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Comparative examination of the soluble muscle proteins of two species of angler-fish (Lophiidae): Lophius piscatorius (L.) and Lophius budegassa (Spinola)
Comp. Biochem. Physiol. Vol. 83B, No. 2, pp. 371-374, 1986
0305-0491/86 $3.00 + 0.00 © 1986 Pergamon Press Ltd
Printed in Great Britain
COMPARATIVE EXAMINATION OF THE SOLUBLE MUSCLE PROTEINS OF TWO SPECIES OF ANGLER-FISH (LOPHIIDAE): LOPHIUS P I S C A T O R I U S (L.) A N D LOPHIUS BUDEGASSA (SPINOLA) W. W. CROZIER Department of Agriculture for Northern Ireland, Fisheries Research Laboratory, 38 Castleroe Road, Coleraine, Co. Londonderry, Northern Ireland (Received 17 July 1985) Abstract--l. A comparative examination of sarcoplasmic proteins of the two nominal European species
of angler-fish, Lophius piscatorius and L. budegassa was carried out using isoelectric focusing techniques. 2. Two protein bands differing in isoelectric point proved diagnostic for L. budegassa (pI 4.40 and pI 5.75) while a third characterized L. piscatorius (pI 4.65). 3. These species-specific protein profiles provide a method of species discrimination independent of morphological criteria. 4. Within-species heterogeneity of banding pattern suggested the presence of polymorphic gene loci of potential use in studies of population structure.
INTRODUCTION
Although the systematics of the family Lophiidae have been extensively studied (Regan, 1903; Caruso, 1981, 1983 and references therein) until recently the relative taxonomic status of even the commoner species of angler-fish remained contended (Le Danois, 1974). This taxonomic uncertainty resulted from the unreliability of morphometric measurements (due to the flaccid condition of these fish) and overlapping of meristic counts (Wheeler et al., 1974; Caruso, 1983). Two species within the genus Lophius are presently described for the North Atlantic (L. piscatorius in northern European waters and L. americanus in North American waters), though formerly only a single species L. piscatorius was recognised (Berrill, 1929). However, most confusion centred on the taxonomic distinction of L. piseatorius and L. budegassa Spinola. Although the latter has its geographical distribution centred in the Mediterranean (Le Danois, 1974; Wheeler, 1978) its range extends well into northern European waters (Wheeler et al., 1974; Fitzmaurice, 1976; Crozier, 1985) thus occurring sympatrically with L. piscatorius. Authors have suggested that only a single species is present throughout the entire European range of Lophius (Lozano y Rey, 1960), or that a morphologically intermediate form occurs in areas of range overlap (Du Buit and Lam Hoai Thong, 1971), though the most recent examination of the problem supports the existence of two species (Caruso, 1983). At present, management of the European fishery for angler-fish is based on a precautionary total allowable catch (1984 T.A.C. 40,000 tonnes), set using historical catch levels, no biological data being available for a scientific stock assessment. This T.A.C. is managed on a joint species basis, L. piscatorius and L. budegassa not being separated in official catch statistics (1984 report of the E.E.C. 371
Scientific and Technical Committee for Fisheries). Pectoral fin ray counts (Guillou and Njock, 1978) and the morphology of the illicium (Caruso, 1983) have been used to achieve separation, but these are difficult to evaluate in small individuals and cannot be used for routine separation as will be required for effective assessment of commercial exploitation of these species. Peritoneum colouration (black in L. budegassa, white in L. piscatorius) appears to provide the most adequate distinction between the species (Guillou and Njock, 1978) but its validity as a taxonomic feature is untested. Furthermore, in several European countries much of the angler-fish catch is landed as tails only (Dupouy, 1984), thus morphological characteristics cannot assist in the evaluation of the relative contributions of either species to the European fishery. The utility of electrophoresis of proteins in determining purely genetic affinities among and within species of plants and animals is well documented (for review see Ferguson, 1980). The difficulty encountered in separation of environmentally induced from genetically controlled variation which inevitably complicates the use of morphological features in taxonomy is avoided by biochemical examination of protein molecules which are directly coded by the genome. Moreover, characterization of protein types can be achieved using small amounts of material and from individuals of any age, often without damage to the specimen which may be required for parallel morphological examination. Analytical isoelectric focusing methods achieve separation of proteins solely on the basis of their net electric charge, electrophoresis being complete when the proteins have reached their isoelectric points (pIs). This method which separates and concentrates proteins characteristically different in pI is capable of resolving proteins differing by 0.0025 units in their pI values (Allen et al., 1974). Thus absolute positions of bands on
372
W.W. CROZIER
protein profiles obtained using isoelectric focusing are reproducible, as distinct from conventional electrophoresis where relative mobilities are timedependent (Jamieson and Turner, 1980). In many instances divergence in the process of speciation has resulted in the accumulation of distinctive genes which allow unequivocal separation of taxonomic units (e.g. McGlade et al., 1983) and enables construction of taxonomic keys which reflect purely genetic characteristics (Mork et at., 1983). The present paper reports comparative electropboretic examination of skeletal muscle proteins of L. piscatorius and L. budegassa in an attempt to verify the presently used methods of species identification, and to provide a method of species discrimination in cases where morphological criteria cannot be applied.
