THOMAS J. MOYLAN and BRUCE D. SIDELL, School of Marine Sciences, University of Maine, Orono, Maine 04469
The six families of the perciform suborder Notothenioidei dominate the fish fauna of the southern ocean. Our work has focused primarily on the Channichthyidae, commonly referred to as icefishes, which are largely endemic to the waters surrounding Antarctica (Dewitt 1971). The 15 known species of the icefishes are unique among adult vertebrates in their complete lack of hemoglobin expression (Ruud 1954). In contrast, the presence of the intracellular oxygen-binding protein myoglobin in these fishes has been a source of some debate. A report by Douglas et al. (1985) found myoglobin in heart ventricles of icefishes, whereas others (Hamoir 1988; Eastman 1990) suggest that myoglobin is absent in icefishes. Using a combined immunological and molecular approach, we have shown that there is extremely variable expression of both myoglobin protein and the mRNA coding for this protein among the Channichthyidae (Sidell et al. 1997). One of the next logical steps was to determine the intracellular concentrations of myoglobin protein and myoglobin mRNA in heart ventricle of these fishes, the primary objective of this study.
Intracellular concentrations of myoglobin in heart ventricles were determined by denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblot analysis. After electrophoresis, gels were stained to reveal protein bands. To identify the myoglobin-associated band, protein from a duplicate gel was immediately electroblotted to a membrane and detected with both monoclonal and polyclonal antihuman myoglobin antibodies as previously described (Sidell et al. 1997). Each gel also contained a standard curve generated by loading known amounts of myoglobin purified from heart ventricle of the notothenioid fish Notothenia coriiceps . Myoglobin concentrations in sample lanes were calculated from the linear relationship between milligram (mg) purified myoglobin standard and integrated area of densitometrically scanned peaks.
This study reports myoglobin protein concentrations in heart ventricles in eight species of icefishes ( Chionodraco rastrospinosus , hamatus , and myersi, Pseudochaenichthys georgianus , Cryodraco antarcticus , Chionobathyscus dewitti , Neopagetopsis ionah , and Chaenodraco wilsoni ) and in two nototheniid species ( Gobionotothen gibberifrons , Trematomus newnesi ). Five additional icefish species lacked detectable myoglobin protein ( Chaenocephalus aceratus , Pagetopsis macropterus and maculatus , Champsocephalus gunnari, and Dacodraco hunteri ), (table). Myoglobin expression was extremely tissue-specific in both icefishes and related red-blooded notothenioid species examined ( G. gibberifrons and T. newnesi ). Myoglobin protein was found only in heart ventricle and was absent from other tissues including the primary oxidative skeletal muscle in these labriform swimmers, the pectoral adductor profundus. Estimates of myoglobin concentration in hearts of channichthyid and nototheniid species are comparable in both families (table) and are similar to values reported for sedentary, benthic fishes from temperate zones (Driedzic and Stewart 1982).
To confirm the pattern of myoglobin protein expression revealed by immunoblot analysis, a myoglobin-specific cDNA probe was used to test definitively for the presence of a messenger RNA (mRNA) coding for myoglobin protein. Total RNA was extracted from heart muscle, separated electrophoretically on agarose gels and transferred to membrane and incubated with a myoglobin-specific cDNA probe (Sidell et al. 1997). As expected, an mRNA of the appropriate size did hybridize to the probe in RNA isolated from heart ventricle of the eight species expressing myoglobin protein. Of the five icefish species lacking myoglobin protein expression, four ( P. macropterus , P. maculatus , D. hunteri , and C. aceratus ) also lack detectable myoglobin mRNA. In the fifth species ( C. gunnari ), an mRNA of the same size as myoglobin mRNA (0.9 kilobases) did hybridize to the myoglobin probe, in low but consistently detectable amounts (Sidell et al. 1997). Thus, for C. gunnari the mechanism(s) responsible for loss of myoglobin protein expression appears to differ from that occurring in the four non-mRNA expressing species.
Slot blot analysis was used to quantify the amount of myoglobin mRNA present in heart ventricle and pectoral adductor tissues. Denatured N. coriiceps myoglobin cDNA insert served as an internal positive control and was used to generate a standard curve within each blot. Myoglobin mRNA concentrations in slots were calculated from the linear relationship between picogram (pg) purified N. coriiceps cDNA standard loaded versus integrated signal-area of densitometrically scanned peaks. Myoglobin mRNA concentrations in heart ventricle for channichthyid species expressing myoglobin protein ranged from 16.22±2.17 to 0.78±0.02 picograms of myoglobin mRNA per milligram of total RNA [pg Mb mRNA (mg total RNA)-1]. The lowest myoglobin mRNA concentrations were found in C. gunnari [0.33±0.09 pg Mb mRNA (mg total RNA)-1], the only icefish species that lacked detectable myoglobin protein while still expressing message. Myoglobin mRNA concentrations for the two red-blooded nototheniid species fell between the high and low values determined for the Channichthyidae (table).
Comparing levels of myoglobin protein versus the corresponding mRNA pool for a given species reveals that the steady-state concentration of myoglobin protein does not parallel the steady-state concentration of myoglobin mRNA (table). The greatest range in myoglobin mRNA levels found in icefishes is the approximately 20-fold difference observed in the congeneric species C. rastrospinosus and C. hamatus . This observed difference in myoglobin mRNA pool is not reflected in the standing stock of myoglobin protein, which is equivalent in hearts of these two species. This result suggests that synthesis and/or turnover of the protein is not coordinately regulated with the mRNA pool. The lack of correlation between myoglobin protein and myoglobin mRNA concentrations in notothenioid fishes suggests protein concentrations are not regulated at the mRNA level.
Phylogenetic analysis possible at this time does not suggest the Channichthyidae are undergoing selective pressure toward a myoglobinless state (Sidell et al. 1997). Indeed, given the seemingly random loss of myoglobin expression in this family, we cannot determine if myoglobin (+) or myoglobin (-) is the most derived state within the Channichthyidae.
We gratefully acknowledge the generous contribution of samples by A.L. DeVries (University of Illinois), G. diPrisco and R. Acierno (Italian National Antarctic Program), T. Iwami (Tokyo Kasei Gakuin University), and H.W. Detrich (Northeastern University). Personnel at the U.S. Antarctic Program's Palmer Station and the masters and crew of R/V Polar Duke provided invaluable support during the course of our work. This work was supported by National Science Foundation grants OPP 92-20775 and OPP 94-21657 to Bruce D. Sidell.
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