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Regulation of Muscle Gene Expression Over the Moult in Crustacea
Comp. Biochem. Physiol. Vol. 117B, No. 3, pp. 323–331, 1997 Copyright 1997 Elsevier Science Inc.
ISSN 0305-0491/97/$17.00 PII S0305-0491(97)00130-2
Regulation of Muscle Gene Expression Over the Moult in Crustacea N. M. Whiteley and A. J. El Haj * School of Biological Sciences, University of Birmingham, Birmingham B15 2TT, U.K. ABSTRACT. Muscle growth in crustacea occurs over the moult and involves a corresponding increase in fractional rates of sarcomeric protein synthesis. Heterologous and homologous cDNA probes for the myofibrillar protein, actin and myosin, have been used to investigate the factors responsible for controlling muscle growth in crustaceans at the molecular level. Factors such as passive stretch, caused by ecdysial expansion; moulting hormones such as ecdysteroids, which regulate the cycle of moult related events; and temperature, an environmental factor known to influence the rates of many biochemical reactions, all play a role in modulating muscle growth in crustacea. In isopod crustaceans, the regulation of muscle growth produces new problems as these crustaceans are characterised by a biphasic moult cycle in which the posterior half of the old exoskeleton is shed hours to days before the anterior half. These animals therefore provide an ideal model for the study of differential muscle growth. In both decapod and isopod crustaceans, transcriptional regulation alone may not be sufficient to account for the the upregulation of sarcomeric protein synthesis during intermittent growth. Both passive stretch and elevated ecdysteroid titre increase protein synthesis rates for actin in lobster muscles, in vivo. These factors may be involved in the promotion of ribosomal activity and translational processing during ecdysial muscle growth. Temperature has a direct effect on protein synthesis rates and levels of actin mRNA expression and may be a principal factor in the control of seasonal moult cycles. comp biochem physiol 117B; 3:323–331, 1997. 1997 Elsevier Science Inc. KEY WORDS. Crustacean, ecdysteroid, muscle, moult cycle, actin, myosin, growth, gene expression
INTRODUCTION Muscle growth in crustaceans is intermittent (9,20,36,37) and closely associated with the moult cycle due to the presence of the rigid calcified exoskeleton. Increases in muscle mass are restricted to the ecdysial period when the old exoskeleton is shed and the new exoskeleton expands in size (9). The increase in size is due to an increase in hydrostatic pressure in the haemolymph (4) resulting from increased rates of water uptake (28). Fractional rates of protein synthesis increase 10-fold in the leg muscles of Carcinus during the premoult stages D3 –4 in preparation for ecdysis, and remain elevated in early postmoult (stages A, B) (11). The postmoult increase in protein synthesis rates coincides with an increase in muscle fibre length due to the addition of sarcomeres and an increase in the number of myofibrils due to longitudinal splitting (9). Protein synthesis rates also increase in the lobster, H. americanus, in both leg, claw, and abdominal muscles during late premoult and early postmoult (8). The elevation in protein synthesis rates is most surprising in the claw muscle, which has been shown in land Address reprint requests to: A. J. El Haj, School of Biological Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K. Tel. 121 414 5459; Fax 121 414 5925; E-mail: [email protected]. Received 20 June 1996; accepted 16 September 1996.
crabs to atrophy by 30–60% prior to ecdysis (37). This moult induced atrophy reduces the muscle mass of the large claw muscle to enable it to be squeezed through the relatively small aperture of the basi-ischial joint. The selective degradation of the claw muscle during premoult is followed by an increase in mass during postmoult (36,31), to sequence. These findings indicate that muscle growth in crustaceans is a complex process involving the interplay of synthesis and degradation rates in a tissue dependent manner that is closely coordinated with the moult cycle. The relationship between synthesis and degradation rates in the muscle tissue during ecdysis, raises some interesting questions as to the nature of the regulatory mechanisms involved in controlling muscle growth. The increase in protein synthesis rates in the claw muscle during premoult atrophy (36) indicates that degradation rates increase to exceed synthesis rates (29). Moult-induced atrophy is confined to the claw muscles and is thought to be regulated by intracellular proteolytic enzymes that increase in activity during premoult (29,30,32). Other factors are also thought to play a regulatory role during muscle growth, such as circulating moulting hormones (8,43) and mechanical stretch resulting from the expansion of the new exoskeleton during the immediate post moult stages (17). However, the mechanisms controlling differential regulation of muscle growth be-
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tween the different muscles is unclear. One avenue of investigation that may help to increase our understanding of the regulatory mechanisms involved includes molecular studies into the expression of actin and myosin genes during muscle growth. A review of the crustacean genes that have been cloned and used to study growth and development in crustaceans has recently been published (7). The object of the present review is to report on the latest developments in the area of molecular regulation of muscle growth by comparing moult related changes in gene expression in the muscles of decapod and isopod crustaceans in response to passive stretch, steroidal action and temperature. MUSCLE GROWTH OVER THE MOULT CYCLE Muscle Growth in Decapod Crustaceans To date, muscle growth studies in crustacea have been restricted to the relatively large decapod crustaceans. In decapods, muscle fibre growth results from an increase in fibre length by addition of new sarcomeres or by lengthening of existing fibres (9,20). Increases in cross-sectional area occurs by longitudinal splitting of existing myofibrils, maintaining a constant fibre number as described in vertebrate striated muscle (14). Over the moult cycle, muscle fibres in the walking leg of the lobster, H. americanus, increase in length in the first few hours of ecdysis (9) accompanied by the longitudinal splitting of larger myofibrils. Fractional rates of total protein synthesis have been measured in the muscles of a variety of decapod crustaceans including the land crab, Gecarcinus lateralis, the shore crab, Carcinus maenas, and the lobsters Homarus gammarus and H. americanus. Early studies in Carcinus, demonstrated a 10-fold increase in protein synthesis rates in the leg muscles during late premoult stages D3 –4, followed by an elevation in rates into early postmoult (stages A, B). This surge in protein synthesis rates, centred around ecdysis, is also found in the leg, claw, and abdominal muscles of H. americanus (12) (Fig. 1). The increase in protein synthesis rates observed in the claw muscle of the land crab Gecarcinus lateralis was thought to be associated with the synthesis of Ca21dependent proteinases for the specific regulation of myofibrillar proteins during atrophy (29,31,32). Growth of this muscle was suggested to occur in postmoult, when a second peak in protein synthesis rates was observed (36). An actin cDNA (HgAct) has been cloned from Homarus gammarus muscle using RT-PCR and degenerate oligonucleotide primers devised from published actin sequences (17). This 736 bp fragment encompasses the majority of the coding region of the actin sequence and recognizes a 1.6 kb mRNA in muscles composed of both fast and slow fibres from the lobster and from Antarctic and temperate isopods (Fig. 2). This clone has been used along with heterologous actin cDNAs from Artemia (24) and the mouse (27), to follow levels of mRNAs for actin in the muscles of a number of decapod crustaceans over the moult, indicating the high
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degree of homology between actin sequences from different species. Initial studies in Carcinus and the freshwater crayfish, Austrpotamobius pallipes revealed that actin mRNA levels increased in the leg muscles during premoult and postmoult (13,43). A much more detailed study in the American lobster, H. americanus, however, has revealed little variation in actin mRNAs in either the claw, leg, or abdominal muscles sampled each day for 40 days, over an entire moult cycle (P. Harrison and A. J. El Haj, unpublished observations). Ribosomal levels in the muscle tissue also remained unchanged over the moult (12). These observations suggest that in lobsters the increase in protein synthesis rates occurred at the level of translation from existing pools of mRNAs rather than by transcriptional regulation via changes in gene expression. However, interspecific variations may occur. Muscle Growth in Isopod Crustaceans Decapod crustaceans are characterised by a moulting pattern in which the entire exoskeleton is shed at one time to allow a general increase in body size over ecdysis. Very little is known about patterns of muscle growth in crustaceans belonging to other Orders that have different moulting patterns. One of the most intriguing in this respect is the biphasic moulting pattern shown by the Order Isopoda. In general, isopods have an elongate body plan and are flattened dorso-ventrally (35). The body is divided into the cephalon, seven thoracic segments, six abdominal segments and the terminal end plate, the telson. The segments are fairly uniform in character and it is difficult to distinguish between the three different sections. During the biphasic moult, the old exoskeleton splits between free thoracic segments four and five and the posterior half of the old exoskeleton is shed, hours to days before the anterior portion. After posterior ecdysis, the posterior portion of the body subsequently increases in size and the epidermis begins to recalcify before the anterior exoskeleton is shed. Therefore, the elongate body increases in size in two distinct phases separated by a period of hours to days. This biphasic pattern of growth includes the muscle groups located in the anterior and posterior segments and, therefore, presents a useful model for studying the role of systemic hormones in regulating differential muscle growth. The functional significance of this moulting pattern is unknown. It may enable isopods to maintain some motility during the moult, and enable the animals to excise themselves from the elongate exoskeleton. The biphasic moult may also be involved, at least in terrestrial isopods, in the mobilisation of calcium during the moult as suggested by Steel (38) who confirmed the presence of Ca21 stores in the white sternal plates of Oniscus asellus. Regardless of the function of the biphasic moult, there are some interesting questions to be addressed concerning the timing and regulation of muscle growth in these animals. The expansion of
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FIG. 1. Fractional rates of protein synthesis (Ks ) (A); and protein synthesis rates per unit of RNA (ribosomal activity) (B) in the leg, abdomen and claw muscle of the lobster H. americanus at three different stages of the moult cycle (a), and in intermoult lobsters after injection with 1026 mol l21 20-hydroxyecdysone (b). Values given as means 1SEM with significant differences from intermoult means at the 95% level of confidence represented by asterisks. [Taken from (18)].
