Transcriptome analysis of the molting gland (Y-organ) from the blackback land crab, Gecarcinus lateralis.

In decapod crustaceans, arthropod steroid hormones or ecdysteroids regulate molting. These hormones are synthesized and released from a pair of molting glands called the Y-organs (YO). Cyclic nucleotide, mTOR, and TGFß/Smad signaling pathways mediate molt cycle-dependent phase transitions in the YO. To further identify the genes involved in the regulation of molting, a YO transcriptome was generated from three biological replicates of intermolt blackback land crab, Gecarcinus lateralis. Illumina sequencing of cDNA libraries generated 227,811,829 100-base pair (bp) paired-end reads; following trimming, 90% of the reads were used for further analyses. The trimmed reads were assembled de novo using Trinity software to generate 288,673 contigs with a mean length of 872 bp and a median length of 1842 bp. Redundancy among contig sequences was reduced by CD-HIT-EST, and the output constituted the baseline transcriptome database. Using Bowtie2, 92% to 93% of the reads were mapped back to the transcriptome. Individual contigs were annotated using BLAST, HMMER, TMHMM, SignalP, and Trinotate, resulting in assignments of 20% of the contigs. Functional and pathway annotations were carried out via gene ontology (GO) and KEGG orthology (KO) analyses; 58% and 44% of the contigs with BLASTx hits were assigned to GO and KO terms, respectively. The gene expression profile was similar to a crayfish YO transcriptome database, and the relative abundance of each contig was highly correlated among the three G. lateralis replicates. Signal transduction pathway orthologs were well represented, including those in the mTOR, TGFß, cyclic nucleotide, MAP kinase, calcium, VEGF, phosphatidylinositol, ErbB, Wnt, Hedgehog, Jak-STAT, and Notch pathways.

Myostatin from the American lobster, Homarus americanus: Cloning and effects of molting on expression in skeletal muscles.

A cDNA encoding a myostatin (Mstn)-like gene from an astacuran crustacean, Homarus americanus, was cloned and characterized. Mstn inhibits skeletal muscle growth in vertebrates and may play a role in crustacean muscle as a suppressor of protein synthesis. Sequence analysis and three-dimensional modeling of the Ha-Mstn protein predicted a high degree of conservation with vertebrate and other invertebrate myostatins. Qualitative polymerase chain reaction (PCR) demonstrated ubiquitous expression of transcript in all tissues, including skeletal muscles. Quantitative PCR analysis was used to determine the effects of natural molting and eyestalk ablation (ESA) on Ha-Mstn expression in the cutter claw (CT) and crusher claw (CR) closer muscles and deep abdominal (DA) muscle. In intermolt lobsters, the Ha-Mstn mRNA level in the DA muscle was significantly lower than the mRNA levels in the CT and CR muscles. Spontaneous molting decreased Ha-Mstn mRNA during premolt, with the CR muscle, which is composed of slow-twitch (S1) fibers, responding preferentially (82% decrease) to the atrophic signal compared to fast fibers in CT (51% decrease) and DA (69% decrease) muscles. However, acute increases in circulating ecdysteroids caused by ESA had no effect on Ha-Mstn mRNA levels in the three muscles. These data indicate that the transcription of Ha-Mstn is differentially regulated during the natural molt cycle and it is an important regulator of protein turnover in molt-induced claw muscle atrophy.

Cloning and tissue expression of eleven troponin-C isoforms in the American lobster, Homarus americanus.

