthesise HUFAs from precursor polyunsaturated fatty acids linolenic acid (18:3 n-3) and linoleic acid (18:2 n-6). This is a complex process that is regulated at several points and catalysed by a series of.

desaturations and elongations. Δ6 desaturase, the first rate-limiting enzyme involved, which can also catalyse the synthesis of docosahexaenoic acid (DHA) from eicosapentaenoic acid (EPA) (Nakamura and Nara, 2003; Tocher et al., 2003; Vagner and Santigosa, 2011). In fish, Δ6 desaturases have been widely studied (Geay et al., 2016; González-Rovira et al., 2009; Rasal et al., 2016; Santigosa et al., 2011), but in other aquatic animals, especially crustaceans, our knowledge of these enzymes is minimal. The first Δ6 fatty acid desaturase (fads) gene in crustaceans was cloned from the hepatopancreas of Chinese mitten crab (Eriocheir sinensis) (Yang et al., 2013), followed by genes in Scylla serrata (Lin et al., 2017) and Litopenaeus vannamei (Chen et al., 2017).

HUFAs are incorporated into phospholipids (PLs), the major components of cell membranes, and Δ6 desaturases are involved in the first rate limiting step of the synthesis of HUFAs, hence their key role in maintaining homeostasis of cellular unsaturated fatty acids. In aquatic poikilotherm animals, the activity of Δ6 desaturase is regulated by many factors including nutrition, salinity, and water temperature (Li et al., 2008; Liu et al., 2017; Tocher et al., 2000; Tocher et al., 2004; Vagner and Santigosa, 2011).

The red claw crayfish, Cherax quadricarinatus, is a crustacean belonging to the order Decapoda, family Parastacidae, that is endemic to freshwater habitats in Australia (Saoud et al., 2013). This species displays a number of physical, biological, and commercial attributes that make it suitable for aquaculture, including rapid growth, and tolerance to relatively high stocking densities and low oxygen concentrations (>1 ppm) (Masser and Rouse, 1993; Wang et al., 2013a, Wang et al., 2013b; Zuo et al., 2015). C. quadricarinatus is native to the tropics, and it has spread to many temperate regions of the world (Karplus et al., 1998; Saoud et al., 2012). This omnivorous shrimp and can make good use of plant oils (Thompson et al., 2010), and its lipid nutrition requirements have been investigated (Hervey et al., 2009; Rodríguez-González et al., 2006; Thompson et al., 2003; Wang et al., 2013a, Wang et al., 2013b), but relatively little is known about the molecular mechanism of high unsaturated fatty acid synthesis in this species. The present study reports the cloning of a Δ6 desaturase-like mRNA from the hepatopancreas of C. quadricarinatus for the first time, along with analysis of its tissue distribution, and the effects of dietary HUFAs and water temperature on its expression. The findings provide insight into the synthesis of high unsaturated fatty acids in C. quadricarinatus and other freshwater crustaceans.