the posterior segments hours to days before the anterior portion suggests differential growth patterns. Study of these animals and their biphasic moult pattern may help to unravel the relationship between the regulation of muscle growth and the timing of ecdysis. Initial studies have been carried out to examine the influence of the biphasic moult on fractional rates of protein synthesis (ks), and mRNA levels for actin, in the ventral longitudinal muscle of the marine isopod, Idotea rescata. This muscle constitutes the main muscle bulk of this species. It is organized segmentally and runs the entire length of the body. Histochemical analysis for myofibrillar ATPase activity in frozen transverse sections of the ventral longitudinal muscle, revealed that this muscle was predominantly composed of fast muscle fibres. The results showed that ks values were higher in the anterior half of the muscle (thoracic muscle segments 1–5) at 1.31% day21, as opposed to the posterior half (thoracic segments 6 and 7 to abdominal segments, 1–6) at 0.31% day21 during half moult when the posterior half of the exoskeleton had been shed (Fig. 3a). This difference continued into postmoult. Absolute rates of protein synthesis (total protein synthesized per day) varied in a similar manner with elevated rates in the anterior portion of the muscle up until 36 hr postmoult when rates were similar in both portions of the muscle (Fig. 3b). The capacity for protein synthesis (RNA : protein ratio) and the protein synthesis rate per unit of RNA or RNA activity also changed over the biphasic moult (Fig. 4a). Measurement of the mRNA levels for actin using the actin cDNA (HgAct) isolated from the lobster, H. gammarus (17) showed that actin mRNA levels remained unchanged in posterior and anterior portions of Idotea muscle over the biphasic moult
(Fig. 5). Myosin HC mRNA levels also remained unchanged as shown in Fig. 5. This indicates that the control of differential production of sarcomeric proteins in the anterior and posterior portions may be due to changes in protein synthesis rates from pools of sarcomeric protein mRNA rather than differential control of transcription rates. FACTORS REGULATING MUSCLE GROWTH Decapod Crustaceans Passive stretch and ecdysteroids have been implicated in playing a role in controlling muscle growth in decapodan crustaceans and in particular, Carcinus maenas and Homarus americanus (10,17,20). Passive stretch results in compensatory fibre lengthening over a period of 2 to 3 weeks (20) with a corresponding increase in actin mRNA levels after 2 weeks accounting for the elevated protein production necessary for new sarcomere assembly (17). A suggestion that ecdysial stretch may play a role in muscle growth in crustacea during the moult has been proposed. However, morphological experiments have shown that muscle fibre lengthening takes place within days, not weeks, and, therefore, passive stretch may not play a major role in the stimulation of muscle growth after the moult. This data demonstrates that crustacean muscle is stretch responsive as found in vertebrates, but the specific role of passive stretch in muscle growth during the moult is unclear. Ecdysteroids have been known to play a role in the regulation of the moult cycle for many decades, but the specific way in which ecdysteroids modulate muscle growth in crustacea has only just been investigated. Ecdysteroid receptor immunoreactivity has been demonstrated in the walking leg
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FIG. 3. (a) Fractional rates of protein synthesis (K s % synthe-
FIG. 2. Autoradiographs of Northern blots showing: (a) total
RNA from the extensor walking leg muscle and gonads of H. gammarus; and (b) total RNA from the body wall muscle of the Antarctic Isopod, Glyptonotus antarcticus (G), and the ventral longitudinal muscle of the temperate isopod, Idotea rescata (I). Both blots were hybridized to the cDNA for actin (HgAct) isolated from H. gammarus muscle (17). In (a), washing under low stringency conditions revealed a hybridization signal at 1.8 and 1.6 kb in the muscle and one band at 1.6 kb in the gonad. Under high stringency wash conditions, no hybridization signal was observed in the gonad but one band at 1.6 kb remained in the muscle. The size of the mRNA corresponds to actin mRNAs detected using a heterologous cDNA for mouse skeletal a-actin isolated from the mouse (27). [Fig. 2(a) was taken from (17)].
sised per day), and (b) absolute rates of protein synthesis (As, total protein synthesised per day), in the longitudinal muscle of Idotea rescata over the biphasic moult. Measurements were taken from pooled muscle samples with the number in each group given above the appropriate moult cycle stage. Halfm. represents isopods in the halfmoult stage of ecdysis when the animals have lost the posterior half of the old exoskeleton but retain the anterior portion. The anterior portion of the muscle was taken as that corresponding to free thoracic segments 1–4, and the posterior portion as muscle corresponding to thoracic segments 5–6, and abdominal segments.
extensor muscle of the lobster by Western blotting and immunocytochemistry (10) using a monoclonal antibody to the ecdysteroid receptor of Drosophila melanogaster developed by Koelle et al. (23). Preliminary observations suggest that receptor immunoreactivity is modulated over the moult with an increase in immunofluorescence in the lobster extensor muscle during premoult. Injections of premoult titres of the ecdysteroid, 20-hydroxyecdysone, into intermoult lobsters results in elevated fractional rates of protein synthesis after 3 days, in leg, claw, and abdominal muscles (8). However, attempts to mimic these effects in vitro have not
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FIG. 4. (a) Summary of the relationship between fractional rates of protein synthesis (K s), the capacity for protein synthesis
(RNA : protein ratio), and the protein synthesis rate per unit of RNA (K RNA), in the anterior and posterior portions of the ventral longitudinal muscle of I. rescata, during intermoult, premoult, and halfmoult. (b) Modification of a diagram taken from (18) to show changes in haemolymph ecdysteroid levels over the biphasic moult in the terrestrial isopod, H. brevicornis. Values for I. rescata have been added to this diagram and are represented by open symbols. E1 and E2 indicate ecdysis, where E1 represents posterior ecdysis and E2, anterior ecdysis. HM represents animals in halfmoult as described in Fig. 3. A, B, postmoult stages; C, intermoult; D0 to D3 premoult stages. Values given as means 6SEM.