Troponin-C is the Ca(2+)-binding subunit of the troponin regulatory complex in striated muscles. As TnC isoforms can influence the Ca(2+)-activation properties of fiber phenotypes, the diversity and tissue distribution of TnC cDNAs were assessed in the American lobster, Homarus americanus. We cloned ten full-length cDNAs and one partial cDNA coding for distinct TnC isoforms. Five were sequenced from expressed sequence tag clones and were designated Ha-TnC(2b)(') (2094 nt, 141 aa and 15.9 kDa), -C(4)(') (1667 nt, 155 aa and 17.3 kDa), -C(5) (2884 nt, 149 aa and 17.3 kDa), -C(6) (2439 nt, 155 nt and 17.4 kDa) and-C(6x) (2171 nt, 154 aa and 16.9 kDa). The remainder were cloned using a combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends: five full-length cDNAs, designated Ha-TnC(1) (814 nt, 150 aa and 17.1 kDa), -C(2a) (639 nt, 152 aa and 17.2 kDa), -C(2b)('') (2136 nt, 155 aa and 17.5 kDa), -C(3) (1046 nt, 150 aa and 16.9 kDa), -C(4)('') (842 nt, 108 aa and 12.1 kDa) and one partial (3') cDNA, designated Ha-TnC(4)(''') (563 nt and 57 aa). Ha-TnC(1), -C(2a), and-C(2b)(') corresponded to lobster TnC sequences in the GenBank protein database (Ha-TnC(1), -C(2a), and-C(2b)). Alternative splicing appeared responsible for TnC(2b)(') and-C(2b)(''); TnC(4)('), -C(4)('') and-C(4)('''); and TnC(6) and-C(6x). The deduced amino acid sequences differed primarily in the terminal regions and EF-hands I and III. Ha-TnC(6x) had a highly divergent 76 aa proline-rich N-terminal sequence. Tissue expression of the Ha-TnC isoforms was analyzed qualitatively by endpoint PCR. Ha-TnC(1), -C(2a), -C(2b)('), -C(2b)('') and-C(3) were expressed primarily in skeletal muscles; Ha-TnC(5) was expressed in heart; and Ha-TnC(4) and-C(6) variants were expressed in muscles and other tissues. The number and diversity of TnC sequences suggest the potential for varying the Ca(2+)-activated properties of the troponin-tropomyosin regulatory complex through differential expression of TnC isoforms.

Twelve actin-encoding cDNAs from the American lobster, Homarus americanus: cloning and tissue expression of eight skeletal muscle, one heart, and three cytoplasmic isoforms.

Lobster muscles express a diverse array of myofibrillar protein isoforms. Three fiber types (fast, slow-twitch or S1, and slow-tonic or S2) differ qualitatively and quantitatively in myosin heavy and light chains, troponin-T, -I, and -C, paramyosin, and tropomyosin variants. However, little is known about the diversity of actin isoforms present in crustacean tissues. In this report we characterized cDNAs that encode twelve actin isoforms in the American lobster, Homarus americanus: eight from skeletal muscle (Ha-ActinSK1-8), one from heart (Ha-ActinHT1), and three cytoplasmic type actins from hepatopancreas (Ha-ActinCT1-3). All twelve cDNAs were products of distinct genes, as indicated by differences in the 3'-untranslated regions (UTRs). The open reading frames specified polypeptides 376 or 377 amino acids in length. Although key amino residues are conserved in the lobster actins, variations in nearby sequences may affect actin polymerization and/or interactions with other myofibrillar proteins. Quantitative reverse transcription-polymerase chain reaction showed muscle fiber type- and tissue-specific expression patterns. Ha-Actin-HT1 was expressed exclusively in heart (87% of the total; 12% of the total was Ha-ActinCT1). Ha-ActinCT1 was expressed in all tissues, while CT2 and CT3 were expressed only in hepatopancreas, with Ha-ActinCT2 as the major isoform (93% of the total). Ha-ActinSK1 and SK2 were the major isoforms (88% and 12% of the total, respectively) in the S1 fibers of crusher claw closer muscle. Fast fibers in the cutter claw closer and deep abdominal muscles differed in SK isoforms. Ha-ActinSK3, SK4, and SK5 were the major isoforms in cutter claw closer muscle (12%, 48%, and 37% of the total, respectively). Ha-ActinSK5 and SK8 were the major isoforms in deep abdominal flexor (31% and 65% of the total, respectively) and extensor (46% and 53% of the total, respectively) muscles, with SK6 and SK7 expressed at low levels. These data indicate that fast fibers in cutter claw and abdominal muscles show a phenotypic plasticity with respect to the expression of actin isoforms and may constitute discrete subtypes that differ in contractile properties.

Muscle-specific calpain is localized in regions near motor endplates in differentiating lobster claw muscles.

