Cloning, Expression, and Localization of Two Types of Fast Skeletal Myosin Heavy Chain Genes From Black Tiger and Pacific White Shrimps

Abstract

The physiology and biochemistry of skeletal muscles in shrimps have been poorly understood compared with those from vertebrates. The present study was conducted focusing on myosin, the major protein in skeletal muscle, from adult specimens of black tiger Penaeus monodon and Pacific white Penaeus vannamei shrimps. Two genes encoding myosin heavy chain (MHC), a large subunit of the myosin molecule, were cloned from abdominal fast skeletal muscle and defined as MHCa and MHCb according to our previous study on kuruma shrimp Marsupenaeus japonicus. Random cloning demonstrated that the MHCb gene (MHCb) was expressed more abundantly than MHCa. The full-length cDNA clones of MHCa and MHCb from black tiger shrimp consisted of 5,926 and 5,914 bp, respectively, which encoded 1,914 and 1,909 amino acids, respectively, whereas those from Pacific white shrimp consisted of 5,923 and 5,908 bp, respectively, which encoded 1,913 and 1,909 amino acids, respectively. Both MHCa and MHCb were considered to be fast muscle type due to their strict localization in fast muscle. The amino acid identities between MHCa and MHCb of black tiger shrimp were 77%, 60%, and 73% in the regions of subfragment-1 (S1), subfragment-2 (S2) and light meromyosin (LMM), respectively, with 71% in total, whereas those of Pacific white shrimp were 78%, 60%, and 73% in the regions of S1, S2, and LMM, respectively, with 72% in total. In situ hybridization and northern blot analysis using different regions from abdominal muscle demonstrated different localizations of MHCa and MHCb transcripts in this muscle, suggesting their distinct physiological functions.

Cloning of Skeletal Myosin Heavy Chain Gene Family From Adult Pleopod Muscle and Whole Larvae of Shrimps

“…It has been confirmed that MHC1 and MHC2 transcripts are localized in adult abdominal muscle of the three shrimp species by in situ hybridization and Northern blot analysis, whereas both transcripts were not detected in adult pleopod muscle (Koyama et al, ). In this study, we cloned MHC s encoding C‐terminal sequences from adult pleopod muscle of the three shrimps by using a degenerate forward primer and GeneRacer 3′ Primer.…”

“…CODEHOP (COnsensus‐DEgenerate Hybrid Oligonucleotide Primers) (Rose et al, ) was used to design degenerate primer F (Table ) based on the deduced amino acid sequences of MHC from fruit fly Drosophila melanogaster (DDBJ/EMBL/GenBank accession number AAA28686) and American lobster fast‐ (U03091) and S 1 slow twitch‐type (AY232598) MHCs. This primer has been demonstrated to amplify DNA fragments encoding MHCs from abdominal muscle of kuruma, black tiger, and Pacific white shrimps as reported previously (Koyama et al, ).…”

“…The pleopod muscles from shrimps were oxidative fibers according to NADH‐diaphorase staining (Koyama et al, ). However, it is unclear whether pleopod muscle was of fast or slow type, because fast‐type oxidative fibers have been observed for decapod crustaceans (Silverman and Charlton, ; Hardy et al, ).…”

“…Two MHC s, MHC1P1 and MHC1P2 , were cloned from postlarvae of black tiger shrimp in our previous study, although their days after metamorphosis were not known (Koyama et al, ). We cloned novel larval‐ and postlarval‐type MHCs from black tiger and Pacific white shrimps in this study.…”

“…To shed light on crustacean skeletal muscle as a comparative model with vertebrate and other invertebrate counterparts, it is useful to investigate primary structures, expression patterns, and tissue distributions of MHCs in shrimps. We reported previously the complete sequences of two MHC genes ( MHC s), MHCa and MHCb , from abdominal muscles of adult specimens of kuruma Marsupenaeus japonicus , black tiger Penaeus monodon and Pacific white Penaeus vannnamei shrimps (Koyama et al, ). MHCa transcripts were localized in flexor muscle, whereas MHCb transcripts were in extensor and flexor muscles.…”