been successful. Levels of mRNAs for actin and myosin sarcomeric proteins also respond to an elevation in ecdysteriod titre by showing transient increases in mRNA levels (A. J. El Haj, unpublished observations). These data suggest that circulating ecdysteroids act directly on the muscle through specific receptors to initiate muscle growth. The lack of any response in vitro may indicate a role for co-factors in the action of ecdysteroids on mRNA and protein synthesis levels in crustacean muscle. The role of one such factor, insulin related peptides, has been investigated using heterologous antibodies and cDNAs to recombinant IGFs (A. J. El Haj and E. S. Chang, unpublished observations). Our recent studies have concentrated on identifying ecdysteroid responsive genes in lobster muscles to establish if the complex
cascade of early and late genes which occurs in insects is also found in crustaceans. Isopod Crustaceans Hormonal control of the biphasic moult cycle has been most extensively studied in the terrestrial isopods. In these animals, the sinus gland is a cephalic neurohaemal organ located posterior to the optic lobe (25,26). Studies on Helleria brevicorrus (18), Oniscus asellus (21), and Idotea rescata (44), have shown that haemolymph ecdysteroid levels are low during intermoult, but concentrations increase to peak values during premoult and fall about 3 days before ecdysis of the posterior exoskeleton (Fig. 4b). A secondary but
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muno-assay, these authors were able to show that CCAP contents of the CNS increased 10-fold, 1–2 days before and during posterior ecdysis. CCAP levels returned to low levels during anterior ecdysis. Immunocytochemistry of premoult animals, localised the increase in CCAP levels to the VNC neural network corresponding to the segments posterior to the line of ecdysis (5). It is possible that neuropeptides such as CCAP secreted in specific thoracic/abdominal segments at certain times of the moult cycle, are involved in the control of this intricate moulting pattern. However, the specific role of ecdysteroids and various neuropeptides in the regulation of moult related changes in muscle ks remains to be investigated. TEMPERATURE EFFECTS ON MUSCLE GROWTH IN CRUSTACEA Protein Synthesis Rates
FIG. 5. Results of slot blot analysis of total RNA from the anterior and posterior segments of the ventral longitudinal muscle in I. rescata taken during halfmoult and 12 and 48 hr postmoult, and hybridized to (a) HgAct, the lobster cDNA for actin, and (b) a cDNA for the fast isoform of myosin heavy chain cloned in H. americanus by Cotton and Mykles (3). The relative abundance of the respective mRNA levels are expressed as the ratio between densitometry readings of the hybridization signal with HgAct or fast MHC against that of 18S rRNA.
smaller peak in haemolymph ecdysteroid levels was measured during half moult shortly after posterior ecdysis in both O. asellus and I. rescata. The pattern of haemolymph ecdysteroid variation in isopods is, therefore, similar to that shown in decapod crustaceans over the moult. Ecdysteroid levels in the anterior and posterior muscle segments did not differ over the biphasic moult (44), indicating that ecdysteroids are not involved in the control of ks during this period. Johnen et al. (21) have recently shown that the crustacean cardioactive peptide (CCAP) may have a function in the regulation of ecdysis behaviour in isopods. By isolating an antibody specific for CCAP in the isopod, Oniscus asellus, and developing a competitive enzyme im-
Fractional rates of protein synthesis (ks ) in the whole bodies and the muscle of intermoult crustaceans vary exponentially with temperature to give a Q10 of approximately 2, which corresponds to temperature-related changes in oxygen consumption rates, suggesting a link between the two variables. A similar response has been described in some fish species (19). In the eurythermal marine isopod, Idotea rescata, the increase in muscle and whole body ks over a 10°C rise in acclimation temperature (Q 10 5 1.99) (Fig. 6), was accompanied by an increase in actin mRNA levels and an increase in RNA activities at a constant RNA : protein ratio (12). This suggests that both rates of transcription and translation are directly related to temperature. Rates of protein degradation may also change with temperature, resulting in an increase in rates of protein turnover with no net protein accretion. Fractional rates of protein synthesis also vary with temperature in the muscle tissue of a seasonally acclimatized crustacean, the freshwater crayfish (A. pallipes), which experiences wide variations in seasonal temperatures (1–21°C) in its natural environment (46). A decrease in water temperature in the autumn/winter, when the animals are in intermoult, is accompanied by an exponential decrease in ks in the leg muscles to give a Q10 value of 5.2 suggesting that there is an acute drop in ks in the winter when crayfish are relatively inactive and are not feeding. The lack of temperature compensation for ks in the leg muscles of freshwater crayfish during the winter is also observed in an Antarctic isopod crustacean, Glyptonotus antarcticus. This benthic isopod lives within a narrow temperature range of 21.89°C in the winter to 0.5°C in the summer, and is therefore strictly stenothermal and extremely intolerant to increases in temperature (1). Physiological studies have shown that this isopod crustacean does not show any metabolic cold adaptation despite the low temperatures of the Southern Ocean, but maintains low rates of oxygen uptake and extremely low rates of whole body protein synthesis (45), in keeping with its relatively inactive and sedentary lifestyle. Glyptonotus
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termoult crayfish resulted in an increase in circulating ecdysteroid levels and subsequent moult within 10 weeks of captivity, one month ahead of the animals in the wild (43). Consequently, low winter temperatures may inhibit moulting activity even though the mechanisms responsible for the morphological, biochemical, and physiological changes over the moult are present and functional in over-wintering animals. It is not known if an increase in moulting frequency results in a concomitant increase in muscle mass over each moult. This relationship remains to be investigated. Myosin Isoforms
FIG. 6. Comparison of the effects of temperature on frac-
tional rates of protein synthesis (k s) in the whole body and ventral longitudinal muscle of I. rescata acclimated to 4° and 14°C. Whole body values given as means 6SEM. Values for k s in the muscle taken from pooled samples with the number of samples given above each data point.