MATERIALS AND METHODS
The angler-fish samples, comprising 147 L. piscatorius (10.9 129.0cm) and 31 L. budegassa (22.7 61.2cm) were obtained from catches taken by commercial fishing vessels in the north-eastern Atlantic and Irish Sea during 1984/1985 (Table 1). Classification of individual fish into species was carried out prior to and independently from the biochemical analysis. This was based on criteria used by Wheeler et al. (1974) and Fitzmaurice (1976), meristic counts, morphology of the illicium and peritoneum colour being examined (Crozier, 1985). Samples of white skeletal muscle were dissected from most fish within 12 hr of capture and stored individually frozen at -80~C, however some fish were stored at -20cC on landing, until transport to the laboratory. Prior to electrophoresis, extraction of soluble proteins was achieved by mechanical homogenization in an equal volume of ice cold 0.01 M Tris HCL, pH 7.0, followed by centrifugation at 3000g for 15min at 4'~C. Isoelectric focusing was carried out on the LKB Ultraphor apparatus using commercially available 1 mm thick polyacrylamide gels (Amopholine PAG plates). Electrophoretic conditions followed manufacturers recommendations. Wide range (pH 3.5 9.5) and narrow range (pH 4.0-6.5) gels were used as appropriate to identify and then closely resolve areas of interest. Isoelectric points of protein bands were obtained by comparing their equilibrium positions with those of reference sets of proteins having known isoelectric points (Pharmacia pI calibration markers). Following electrofocusing, proteins were fixed in 0.7 M trichloroacetic acid/0.16 M sulphosalicylicacid and visualized by staining with 0.1% Coomassie Brillant Blue R.
RESULTS
Separations of muscle extracts in isoelectric focusing gels of pH range 3.5-9.5 showed in excess of 50 bands (Fig. 1). While several of the major and many of the minor bands were common to all individuals, the samples fell into two discrete groups, separable on
the basis of presence or absence of major bands A and B respectively. On comparison of individual samples with the initial morphological classification these groups corresponded exactly to L. piscatorius and L. budegassa. Thus band A was common to all L. piscatorius examined but absent in L. budegassa, while band B was fixed in L. budegassa alone. No intermediate electrophoretic profiles were observed. In order to further examine these differences individuals were subjected to isoelectric focusing using narrow pH range gels (pH 4.0-6.5), thus achieving greater resolution of the anodal part of the pH gradient. The resultant protein profiles are illustrated in Fig. 2. As before bands A and B separate the two Lophius species, thus confirming their validity as species-specific genetic markers. In addition the increased resolution revealed a further band (C) present only in individuals of L. budegassa. Thus bands B and C biochemically define L. budegassa, while band A defines L. piscatorius. The isoelectric points of these bands were estimated as: pH 4.40 (band B); pH 4.65 (band A); pH 5.75 (band C). Side by side electrophoretic comparisons of samples from the various sampling locations did not reveal any consistent inter-regional differences in banding pattern within either species. However, differences among individuals in presence or absence of several bands were detected within both species (Figs 1 and 2). These heterogeneities were unrelated to area of capture, age or sex of individuals and suggest the segregation of several polymorphic gene loci within L. piscatorius and L. budegassa. Given the acknowledged danger of artefactual heterogeneity on IEF profiles of samples subject to differing storage/extraction procedures (Righetti and Drysdale, 1976; King and Moffett, 1984) it is essential that non-genetic variation be identified prior to concluding that any heterogeneity has a genetic basis. Because of the differing sampling procedures necessary in the present investigation control experiments were carried out to determine whether time period between capture and sampling, and length of frozen storage altered protein profiles. The only detectable heterogeneity among samples subject to differing treatment was a slight shift towards the low pH end of the gel (more electropositive) of the entire banding pattern following prolonged (10 months) frozen storage. The agreement of the biochemical with the morphological distinction between the two species supports the validity of the morphological features used for the initial identification. In this respect the colour of the peritoneum provides the most convenient and indeed the most accurate method of separation, no intermediate colouration being observed among the 178 individuals examined.