Calpains are Ca2+-dependent proteinases that mediate protein turnover in crustacean skeletal muscles. We used an antibody directed against lobster muscle-specific calpain (Ha-CalpM) to examine its distribution in differentiating juvenile lobster claw muscles. These muscles are comprised of both fast and slow fibers early in development, but become specialized into predominantly fast or exclusively slow muscles in adults. The transition into adult muscle types requires that myofibrillar proteins specific for fast or slow muscles to be selectively removed and replaced by the appropriate proteins. Using immunohistochemistry, we observed a distinct staining pattern where staining was preferentially localized in the fiber periphery along one side of the fiber. Immunolabeling with an antibody directed against synaptotagmin revealed that the calpain staining was greatest in the cytoplasm adjacent to synaptic terminals. In complementary analyses, we used sequence-specific primers with real-time PCR to quantify the levels of Ha-CalpM in whole juvenile claw muscles. These expression levels were not significantly different between cutter and crusher claws, but were positively correlated with the expression of fast myosin heavy chain. The anatomical localization of Ha-CalpM near motor endplates, coupled with the correlation with fast myofibrillar gene expression, suggests a role for this intracellular proteinase in fiber type switching.

Myofibrillar gene expression in differentiating lobster claw muscles.

Lobster claw muscles undergo a process of fiber switching during development, where isomorphic muscles containing a mixture of both fast and slow fibers, become specialized into predominantly fast, or exclusively slow, muscles. Although this process has been described using histochemical methods, we lack an understanding of the shifts in gene expression that take place. In this study, we used several complementary techniques to follow changes in the expression of a number of myofibrillar genes in differentiating juvenile lobster claw muscles. RNA probes complementary to fast and slow myosin heavy chain (MHC) mRNA were used to label sections of 7th stage (approximately 3 months old) juvenile claw muscles from different stages of the molt cycle. Recently molted animals (1-5 days postmolt) had muscles with distinct regions of fast and slow gene expression, whereas muscles from later in the molt cycle (7-37 days postmolt) had regions of fast and slow MHC expression that were co-mingled and indistinct. Real-time PCR was used to quantify several myofibrillar genes in 9th and 10th stages (approximately 6 months old) juvenile claws and showed that these genes were expressed at significantly higher levels in the postmolt claws, as compared with the intermolt and premolt claws. Finally, Western blot analyses of muscle fibers from juvenile lobsters approximately 3 to 30 months in age showed a shift in troponin-I (TnI) isoform expression as the fibers differentiated into the adult phenotypes, with expression of the adult fast fiber TnI pattern lagging behind the adult slow fiber TnI pattern. Collectively, these data show that juvenile and adult fibers differ both qualitatively and quantitative in the expression of myofibrillar proteins and it may take as much as 2 years for juvenile fibers to achieve the adult phenotype.

Ecdysteroid-responsive genes, RXR and E75, in the tropical land crab, Gecarcinus lateralis: differential tissue expression of multiple RXR isoforms generated at three alternative splicing sites in the hinge and ligand-binding domains.

In order to study the potential role of the steroid molting hormone (20-hydroxyecdysone) in regulating molt-induced claw muscle atrophy, full-length cDNAs encoding retinoid-X receptor (Gl-RXR) and E75 early ecdysone inducible gene (Gl-E75) were obtained from land crab (Gecarcinus lateralis) skeletal muscle mRNA using RT-PCR and 3' and 5' RACE. Gl-E75A (3528bp), which encoded a protein of 828 amino acids, had highest sequence identity to Me-E75A from a shrimp (Metapenaeus ensis). It was expressed in skeletal muscle and gonads. The deduced amino acid sequence of Gl-RXR was highly similar to that of the fiddler crab RXR (Up-RXR) and insect ultraspiracle (USP). Nine variant sequences occurred in Gl-RXR mRNAs at three alternative splicing sites, one in the "T box" in the linker D domain and two in the ligand-binding domain (LBD). The three T-box variants, termed T(+8), T(+7), and T(+12), contained insertions of 8, 7, or 12 amino acids, respectively. Four variants were generated at the first site in the LBD. Two of the LBD site 1 variants differed in the presence (+33) or absence (-33) of a 33-amino acid sequence; the other two were LBD truncations with or without the 33 amino acid sequence (+33DeltaE/F and -33DeltaE/F, respectively). Two variants differing in the presence (+35) or absence (-35) of a 35-amino acid sequence were generated at the second site in the LBD. The Gl-RXRa isoform (1516 bp) with the longest open reading frame (+12/+33/+35) encoded a protein of 436 amino acids. Thoracic muscle expressed only isoforms with the T(+12) sequence. In contrast, claw muscle expressed isoforms with T(+7) or T(+12) and fewer isoforms with T(+8). Ovary and testis expressed a greater number of RXR isoforms than skeletal muscle. All tissues expressed full-length and truncated RXR isoforms. These data suggest that differences in response of claw and thoracic muscles to elevated ecdysteroid are due in part to differences in the expression of RXR isoforms.