shares many characteristics of other benthic marine Antarctic species in that they are slow growing with low moulting frequencies and have a tendency to gigantism as animals range from 9 to 120 mm in length, which is relatively large for isopod crustaceans. Moult Frequency In addition to the direct effects of temperature on rates of protein turnover in the muscles of intermoult crustaceans, rising temperature has also been shown to increase the frequency of the moult and, therefore, growth rates. Early studies on moulting behaviour in crustaceans, found that an increase in temperature led to an increase in the number of moults (2,39), and to a decrease in the duration of each moult cycle (33). Alternatively, a decrease in temperature reduced the incidence of moulting (6,34). Temperature also plays an important role in the control of seasonal moult cycles. In the temperate freshwater crayfish, A. pallipes, an increase in water temperature from 6° to 16°C initiated the moult in intermoult animals brought into the laboratory in January. In the wild, these animals moult between May and September, when water temperatures range between 16° and 21°C. Exposure to higher temperatures in winter in-
Temperature acclimation in fish is followed by a number of partial capacity adaptations for metabolic processes in the muscle, such as muscle contraction and swimming performance (22). In Antarctic fish, the adaptation to low temperatures involves a change in the myofibrillar apparatus to give a specific ATPase activity which may be achieved by changes in gene expression of the protein isoforms (16). Changes in isoform expression are also observed in the red and white muscles of eurythermal fish acclimated to a range of environmental temperatures (15,40,41). Multiple isoforms of both regulatory and contractile proteins have been identified in fast and slow muscles from a variety of crustaceans [cf. (3)], but relatively little is known about the effects of environmental temperature on the expression of these isoforms. In the carp, Cyprinus carpio, three myosin heavy chain isoforms are expressed in an acclimation temperature-dependent manner, encoded by multiple genes (41). Initial studies have used SDS PAGE analysis to separate the different isoforms present in the muscles of various crustaceans over a range of temperatures. Fast muscle fibres from the temperate isopod, Idotea rescata, acclimated at 4° and 15°C, the Antarctic isopod Glyptonotus held at 0°C and H. americanus at 15°C had similar myosin HC isoforms (12). An examination of the myosin light chains are required to fully characterise potential differences in myosin protein isoforms between crustaceans living in different thermal regimes. However, a molecular characterisation of myosin heavy chains has been initiated in the giant Antarctic isopod Glyptonotus antarcticus. In recent experiments, degenerate oligonucleotide primers have been developed and used in conjunction with RT-PCR of total RNA from the abdominal muscle of Glyptonotus, to isolate a PCR product of 544 bp in length (N. M. Whiteley and A. J. El Haj, unpublished observations). Subsequent cloning and sequencing of this cDNA has revealed 60–75% homology with myosin heavy chain clones from a range of different animals including invertebrates such as Drosophila, C. elegans, and A. irradians to vertebrates such as Rattus norvegicus. This cDNA for myosin HC hybridized to a mRNA transcript of 6.6 kb in size corresponding to myosin HC mRNAs (3). Subsequently
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this cDNA will be used to screen a genomic DNA library isolated from Glyptonotus, to further characterise the gene or gene family for myosin HC. SUMMARY Regulation of muscle growth in crustaceans is a complex process involving fine control of muscle protein synthesis and degradation rates. The mechanisms regulating this relationship appear to be tissue specific, involving a combination of translational processing and transcriptional regulation that may vary from species to species. Ecdysteroid hormones and passive stretch have been implicated in the control of cyclical muscle growth in crustaceans. Recent studies on intermoult lobsters have demonstrated that both factors can increase protein synthesis rates and fibre lengthening in the leg, claw, and abdominal muscles, but their mode of action over the moult cycle remains to be determined. The biphasic moult pattern in isopod crustaceans is characterised by differential muscle growth due to a variation in protein synthesis rates between anterior and posterior muscle segments during halfmoult and immediate postmoult stages. Ecdysteroid levels in the two muscle sections remain unchanged over the moult but investigations into cardioactive peptides suggest that neuropeptides and the CNS may play a more critical role in the regulation of the biphasic moult. Temperature has a direct effect on protein synthesis rates, giving a Q10 value of 1.99 in I. rescata between 4° and 14°C, and is important in the environmental regulation of the seasonal moult. The temperature related increase in both whole body and muscle rates of protein synthesis, however, is thought to be associated with equal increases in degradation rates. In some crustacea, temperature may, therefore, play a more fundamental role in the control of muscle growth by promoting the moult, increasing moulting frequency and increasing the opportunity for accretion of sarcomeric proteins. We would like to thank the following people for their help and collaboration during the studies reported in this review: Prof. E. Chang for providing research facilities in this laboratory at the Bodega Marine Laboratory, University of California at Davis, for the studies on Idotea, Prof. Ted Taylor, Dr. Paul Harrison, Sharon Taylor, and Sue Clark for their contributions to this work at the University of Birmingham, and to Prof. Andrew Clarke and Dr. Lloyd Peck for their collaboration during the research on the Antarctic isopod, Glyptonotus antarcticus. We are grateful to NERC, AFRC, The Royal Society, and The Linnean Society, U.K., for financial support.
References 1. Arnaud, P.M. Essai de synthe`se des particularities eco-biologiques (adapations) des inverte´bre´s benthiques antarctique. Oceanis. 11:117–124;1985. 2. Balesdent-Marquet, M.L. Inflence de la temperature sur la mue l’activite´ ovarienne et les characte`res sexuelstemporairies du Crustace´ Isopode Asellus acquaticus. L. Compt. Rend. 240: 1372–1374;1955.
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3. Cotton, J.L.S.; Mykles D.L. Cloning of a crustacean myosin heavy chain isoform: Exclusive expression in fast muscle. J. Exp. Zool. 267:578–586;1993. 4. deFur, P.L.; Mangum, C.P.; McMahon, B.R. Cardiovascular and ventilatory changes during ecdysis in the blue crab, Callinectes sapidus Rathburn. J. Crust. Biol. 5:207–215;1985. 5. Dircksen, H.; Johnen, C.; Gliscynski. U.; Nussbaum, T. Moult cycle related changes in haemolymph ecdysteroids and CNSimmunoreactivity to crustacean cardioactive peptide in an isopod. The Biology of Crustacea, Meeting abstract no. 19; 1996. 6. Drach, P. Pe´riodes de mue chez un Crustace´ a` croissance lente (Xantho floridus Montagu) dans le district septentrional de son dire de re´partition. Compt. Rendu. 228:128–130;1949. 7. El Haj, A.J. Crustacean genes involved in growth. In: Ennion, S.; Goldspink, G. (eds). Gene Regulation in Aquatic Organisms. SEB Seminar Series. Cambridge: Cambridge University Press; 1996:94–112. 8. El Haj, A.J.; Clarke, S.; Harrison, P.; E.S. Chang. In vivo muscle synthesis rates in the American lobster, Homarus americanus, during the moult cycle and in response to 20-hydroxyecdysone. J. Exp. Biol. 199:579–585;1996. 9. El Haj, A.J.; Govind, C.K.; Houlihan D.F. Growth of lobster leg muscle fibers over intermoult and moult. J. Crust. Biol. 4: 536–545;1984. 10. El Haj, A.J.; Harrison, P.; Chang, E.S. Localization of ecdysteroid receptors in eyestalk and muscle tissue of the American lobster. J. Exp. Zool. 270:343–349;1994. 11. El Haj, A.J.; Houlihan, D.F. In vitro and in vivo protein synthesis rates in a crustacean muscle during the moult cycle. J. Exp. Biol. 127:413–426;1987. 12. El Haj, A.J.; Whiteley, N.M. Molecular regulation of muscle growth in Crustacea. J. Mar. Biol. Assoc. U.K. 1997:77;95– 106. 13. El Haj, A.J., Whiteley, N.M.; Harrison, P. Molecular regulation of muscle growth over the crustacean moult cycle. In: El Haj, A.J. (ed). Molecular Biology of Muscle. SEB Seminar Series No. 46. Cambridge: Cambridge University Press; 1992: 151–165. 14. Goldspink, G. Growth of muscle. In: Goldspink, D.F. (ed.) Development and Specialization of Skeletal Muscle. SEB Seminar Series No. 7. Cambridge: Cambridge University Press; 1980. 15. Goldspink, G. Adaptation of fish to different environmental temperatures by qualitative and quantitative changes in gene expression. J. Therm. Biol. 20:167–174;1995. 16. Goldspink, G.; Turray, L.; Hansen, E.; Ennion, S.; Gerlach, G. Switches in fish myosin genes induced by environment temperature in muscle of the carp. In: El Haj, A.J. (ed). Molecular Biology of Muscle. SEB Symposium Series No. 46. Cambridge: Company of Biologists; 1992:139–149. 17. Harrison, P.; El Haj, A.J. Actin mRNA levels and myofibrillar growth in leg muscles of the European lobster (H. gammarus) in response to passive stretch. Mol. Mar. Biol. Biotech. 3:35– 42;1994. 18. Hoarau, F.; Hirn, M. Evolution du taux des ecdyste´roids au cours du cycle de mue chez Helleria brevicornis Ebner (Isopode terrestre). C. R. Acad. Sci. Paris Ser. D 286:1443–1446;1978. 19. Houlihan, D.F. Protein turnover in ectotherms, relation to energetics. In: Gilles, R. (ed). Comparative Environmental Physiology. Berlin: Springer Verlag; 1991:1–44. 20. Houlihan, D.F.; El Haj, A.J. An analysis of muscle growth. In: Wenner, A. M. (ed). Factors in Adult Growth Amsterdam: A. A. Blaskema Press; 1985. 21. Johnen, C.; Vongliscynski, U.; Dircksen, H. Changes in hemolymph ecdysteroid levels and CNS contents of crustacean