Table 1. Samples of Lophius examined in the present investigation Date
Location
22.6.84 June 1984 5.7.84 18.10.84 15.11.84 9.5.85
West Scotland Irish Sea Irish Sea South-west Ireland Irish Sea West Ireland
A p p r o x i m a t e position 56"20'N 53"20' 54'20' 51"20' 53"45' 54'~30 '
7 50'W 550' 455' 11"30' 530' 9"25 '
L. piscatorius 17 21 50
L. budegassa 19
I1 48 11 147
1 31
Electrofocusing of Lophius muscle proteins
373
pH 3.5
C-
Origi
pH 9.5 b
p
p
p
p
b
p
b
p
b
p
p
b
b
Fig. 1. General protein patterns of skeletal muscle extracts of two Lophius species separated by isoelectric focusing in a pH gradient 3.5-9.5. Samples: (a) Lophius piscatorius; (b) L. budegassa.
~-~
pH 4.0
Origi
pH 6.5 p
p
p
b
p
b
p
b
p
p
b
p
p
p
Fig. 2. General protein profiles of Lophius spp. separated by isoelectric focusing in a pH gradient 4.0-6.5. Labelling as Fig. 1. Band C is indicated for individuals of L. budegassa.
DISCUSSION The advantage of using biochemical characteristics to identify species is that they can provide a genetic base, allowing verification on organismal differences. This approach has been applied successfully to .aid
resolution of taxonomic difficulties in many organisms including tapeworms (Bylund and Djupsund, 1977), nematodes (Fleming and Marks, 1982) and fishes, including eels (Jamieson and Turner, 1980), coregonids (Ferguson et al., 1978) and redfish (McGlade et al., 1983). Further applications utilize
374
W.W. CROZIER
specificity of proteins at the species level to identify fillets of marine fish (Lundstrom, 1977) and to determine the species composition of pelagic samples of fish eggs (Mork et al., 1983). The results reported here have direct applicability to the future scientific assessment of the increasingly important European fishery for Lophius spp., as biochemical examination of muscle proteins will offer a convenient and reliable method for determining the relative contributions of L. piscatorius and L. budegassa to total landings of angler-fish. Lundstrom (1981) using agarose and polyacrylamide gels examined skeletal muscle extracts of Lophius americanus by isoelectric focusing. Although homologies between particular protein bands cannot be established without direct side by side electrophoretic comparison, examination of the illustrations given in Lundstrom (1981) suggests that while L. americanus display many bands in c o m m o n with L. piscatorius and L. budegassa they possess distinctive (and possibly species-specific) bands in the pH range 3.5-4.5. Lundstrom (1981) reported reproducible differences among individuals in respect of banding pattern (ten putative genotypes being identified) suggestive o f the presence of polymorphic gene loci in this species. The present research permits for the first time definitive separation of L. piscatorius and L. budegassa at the biochemical level. The species-specific protein profiles described here can be viewed as evidence that these two lophiid angler-fishes have diverged sufficiently from their c o m m o n ancestor to have accumulated distinctive genes. A comparative examination of the genetic expression of a range of enzymatic proteins presently being carried out by the author may well reveal further evidence of genetic divergence between these species, gene frequency data allowing calculation of genetic divergence and estimation of approximate time of divergence (Nei, 1975). The suspected presence of polymorphic gene loci in both European angler-fish species warrants further investigation, as these would potentially serve as genetic markers in an examination of population structure. Acknowledgements--The author wishes to thank Mr L. Girvan and Dr R. Grainger for assistance with obtaining samples of Lophius. Dr D. King made useful comments on a preliminary draft of the manuscript. The provision of a research grant by the Jeffreys Association Limited is gratefully acknowledged. REFERENCES
Allen R. C,, Harley R, A. and Talamo R. C. (1974) A new method for determination of alpha-l-antitrypsin phenotypes using isoelectric focusing on polyacrylamide gel slabs. Amer. J. clin. Pathol. 62, 732 739. Berrill N. J. (1929) The validity of Lophius americanus Val. as a species distinct from L. piscatorius Linn., with notes on the rate of development. Contrih. Can. Biol. Fish. 12, 145-151. Bylund G. and Djupsund B. M. (1977) Protein profiles as an aid to taxonomy in the genus Diphyllobothrium. Z. Parasitenk. 51, 241-247. Caruso J. M. (1981) The systematics and distribution of the tophiid angler-fishes : I. A revision of the genus Lophiodes with the description of two new species. Copeia 3, 522-549.