Eyestalk ablation has little effect on actin and myosin heavy chain gene expression in adult lobster skeletal muscles.

The organization of skeletal muscles in decapod crustaceans is significantly altered during molting and development. Prior to molting, the claw muscles atrophy dramatically, facilitating their removal from the base of the claw. During development, lobster claw muscles exhibit fiber switching over several molt cycles. Such processes may be influenced by the secretion of steroid molting hormones, known collectively as ecdysteroids. To assay the effects of these hormones, we used eyestalk ablation to trigger an elevation of circulating ecdysteroids and then quantified myofibrillar mRNA levels with real-time PCR and myofibrillar protein levels by SDS-PAGE. Levels of myosin heavy chain (MHC) and actin proteins and the mRNA encoding them were largely unaffected by eyestalk ablation, but in muscles from intact animals, myofibrillar gene expression was modestly elevated in premolt and postmolt animals. In contrast, polyubiquitin mRNA was significantly elevated (about 2-fold) in claw muscles from eyestalk-ablated animals with elevated circulating ecdysteroids. Moreover, patterns of MHC and actin gene expression are significantly different among slow and fast claw muscles. Consistent with these patterns, the three muscle types differed in the relative amounts of myosin heavy chain and actin proteins. All three muscles also co-expressed fast and slow myosin isoforms, even in fibers that are generally regarded as exclusively fast or slow. These results are consistent with other recent data demonstrating co-expression of myosin isoforms in lobster muscles.

Fiber polymorphism in skeletal muscles of the American lobster, Homarus americanus: continuum between slow-twitch (S1) and slow-tonic (S2) fibers.

In recent years, an increasing number of studies has reported the existence of single fibers expressing more than one myosin heavy chain (MHC) isoform at the level of fiber proteins and/or mRNA. These mixed phenotype fibers, often termed hybrid fibers, are currently being recognized as the predominant fiber type in many muscles, and the implications of these findings are currently a topic of great interest. In a recent study, we reported single fibers from the cutter claw closer muscle of lobsters that demonstrated a gradation between the slow-twitch (S1) and slow-tonic (S2) muscle phenotype. In the present study, we focused on S1 and S2 fibers from the superficial abdominal muscles of the lobster as a model to study the continuum among muscle fiber types. Complementary DNAs (cDNA) encoding an S2 isoform of myosin heavy chain (MHC) and an S2 isoform of tropomyosin (Tm) were isolated from the superficial abdominal flexor muscles of adult lobsters. These identified sequences were used to design PCR primers used in conjunction with RT-PCR and real-time PCR to measure expression levels of these genes in small muscle samples and single fibers. The relative expression of the corresponding S1 MHC and S1 Tm isoforms was measured in the same samples with PCR primers designed according to previously identified sequences. In addition, we measured the relative proportions of MHC, troponin (Tn) T and I protein isoforms present in the same samples to examine the correlation of these proteins with one another and with the MHC and Tm mRNAs. These analyses revealed significant correlations among the different myofibrillar proteins, with the S1 and S2 fibers being characterized by a whole assemblage of myofibrillar isoforms. However, they also showed that small muscle samples, and more importantly single fibers, existed as a continuum from one phenotype to another. Most fibers possessed mixtures of mRNA for MHC isoforms that were unexpected based on protein analysis. These findings illustrate that muscle fibers in general may possess a phenotype that is intermediate between the extremes of 'pure' fiber types, not only at the MHC level but also in terms of whole myofibrillar assemblages. This study supports and extends our recent observations of mixed phenotype fibers in lobster claw and leg muscles. The existence of single fiber polymorphism in an invertebrate species underscores the generality of the phenomenon in skeletal muscles and emphasizes the need for an understanding of the proximal causes and physiological consequences of these intermediate fiber types.

