Abstract
Simple SummaryIn the age of shrinking stocks and intensive animal production alternative raw materials are in the eye of the scientists. Here, we aimed to identify molecular tools to evaluate the quality of spiny-cheek crayfish meat. Results showed a set of genes that were steadily or deferentially expressed between seasons and sexes. Additionally, the authors revealed genes involved in molting cycle and muscle growth/atrophy of the crayfish. The suite of molecular tools developed in the study have potential for the profiling of crayfish raw material quality in the food sector.Real-time quantitative reverse transcription PCR (RT-qPCR) is a sensitive and broadly used technique of assessing gene activity. To obtain a reliable result, stably expressed reference genes are essential for normalization of transcripts in various samples. To our knowledge, this is the first systematic analysis of reference genes for normalization of RT-qPCR data in spiny-cheek crayfish (Faxonius limosus). In this study, expression of five candidate reference genes (actb, β-actin; gapdh, glyceraldehyde-3-phosphate dehydrogenase; eif, eukaryotic translation initiation factor 5a; ef-1α, elongation factor-1α; and tub, α-tubulin) in muscle samples from male and female F. limosus in spring and autumn was analyzed. Additionally, the most stable reference genes were used for accurate normalization of five target genes, i.e., tnnc, troponin c; ak, arginine kinase; fr, ferritin; ccbp-23, crustacean calcium-binding protein 23; and actinsk8, skeletal muscle actin 8. Results obtained using the geNorm and NormFinder algorithms showed high consistency, and differences in the activity of the selected actb with eif genes were successfully identified. The spring and autumn activities of the target genes (except ak) in the muscle tissue of males and females differed significantly, showing that both sexes are immensely involved in an array of breeding behaviors in spring, and females intensively recover in the autumn season. Characterization of first reference genes in spiny-cheek crayfish will facilitate more accurate and reliable expression studies in this key species.
Highlights
The food sector usually assess quality parameters of raw material by means of basic chemical analyses and based on processingAnimals 2020, 10, 1140; doi:10.3390/ani10071140 www.mdpi.com/journal/animals and sensory parameters [1]
All assembled partial nucleotide sequences of the candidate reference and target genes were deposited in GenBank under the following names and numbers: eukaryotic translation initiation factor 5a (MN958081), elongation factor-1α (MN958082), α-tubulin (MN958083), glyceraldehyde-3-phosphate dehydrogenase (MN958084), β-actin (MN958085), arginine kinase (MN958080), ferritin (MK246411), crustacean calcium-binding protein 23 (MK246412), troponin c (MK246413) and skeletal muscle actin 8 (MK246414)
F. limosus showed a close clustering with the gapdh gene from the Cambaridae (P. clarkii, Cambarus cymatilis) and Astacidae (Pacifastacus leniusculus) families (Figure 2)
Summary
The food sector usually assess quality parameters of raw material by means of basic chemical analyses (e.g., protein and mineral content, composition of fatty acids) and based on processingAnimals 2020, 10, 1140; doi:10.3390/ani10071140 www.mdpi.com/journal/animals (shear force value, drip loss) and sensory parameters (texture, juiciness) [1]. Novel analytic approaches employ molecular methods to provide information about changes occurring at the cellular level [2], which affect raw material quality Such analyses can employ the commonly used technique of real-time PCR, which allows for measuring of the activity of genes encoding proteins involved in key metabolic pathways, including development and function of muscles, and affecting the quality of meat [3,4,5]. Other examples of genes and their protein products with an impact on muscle function include arginine kinase, which regulates energy generation [7], as well as troponin c and actin, which enable muscle contraction in common shore crab (Carcinus maenasi) [8,9] As for the latter pair of genes, it has been demonstrated that low water temperature in autumn and winter reduces their expression, which in turn results in poorer growth of the muscle tissue. For the latter group of animals a set of suitable reference genes have not been characterized that could potentially be used in other studies, such as environmental, epidemiological or behavioral
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