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cardioactive peptide immunoreactivity during the molt cycle of the isopod Oniscus asellus. Neth. J. Zool. 45(1–2):38–40 Johnston, I.A. Cold adaptation in marine organisms. Phil. Trans. R. Soc. Ser. B 326:655 –667;1990. Koelle, M.R.; Talbot, W.S.; Segraves, W.A.; Bender, M.T.; Cherbas, P.; Hogness, D.S. The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell 67:59–77;1991. Macias, M.T.; Sastre, L. Molecular cloning and expression of four actin isoforms during Artemia development. Nucleic Acids Res. 18:5219 –5225;1990. Martin, G. Etude ultrastructurale de la re´ge´ne´ration de la glande du sinus chez l’oniscoide Porcellio dilatatus Brandt: Donne´es comple´mentaires sur l’ origine des terminaisons de cet organe neurohe´mal. J. Physiol. (Paris) 78:73–81;1982. Martin, G.; Maissiat, R.; Girard, P. Ultrastructure of the sinus gland and lateral cephalic nerve plexus in the isopod Ligia oceanica (Crustacea, Oniscoidea). Gen. Comp. Endocrinol. 52:28–50;1983. Minty, A.J.; Caravatti, M.; Robert, B.; Cohen, A.; Daubas, P.; Weydert, A.; Gros, F.; Buckingham, M. Mouse actin mRNAs. J. Biol. Chem. 256:1008 –1014;1981. Mykles, D.L. The mechanism of fluid absorption at ecdysis in the American lobster, Homarus americanus. J. Exp. Biol. 84: 89–101;1980. Mykles, D.L. Getting out of a tight squeeze-enzymic regulation of claw muscle atrophy in molting. Am Zool. 32:485– 494;1992. Mykles, D.L. Lobster muscle proteasome and the degradation of myofibrillar proteins. Enzyme Protein. 476(4–6):220–231; 1993. Mykles D.L.; Skinner, D.M. Crustacean muscle; atrophy and regeneration during moulting. In: Iwarog, B.M.; Levine, R.J.C.; Dewey, M.M. (eds). Basic Biology of Muscles: A Comparative Approach. New York: Raven Press; 1982:337–357. Mykles, D.L.; Skinner, D.M. Atrophy of crustacean somatic muscle and the proteinases that do the job. A review. J. Crust. Biol. 10:577–594;1990. Passano, L.M. Molting and its control. In: Waterman, T.H. (ed). The physiology of Crustacea. I. Metabolism and Growth. New York: Academic Press; 1960:473–560. Roberts, J.L. Thermal acclimation of metabolism in the crab Pachygrapsus crassipes Randall I. The influence of body size, starvation and molting. Physiol. Zool. 30:232–242;1957.
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35. Schram, F.R. Crustacea. Oxford: Oxford University Press; 1986. 36. Skinner, D.M. Amino acid incorporation into protein during the moult cycle of the land crab, Gecarcinus lateralis. J. Exp. Zool. 226–233;1965. 37. Skinner, D.M. Breakdown and reformation of somatic muscle during the moult cycle of the land crab, Gecarcinus lateralis. J. Exp. Zool. 163:115–124;1966. 38. Steel, C.G.H. Stages of the intermoult cycle in the terrestrial isopod, Oniscus asellus and their relation to biphasic cuticle secretion. Can. J. Zool. 60:429–437;1982. 39. Travis, D.F. The molting cycle of the spiny lobster Panulirus argus Latreille. I. Molting and growth in laboratory maintained individuals. Biol. Bull. 107:433–450;1954. 40. Watabe, S.; Imai, J.I.; Nakaya, M.; Hirayama, Y., Okamoto, Y.; Masaki, H.; Uozumi, T.; Hirono, I; Aoki, T. Temperatureacclimation induces light-meromyosin isoforms with different primary structures in carp fast skeletal muscle. Biochem. Biophys. Res. Com. 208:118 –125;1995. 41. Watabe, S.; Hirayama, Y.; Kikuchi, K.; Kakinuma, M.; Nakaya, M. An acclimation temperature-dependent expression of carp myosin isoforms. SEB abstract. C1.6; 1996. 42. Whiteley, N.M.; Harrison, P.; Taylor E.W.; El Haj A.J. The influence of environmentally induced changes in haemolymph ecdysteroid titre on the regulation of muscle growth in freshwater crayfish. Xth Ecdysone Workshop; 1992. (Abstract) 43. Whiteley, N.M.; Taylor, E.W.; El Haj, A.J. Actin gene expression during muscle growth in Carcinus maenas. J. Exp. Biol. 167:277–284;1992. 44. Whiteley, N.M., Taylor, S.E., Chang, E.S. and El Haj, A.J. Muscle growth in an isopod crustacean over the biphasic moult. Physiol. Zool. 68(4):148;1995. 45. Whiteley, N.M.; Taylor, E.W.; El Haj, A.J. A comparison of the metabolic cost of protein synthesis in stenothermal and eurythermal isopod crustaceans. Am. J. Physiol. 40(5):1295– 1303;1996. 46. Whiteley, N.M.; Taylor, E.W.; deSouza, S.C.R.; El Haj, A.J. Seasonal and moult related changes in haemolymph oxygen and acid-base levels in a wild population of Austropotamobius pallipes (Lereboullet). Freshwater Crayfish IX:189–199;1993.
ISSN 0305-0491/97/$17.00 PII S0305-0491(97)00130-2
Regulation of Muscle Gene Expression Over the Moult in Crustacea N. M. Whiteley and A. J. El Haj * School of Biological Sciences, University of Birmingham, Birmingham B15 2TT, U.K. ABSTRACT. Muscle growth in crustacea occurs over the moult and involves a corresponding increase in fractional rates of sarcomeric protein synthesis. Heterologous and homologous cDNA probes for the myofibrillar protein, actin and myosin, have been used to investigate the factors responsible for controlling muscle growth in crustaceans at the molecular level. Factors such as passive stretch, caused by ecdysial expansion; moulting hormones such as ecdysteroids, which regulate the cycle of moult related events; and temperature, an environmental factor known to influence the rates of many biochemical reactions, all play a role in modulating muscle growth in crustacea. In isopod crustaceans, the regulation of muscle growth produces new problems as these crustaceans are characterised by a biphasic moult cycle in which the posterior half of the old exoskeleton is shed hours to days before the anterior half. These animals therefore provide an ideal model for the study of differential muscle growth. In both decapod and isopod crustaceans, transcriptional regulation alone may not be sufficient to account for the the upregulation of sarcomeric protein synthesis during intermittent growth. Both passive stretch and elevated ecdysteroid titre increase protein synthesis rates for actin in lobster muscles, in vivo. These factors may be involved in the promotion of ribosomal activity and translational processing during ecdysial muscle growth. Temperature has a direct effect on protein synthesis rates and levels of actin mRNA expression and may be a principal factor in the control of seasonal moult cycles. comp biochem physiol 117B; 3:323–331, 1997. 1997 Elsevier Science Inc. KEY WORDS. Crustacean, ecdysteroid, muscle, moult cycle, actin, myosin, growth, gene expression
INTRODUCTION Muscle growth in crustaceans is intermittent (9,20,36,37) and closely associated with the moult cycle due to the presence of the rigid calcified exoskeleton. Increases in muscle mass are restricted to the ecdysial period when the old exoskeleton is shed and the new exoskeleton expands in size (9). The increase in size is due to an increase in hydrostatic pressure in the haemolymph (4) resulting from increased rates of water uptake (28). Fractional rates of protein synthesis increase 10-fold in the leg muscles of Carcinus during the premoult stages D3 –4 in preparation for ecdysis, and remain elevated in early postmoult (stages A, B) (11). The postmoult increase in protein synthesis rates coincides with an increase in muscle fibre length due to the addition of sarcomeres and an increase in the number of myofibrils due to longitudinal splitting (9). Protein synthesis rates also increase in the lobster, H. americanus, in both leg, claw, and abdominal muscles during late premoult and early postmoult (8). The elevation in protein synthesis rates is most surprising in the claw muscle, which has been shown in land Address reprint requests to: A. J. El Haj, School of Biological Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K. Tel. 121 414 5459; Fax 121 414 5925; E-mail: [email protected]. Received 20 June 1996; accepted 16 September 1996.