Analysis of myofibrillar proteins and transcripts in adult skeletal muscles of the American lobster Homarus americanus: variable expression of myosins, actin and troponins in fast, slow-twitch and slow-tonic fibres.

Skeletal muscles are diverse in their contractile properties, with many of these differences being directly related to the assemblages of myofibrillar isoforms characteristic of different fibers. Crustacean muscles are similar to other muscles in this respect, although the majority of information about differences in muscle organization comes from vertebrate species. In the present study, we examined the correlation between myofibrillar protein isoforms and the patterns of myofibrillar gene expression in fast, slow-phasic (S(1)) and slow-tonic (S(2)) fibers of the American lobster Homarus americanus. SDS-PAGE and western blotting were used to identify isoform assemblages of myosin heavy chain (MHC), P75, troponin T (TnT) and troponin I (TnI). RT-PCR was used to monitor expression of fast and slow (S(1)) MHC, P75 and actin in different fiber types, and the MHC and actin levels were quantified by real-time PCR. Fast and slow fibers from the claw closers predominantly expressed fast and S(1) MHC, respectively, but also lower levels of the alternate MHC. By contrast, fast fibers from the deep abdominal muscle expressed fast MHC exclusively. In addition, slow muscles expressed significantly higher levels of actin than fast fibers. A distal bundle of fibers in the cutter claw closer muscle was found to be composed of a mixture of S(1) and S(2) fibers, many of which possessed a mixture of S(1) and S(2) MHC isoforms. This pattern supports the idea that S(1) and S(2) fibers represent extremes in a continuum of slow muscle phenotype. Overall, these patterns demonstrate that crustacean skeletal muscles cannot be strictly categorized into discrete fiber types, but a muscle's properties probably represent a point on a continuum of fiber types. This trend may result from differences in innervation pattern, as each muscle is controlled by a unique combination of phasic, tonic or both phasic and tonic motor nerves. In this respect, future studies examining how muscle phenotype correlates with innervation pattern may help account for variation in crustacean fiber types.

Molt cycle-dependent molecular chaperone and polyubiquitin gene expression in lobster.

Lobster claw muscle undergoes atrophy in correlation with increasing ecdysteroid (steroid molting hormone) titers during premolt. In vivo molecular chaperone (constitutive heat shock protein 70 [Hsc70], heat shock protein 70 [Hsp70], and Hsp90) and polyubiquitin messenger ribonucleic acid (mRNA) levels were examined in claw and abdominal muscles from individual premolt or intermolt lobsters. Polyubiquitin gene expression was assayed as a marker for muscle atrophy. Both Hsc70 and Hsp90 mRNA levels were significantly induced in premolt relative to intermolt lobster claw muscle, whereas Hsp70 mRNA levels were not. Hsp90 gene expression was significantly higher in premolt claw muscle when compared with abdominal muscle. Polyubiquitin mRNA levels were elevated in premolt when compared with intermolt claw muscle and significantly elevated relative to premolt abdominal muscle.

Cloning of a muscle-specific calpain from the American lobster Homarus americanus: expression associated with muscle atrophy and restoration during moulting.

A cDNA (1977 bp) encoding a crustacean calpain (Ha-CalpM; GenBank accession no. AY124009) was isolated from a lobster fast muscle cDNA library. The open reading frame specified a 575-amino acid (aa) polypeptide with an estimated mass of 66.3 kDa. Ha-CalpM shared high identity with other calpains in the cysteine proteinase domain (domain II; aa 111-396) and domain III (aa 397-575), but most of the N-terminal domain (domain I; aa 1-110) was highly divergent. Domain II contained the cysteine, histidine and asparagine triad essential for catalysis, as well as two conserved aspartate residues that bind Ca(2+). In domain III an acidic loop in the C2-like region, which mediates Ca(2+)-dependent phospholipid binding, had an expanded stretch of 17 aspartate residues. Ha-CalpM was classified as a non-EF-hand calpain, as it lacked domain IV, a calmodulin-like region containing five EF-hand motifs. Northern blot analysis, relative reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR showed that Ha-CalpM was highly expressed in skeletal muscles, but at much lower levels in heart, digestive gland, intestine, integument, gill, nerve cord/thoracic ganglion and antennal gland. An antibody raised against a unique N-terminal sequence recognized a 62 kDa isoform in cutter claw and crusher claw closer muscles and a 68 kDa isoform in deep abdominal muscle. Ha-CalpM was distributed throughout the cytoplasm, as well as in some nuclei, of muscle fibers. Purification of Ha-CalpM showed that the 62 kDa and 68 kDa isoforms co-eluted from gel filtration and ion exchange columns at positions consistent with those of previously described Ca(2+)-dependent proteinase III (CDP III; 59 kDa). Ha-CalpM mRNA and protein did not change during the moulting cycle. The muscle-specific expression of Ha-CalpM and the ability of Ha-CalpM/CDP III to degrade myofibrillar proteins suggest that it is involved in restructuring and/or maintaining contractile structures in crustacean skeletal muscle.

Ubiquitin and actin expression in claw muscles of land crab, Gecarcinus lateralis, and American lobster, Homarus americanus: differential expression of ubiquitin in two slow muscle fiber types during molt-induced atrophy.

The closer muscle of large-clawed decapod crustaceans undergoes a proecdysial (premolt) atrophy to facilitate withdrawal of the appendage at ecdysis. This atrophy involves the activation of both calcium-dependent (calpains) and ubiquitin (Ub)/proteasome-dependent proteolytic systems that break down proteins to reduce muscle mass. Moreover, the large slow-twitch (S(1)) fibers undergo a greater atrophy than the small slow-tonic (S(2)) fibers. Both polyUb mRNA and Ub-protein conjugates increase during claw muscle atrophy. In this study in situ hybridization and RT-PCR were used to determine the temporal and spatial expression of polyUb and alpha-actin. A cDNA encoding the complete sequence of lobster muscle alpha-actin was characterized; a probe synthesized from the cDNA provided a positive control for optimizing RT-PCR and in situ hybridization. PolyUb was expressed at low levels in claw closer muscle from anecdysial (intermolt) land crab. By early proecdysis (premolt; stage D(0)), polyUb mRNA levels increased in medial fibers that insert along the midline of the apodeme, with greater expression in S(1) than S(2), while levels remained low in peripheral fibers. By late proecdysis, polyUb mRNA decreased in central fibers, while mRNA increased in peripheral S(1) fibers. In contrast, alpha-actin was expressed in lobster claw muscles at relatively constant levels during the intermolt cycle. These results suggest that Ub/proteasome-dependent proteolysis contributes to enhanced turnover of myofibrillar proteins during claw closer muscle atrophy. Furthermore, atrophy is not synchronous within the muscle; it begins in medial fibers and then progresses peripherally.

Myofibrillar protein isoform expression is correlated with synaptic efficacy in slow fibres of the claw and leg opener muscles of crayfish and lobster.

In the crayfish and lobster opener neuromuscular preparations of the walking legs and claws, there are regional differences in synaptic transmission even though the entire muscle is innervated by a single excitatory tonic motor neuron. The innervation of the proximal fibres produced larger excitatory postsynaptic potentials (EPSPs) than those of the central fibres. The amplitudes of the EPSPs in the distal fibres were intermediate between those of the proximal and central regions. These differences in EPSP amplitudes were correlated with differences in short-term facilitation between the three regions. When given a 10- or 20-pulse train of stimuli, the proximal fibres showed greater short-term facilitation initially, often followed by a maximization of short-term facilitation towards the end of a train. In contrast, the central fibres showed a linear increase in short-term facilitation throughout a stimulus train. The distal fibres showed intermediate short-term facilitation compared with the other two regions. Analysis of myofibrillar isoforms showed that levels of troponin-T(1) (TnT(1)), a 55 kDa isoform expressed in slow-tonic (S(2)) fibres, were correlated with synaptic properties. Proximal fibres had the highest levels of TnT(1), with lower levels in distal fibres; central fibres lacked TnT(1), which is characteristic of slow-twitch (S(1)) fibres. In addition, differences in troponin-I isoforms correlated with TnT(1) levels between the proximal, central and distal regions. The correlation between slow fibre phenotype and strength of innervation suggests a relationship between synaptic structure and expression of troponin isoforms.