crabs to atrophy by 30–60% prior to ecdysis (37). This moult induced atrophy reduces the muscle mass of the large claw muscle to enable it to be squeezed through the relatively small aperture of the basi-ischial joint. The selective degradation of the claw muscle during premoult is followed by an increase in mass during postmoult (36,31), to sequence. These findings indicate that muscle growth in crustaceans is a complex process involving the interplay of synthesis and degradation rates in a tissue dependent manner that is closely coordinated with the moult cycle. The relationship between synthesis and degradation rates in the muscle tissue during ecdysis, raises some interesting questions as to the nature of the regulatory mechanisms involved in controlling muscle growth. The increase in protein synthesis rates in the claw muscle during premoult atrophy (36) indicates that degradation rates increase to exceed synthesis rates (29). Moult-induced atrophy is confined to the claw muscles and is thought to be regulated by intracellular proteolytic enzymes that increase in activity during premoult (29,30,32). Other factors are also thought to play a regulatory role during muscle growth, such as circulating moulting hormones (8,43) and mechanical stretch resulting from the expansion of the new exoskeleton during the immediate post moult stages (17). However, the mechanisms controlling differential regulation of muscle growth be-
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tween the different muscles is unclear. One avenue of investigation that may help to increase our understanding of the regulatory mechanisms involved includes molecular studies into the expression of actin and myosin genes during muscle growth. A review of the crustacean genes that have been cloned and used to study growth and development in crustaceans has recently been published (7). The object of the present review is to report on the latest developments in the area of molecular regulation of muscle growth by comparing moult related changes in gene expression in the muscles of decapod and isopod crustaceans in response to passive stretch, steroidal action and temperature. MUSCLE GROWTH OVER THE MOULT CYCLE Muscle Growth in Decapod Crustaceans To date, muscle growth studies in crustacea have been restricted to the relatively large decapod crustaceans. In decapods, muscle fibre growth results from an increase in fibre length by addition of new sarcomeres or by lengthening of existing fibres (9,20). Increases in cross-sectional area occurs by longitudinal splitting of existing myofibrils, maintaining a constant fibre number as described in vertebrate striated muscle (14). Over the moult cycle, muscle fibres in the walking leg of the lobster, H. americanus, increase in length in the first few hours of ecdysis (9) accompanied by the longitudinal splitting of larger myofibrils. Fractional rates of total protein synthesis have been measured in the muscles of a variety of decapod crustaceans including the land crab, Gecarcinus lateralis, the shore crab, Carcinus maenas, and the lobsters Homarus gammarus and H. americanus. Early studies in Carcinus, demonstrated a 10-fold increase in protein synthesis rates in the leg muscles during late premoult stages D3 –4, followed by an elevation in rates into early postmoult (stages A, B). This surge in protein synthesis rates, centred around ecdysis, is also found in the leg, claw, and abdominal muscles of H. americanus (12) (Fig. 1). The increase in protein synthesis rates observed in the claw muscle of the land crab Gecarcinus lateralis was thought to be associated with the synthesis of Ca21dependent proteinases for the specific regulation of myofibrillar proteins during atrophy (29,31,32). Growth of this muscle was suggested to occur in postmoult, when a second peak in protein synthesis rates was observed (36). An actin cDNA (HgAct) has been cloned from Homarus gammarus muscle using RT-PCR and degenerate oligonucleotide primers devised from published actin sequences (17). This 736 bp fragment encompasses the majority of the coding region of the actin sequence and recognizes a 1.6 kb mRNA in muscles composed of both fast and slow fibres from the lobster and from Antarctic and temperate isopods (Fig. 2). This clone has been used along with heterologous actin cDNAs from Artemia (24) and the mouse (27), to follow levels of mRNAs for actin in the muscles of a number of decapod crustaceans over the moult, indicating the high
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degree of homology between actin sequences from different species. Initial studies in Carcinus and the freshwater crayfish, Austrpotamobius pallipes revealed that actin mRNA levels increased in the leg muscles during premoult and postmoult (13,43). A much more detailed study in the American lobster, H. americanus, however, has revealed little variation in actin mRNAs in either the claw, leg, or abdominal muscles sampled each day for 40 days, over an entire moult cycle (P. Harrison and A. J. El Haj, unpublished observations). Ribosomal levels in the muscle tissue also remained unchanged over the moult (12). These observations suggest that in lobsters the increase in protein synthesis rates occurred at the level of translation from existing pools of mRNAs rather than by transcriptional regulation via changes in gene expression. However, interspecific variations may occur. Muscle Growth in Isopod Crustaceans Decapod crustaceans are characterised by a moulting pattern in which the entire exoskeleton is shed at one time to allow a general increase in body size over ecdysis. Very little is known about patterns of muscle growth in crustaceans belonging to other Orders that have different moulting patterns. One of the most intriguing in this respect is the biphasic moulting pattern shown by the Order Isopoda. In general, isopods have an elongate body plan and are flattened dorso-ventrally (35). The body is divided into the cephalon, seven thoracic segments, six abdominal segments and the terminal end plate, the telson. The segments are fairly uniform in character and it is difficult to distinguish between the three different sections. During the biphasic moult, the old exoskeleton splits between free thoracic segments four and five and the posterior half of the old exoskeleton is shed, hours to days before the anterior portion. After posterior ecdysis, the posterior portion of the body subsequently increases in size and the epidermis begins to recalcify before the anterior exoskeleton is shed. Therefore, the elongate body increases in size in two distinct phases separated by a period of hours to days. This biphasic pattern of growth includes the muscle groups located in the anterior and posterior segments and, therefore, presents a useful model for studying the role of systemic hormones in regulating differential muscle growth. The functional significance of this moulting pattern is unknown. It may enable isopods to maintain some motility during the moult, and enable the animals to excise themselves from the elongate exoskeleton. The biphasic moult may also be involved, at least in terrestrial isopods, in the mobilisation of calcium during the moult as suggested by Steel (38) who confirmed the presence of Ca21 stores in the white sternal plates of Oniscus asellus. Regardless of the function of the biphasic moult, there are some interesting questions to be addressed concerning the timing and regulation of muscle growth in these animals. The expansion of
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FIG. 1. Fractional rates of protein synthesis (Ks ) (A); and protein synthesis rates per unit of RNA (ribosomal activity) (B) in the leg, abdomen and claw muscle of the lobster H. americanus at three different stages of the moult cycle (a), and in intermoult lobsters after injection with 1026 mol l21 20-hydroxyecdysone (b). Values given as means 1SEM with significant differences from intermoult means at the 95% level of confidence represented by asterisks. [Taken from (18)].
the posterior segments hours to days before the anterior portion suggests differential growth patterns. Study of these animals and their biphasic moult pattern may help to unravel the relationship between the regulation of muscle growth and the timing of ecdysis. Initial studies have been carried out to examine the influence of the biphasic moult on fractional rates of protein synthesis (ks), and mRNA levels for actin, in the ventral longitudinal muscle of the marine isopod, Idotea rescata. This muscle constitutes the main muscle bulk of this species. It is organized segmentally and runs the entire length of the body. Histochemical analysis for myofibrillar ATPase activity in frozen transverse sections of the ventral longitudinal muscle, revealed that this muscle was predominantly composed of fast muscle fibres. The results showed that ks values were higher in the anterior half of the muscle (thoracic muscle segments 1–5) at 1.31% day21, as opposed to the posterior half (thoracic segments 6 and 7 to abdominal segments, 1–6) at 0.31% day21 during half moult when the posterior half of the exoskeleton had been shed (Fig. 3a). This difference continued into postmoult. Absolute rates of protein synthesis (total protein synthesized per day) varied in a similar manner with elevated rates in the anterior portion of the muscle up until 36 hr postmoult when rates were similar in both portions of the muscle (Fig. 3b). The capacity for protein synthesis (RNA : protein ratio) and the protein synthesis rate per unit of RNA or RNA activity also changed over the biphasic moult (Fig. 4a). Measurement of the mRNA levels for actin using the actin cDNA (HgAct) isolated from the lobster, H. gammarus (17) showed that actin mRNA levels remained unchanged in posterior and anterior portions of Idotea muscle over the biphasic moult
(Fig. 5). Myosin HC mRNA levels also remained unchanged as shown in Fig. 5. This indicates that the control of differential production of sarcomeric proteins in the anterior and posterior portions may be due to changes in protein synthesis rates from pools of sarcomeric protein mRNA rather than differential control of transcription rates. FACTORS REGULATING MUSCLE GROWTH Decapod Crustaceans Passive stretch and ecdysteroids have been implicated in playing a role in controlling muscle growth in decapodan crustaceans and in particular, Carcinus maenas and Homarus americanus (10,17,20). Passive stretch results in compensatory fibre lengthening over a period of 2 to 3 weeks (20) with a corresponding increase in actin mRNA levels after 2 weeks accounting for the elevated protein production necessary for new sarcomere assembly (17). A suggestion that ecdysial stretch may play a role in muscle growth in crustacea during the moult has been proposed. However, morphological experiments have shown that muscle fibre lengthening takes place within days, not weeks, and, therefore, passive stretch may not play a major role in the stimulation of muscle growth after the moult. This data demonstrates that crustacean muscle is stretch responsive as found in vertebrates, but the specific role of passive stretch in muscle growth during the moult is unclear. Ecdysteroids have been known to play a role in the regulation of the moult cycle for many decades, but the specific way in which ecdysteroids modulate muscle growth in crustacea has only just been investigated. Ecdysteroid receptor immunoreactivity has been demonstrated in the walking leg
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FIG. 3. (a) Fractional rates of protein synthesis (K s % synthe-
FIG. 2. Autoradiographs of Northern blots showing: (a) total
RNA from the extensor walking leg muscle and gonads of H. gammarus; and (b) total RNA from the body wall muscle of the Antarctic Isopod, Glyptonotus antarcticus (G), and the ventral longitudinal muscle of the temperate isopod, Idotea rescata (I). Both blots were hybridized to the cDNA for actin (HgAct) isolated from H. gammarus muscle (17). In (a), washing under low stringency conditions revealed a hybridization signal at 1.8 and 1.6 kb in the muscle and one band at 1.6 kb in the gonad. Under high stringency wash conditions, no hybridization signal was observed in the gonad but one band at 1.6 kb remained in the muscle. The size of the mRNA corresponds to actin mRNAs detected using a heterologous cDNA for mouse skeletal a-actin isolated from the mouse (27). [Fig. 2(a) was taken from (17)].
sised per day), and (b) absolute rates of protein synthesis (As, total protein synthesised per day), in the longitudinal muscle of Idotea rescata over the biphasic moult. Measurements were taken from pooled muscle samples with the number in each group given above the appropriate moult cycle stage. Halfm. represents isopods in the halfmoult stage of ecdysis when the animals have lost the posterior half of the old exoskeleton but retain the anterior portion. The anterior portion of the muscle was taken as that corresponding to free thoracic segments 1–4, and the posterior portion as muscle corresponding to thoracic segments 5–6, and abdominal segments.
extensor muscle of the lobster by Western blotting and immunocytochemistry (10) using a monoclonal antibody to the ecdysteroid receptor of Drosophila melanogaster developed by Koelle et al. (23). Preliminary observations suggest that receptor immunoreactivity is modulated over the moult with an increase in immunofluorescence in the lobster extensor muscle during premoult. Injections of premoult titres of the ecdysteroid, 20-hydroxyecdysone, into intermoult lobsters results in elevated fractional rates of protein synthesis after 3 days, in leg, claw, and abdominal muscles (8). However, attempts to mimic these effects in vitro have not
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FIG. 4. (a) Summary of the relationship between fractional rates of protein synthesis (K s), the capacity for protein synthesis
(RNA : protein ratio), and the protein synthesis rate per unit of RNA (K RNA), in the anterior and posterior portions of the ventral longitudinal muscle of I. rescata, during intermoult, premoult, and halfmoult. (b) Modification of a diagram taken from (18) to show changes in haemolymph ecdysteroid levels over the biphasic moult in the terrestrial isopod, H. brevicornis. Values for I. rescata have been added to this diagram and are represented by open symbols. E1 and E2 indicate ecdysis, where E1 represents posterior ecdysis and E2, anterior ecdysis. HM represents animals in halfmoult as described in Fig. 3. A, B, postmoult stages; C, intermoult; D0 to D3 premoult stages. Values given as means 6SEM.
been successful. Levels of mRNAs for actin and myosin sarcomeric proteins also respond to an elevation in ecdysteriod titre by showing transient increases in mRNA levels (A. J. El Haj, unpublished observations). These data suggest that circulating ecdysteroids act directly on the muscle through specific receptors to initiate muscle growth. The lack of any response in vitro may indicate a role for co-factors in the action of ecdysteroids on mRNA and protein synthesis levels in crustacean muscle. The role of one such factor, insulin related peptides, has been investigated using heterologous antibodies and cDNAs to recombinant IGFs (A. J. El Haj and E. S. Chang, unpublished observations). Our recent studies have concentrated on identifying ecdysteroid responsive genes in lobster muscles to establish if the complex
cascade of early and late genes which occurs in insects is also found in crustaceans. Isopod Crustaceans Hormonal control of the biphasic moult cycle has been most extensively studied in the terrestrial isopods. In these animals, the sinus gland is a cephalic neurohaemal organ located posterior to the optic lobe (25,26). Studies on Helleria brevicorrus (18), Oniscus asellus (21), and Idotea rescata (44), have shown that haemolymph ecdysteroid levels are low during intermoult, but concentrations increase to peak values during premoult and fall about 3 days before ecdysis of the posterior exoskeleton (Fig. 4b). A secondary but
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muno-assay, these authors were able to show that CCAP contents of the CNS increased 10-fold, 1–2 days before and during posterior ecdysis. CCAP levels returned to low levels during anterior ecdysis. Immunocytochemistry of premoult animals, localised the increase in CCAP levels to the VNC neural network corresponding to the segments posterior to the line of ecdysis (5). It is possible that neuropeptides such as CCAP secreted in specific thoracic/abdominal segments at certain times of the moult cycle, are involved in the control of this intricate moulting pattern. However, the specific role of ecdysteroids and various neuropeptides in the regulation of moult related changes in muscle ks remains to be investigated. TEMPERATURE EFFECTS ON MUSCLE GROWTH IN CRUSTACEA Protein Synthesis Rates
FIG. 5. Results of slot blot analysis of total RNA from the anterior and posterior segments of the ventral longitudinal muscle in I. rescata taken during halfmoult and 12 and 48 hr postmoult, and hybridized to (a) HgAct, the lobster cDNA for actin, and (b) a cDNA for the fast isoform of myosin heavy chain cloned in H. americanus by Cotton and Mykles (3). The relative abundance of the respective mRNA levels are expressed as the ratio between densitometry readings of the hybridization signal with HgAct or fast MHC against that of 18S rRNA.
smaller peak in haemolymph ecdysteroid levels was measured during half moult shortly after posterior ecdysis in both O. asellus and I. rescata. The pattern of haemolymph ecdysteroid variation in isopods is, therefore, similar to that shown in decapod crustaceans over the moult. Ecdysteroid levels in the anterior and posterior muscle segments did not differ over the biphasic moult (44), indicating that ecdysteroids are not involved in the control of ks during this period. Johnen et al. (21) have recently shown that the crustacean cardioactive peptide (CCAP) may have a function in the regulation of ecdysis behaviour in isopods. By isolating an antibody specific for CCAP in the isopod, Oniscus asellus, and developing a competitive enzyme im-
Fractional rates of protein synthesis (ks ) in the whole bodies and the muscle of intermoult crustaceans vary exponentially with temperature to give a Q10 of approximately 2, which corresponds to temperature-related changes in oxygen consumption rates, suggesting a link between the two variables. A similar response has been described in some fish species (19). In the eurythermal marine isopod, Idotea rescata, the increase in muscle and whole body ks over a 10°C rise in acclimation temperature (Q 10 5 1.99) (Fig. 6), was accompanied by an increase in actin mRNA levels and an increase in RNA activities at a constant RNA : protein ratio (12). This suggests that both rates of transcription and translation are directly related to temperature. Rates of protein degradation may also change with temperature, resulting in an increase in rates of protein turnover with no net protein accretion. Fractional rates of protein synthesis also vary with temperature in the muscle tissue of a seasonally acclimatized crustacean, the freshwater crayfish (A. pallipes), which experiences wide variations in seasonal temperatures (1–21°C) in its natural environment (46). A decrease in water temperature in the autumn/winter, when the animals are in intermoult, is accompanied by an exponential decrease in ks in the leg muscles to give a Q10 value of 5.2 suggesting that there is an acute drop in ks in the winter when crayfish are relatively inactive and are not feeding. The lack of temperature compensation for ks in the leg muscles of freshwater crayfish during the winter is also observed in an Antarctic isopod crustacean, Glyptonotus antarcticus. This benthic isopod lives within a narrow temperature range of 21.89°C in the winter to 0.5°C in the summer, and is therefore strictly stenothermal and extremely intolerant to increases in temperature (1). Physiological studies have shown that this isopod crustacean does not show any metabolic cold adaptation despite the low temperatures of the Southern Ocean, but maintains low rates of oxygen uptake and extremely low rates of whole body protein synthesis (45), in keeping with its relatively inactive and sedentary lifestyle. Glyptonotus
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termoult crayfish resulted in an increase in circulating ecdysteroid levels and subsequent moult within 10 weeks of captivity, one month ahead of the animals in the wild (43). Consequently, low winter temperatures may inhibit moulting activity even though the mechanisms responsible for the morphological, biochemical, and physiological changes over the moult are present and functional in over-wintering animals. It is not known if an increase in moulting frequency results in a concomitant increase in muscle mass over each moult. This relationship remains to be investigated. Myosin Isoforms
FIG. 6. Comparison of the effects of temperature on frac-
tional rates of protein synthesis (k s) in the whole body and ventral longitudinal muscle of I. rescata acclimated to 4° and 14°C. Whole body values given as means 6SEM. Values for k s in the muscle taken from pooled samples with the number of samples given above each data point.
shares many characteristics of other benthic marine Antarctic species in that they are slow growing with low moulting frequencies and have a tendency to gigantism as animals range from 9 to 120 mm in length, which is relatively large for isopod crustaceans. Moult Frequency In addition to the direct effects of temperature on rates of protein turnover in the muscles of intermoult crustaceans, rising temperature has also been shown to increase the frequency of the moult and, therefore, growth rates. Early studies on moulting behaviour in crustaceans, found that an increase in temperature led to an increase in the number of moults (2,39), and to a decrease in the duration of each moult cycle (33). Alternatively, a decrease in temperature reduced the incidence of moulting (6,34). Temperature also plays an important role in the control of seasonal moult cycles. In the temperate freshwater crayfish, A. pallipes, an increase in water temperature from 6° to 16°C initiated the moult in intermoult animals brought into the laboratory in January. In the wild, these animals moult between May and September, when water temperatures range between 16° and 21°C. Exposure to higher temperatures in winter in-
Temperature acclimation in fish is followed by a number of partial capacity adaptations for metabolic processes in the muscle, such as muscle contraction and swimming performance (22). In Antarctic fish, the adaptation to low temperatures involves a change in the myofibrillar apparatus to give a specific ATPase activity which may be achieved by changes in gene expression of the protein isoforms (16). Changes in isoform expression are also observed in the red and white muscles of eurythermal fish acclimated to a range of environmental temperatures (15,40,41). Multiple isoforms of both regulatory and contractile proteins have been identified in fast and slow muscles from a variety of crustaceans [cf. (3)], but relatively little is known about the effects of environmental temperature on the expression of these isoforms. In the carp, Cyprinus carpio, three myosin heavy chain isoforms are expressed in an acclimation temperature-dependent manner, encoded by multiple genes (41). Initial studies have used SDS PAGE analysis to separate the different isoforms present in the muscles of various crustaceans over a range of temperatures. Fast muscle fibres from the temperate isopod, Idotea rescata, acclimated at 4° and 15°C, the Antarctic isopod Glyptonotus held at 0°C and H. americanus at 15°C had similar myosin HC isoforms (12). An examination of the myosin light chains are required to fully characterise potential differences in myosin protein isoforms between crustaceans living in different thermal regimes. However, a molecular characterisation of myosin heavy chains has been initiated in the giant Antarctic isopod Glyptonotus antarcticus. In recent experiments, degenerate oligonucleotide primers have been developed and used in conjunction with RT-PCR of total RNA from the abdominal muscle of Glyptonotus, to isolate a PCR product of 544 bp in length (N. M. Whiteley and A. J. El Haj, unpublished observations). Subsequent cloning and sequencing of this cDNA has revealed 60–75% homology with myosin heavy chain clones from a range of different animals including invertebrates such as Drosophila, C. elegans, and A. irradians to vertebrates such as Rattus norvegicus. This cDNA for myosin HC hybridized to a mRNA transcript of 6.6 kb in size corresponding to myosin HC mRNAs (3). Subsequently
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this cDNA will be used to screen a genomic DNA library isolated from Glyptonotus, to further characterise the gene or gene family for myosin HC. SUMMARY Regulation of muscle growth in crustaceans is a complex process involving fine control of muscle protein synthesis and degradation rates. The mechanisms regulating this relationship appear to be tissue specific, involving a combination of translational processing and transcriptional regulation that may vary from species to species. Ecdysteroid hormones and passive stretch have been implicated in the control of cyclical muscle growth in crustaceans. Recent studies on intermoult lobsters have demonstrated that both factors can increase protein synthesis rates and fibre lengthening in the leg, claw, and abdominal muscles, but their mode of action over the moult cycle remains to be determined. The biphasic moult pattern in isopod crustaceans is characterised by differential muscle growth due to a variation in protein synthesis rates between anterior and posterior muscle segments during halfmoult and immediate postmoult stages. Ecdysteroid levels in the two muscle sections remain unchanged over the moult but investigations into cardioactive peptides suggest that neuropeptides and the CNS may play a more critical role in the regulation of the biphasic moult. Temperature has a direct effect on protein synthesis rates, giving a Q10 value of 1.99 in I. rescata between 4° and 14°C, and is important in the environmental regulation of the seasonal moult. The temperature related increase in both whole body and muscle rates of protein synthesis, however, is thought to be associated with equal increases in degradation rates. In some crustacea, temperature may, therefore, play a more fundamental role in the control of muscle growth by promoting the moult, increasing moulting frequency and increasing the opportunity for accretion of sarcomeric proteins. We would like to thank the following people for their help and collaboration during the studies reported in this review: Prof. E. Chang for providing research facilities in this laboratory at the Bodega Marine Laboratory, University of California at Davis, for the studies on Idotea, Prof. Ted Taylor, Dr. Paul Harrison, Sharon Taylor, and Sue Clark for their contributions to this work at the University of Birmingham, and to Prof. Andrew Clarke and Dr. Lloyd Peck for their collaboration during the research on the Antarctic isopod, Glyptonotus antarcticus. We are grateful to NERC, AFRC, The Royal Society, and The Linnean Society, U.K., for financial support.
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