Caruso J. H. (1983) The systematics and distribution of the lophiid angler-fishes. II. Revisions of the genera Lophiomus and Lophius. Copeia i, 11 30. Crozier W. W. (1985) Observations on the distribution of Lophius budegassa Spinola, 1807 in the waters around Northern Ireland. Ir. Nat. J. 21, 410-412. Du Buit M. H. and Lam Hoai Thong (1971) Quelques notes sur le filament p~cheur (illicium) des baudroies (Lophius" spp.) d+barqu6es fi Concarneau. Tray. Lab. Biol. halieut. Univ. Rennes. 5, 45-47. Dupouy N. (1984) Relation entre la longeur totale ct quelques measures caract6ristiques de la t6te de la baudroie (Lophius piscatorius). I.C.E.S.C.M. 1984/G: 12, 6pp. Ferguson A. (1980) Biochemical Systematics and Ez:olution. Blackie, Glasgow. Ferguson A., Himberg K. J. M. and Savardson G. (1978) Systematics of the Irish pollan (Coregonus pollan Thompson). An electrophoretic comparison with other holarctic Coregoinae. J, Fish Biol. 12, 221 233. Fitzmaurice P. (1976) Lophius budegassa Spinola 1807 from Irish inshore waters, lr. Nat. J. 18, 279 280. Fleming C. C. and Marks R. J. (1982) A method for the quantitative estimation of Globodera rostochiensis and Globodera pallida in mixed-species samples. Rec. Agric. Res., Dep. Agric. N. Ireland 30, 67 70. Guillou A. and Njock J. C. (1978) Analyse des structures de la p~che dans les ports de la c6te atlantique francaise de 1961 ~i 1975 et des incidences du chalutage sur les stocks de principles esp6ces concernees par cette activit6 dans les mers adjacentes. Rev. Tray. lnst. P~ches Mar. 42, 17-164. Jamieson A. and Turner R. J. (1980) Muscle protein differences in two eels Anguilla anguilla (Linneaeus) and Anguilla rostrata (Le Seuer). Biol. J. Linn. Soc. 13, 41 45. King D. P. F. and Moffett I. J. J. (1984) Evaluation of isoelectric focusing of sarcoplasmic proteins as a means of separating stocks of Atlantic herring (Clupea harengus L.) in the north Irish Sea. Rec. Agric. Res., Dep. Agric. N. Ireland 30, 67-70. LeDanois Y. (1974) Etude ost6o-myologique et r6vision systematique de la famille des lophiidae (Pediculates Haplopt~rygiens). Mem. Mus. nat. Hist. nat. Paris 19, 127. Lozano y Rey D. L. (1960) Peces Fisoctistos, tercera parte. Mere. Real Acad. cienc, exact. Fis. nat. Mad. 25, 1 613. Lundstrom R. C. (1977) Identification of fish species by thin-layer polyacrylamide gel isoelectric focusing. Fish. Bull. Wash. 75, 571-576. Lundstrom R. C. (1981) Fish species identification by isoelectric focusing: sarcoplasmic protein polymorphism in monkfish (Lophius americanus). J. Ass. 0[Ii Anal. Chem. 64, 32-37. McGlade J. M., Annand M. C. and Kenchington T. J. (1983) Electrophoretic identification of Sebastes and Helicolenus in the north western Atlantic. Can. J. Fish. Aquat. Sci. 40, 1861-1870. Mork J., Solemdal P. and Sundnes G. (1983) Identification of marine fish eggs: a biochemical genetics approach. Can. J. Fish. Aquat. Sci. 40, 361 369. Nei M. (1975) Molecular Population Genetics and Evolution. North-Holland, Amsterdam. Regan C. T. (1903) A revision of the fishes of the family Lophiidae. Ann. Mag. Nat. Hist. Series 7, 11, 277 285. Righetti P. G. and Drysdale J. W. (1976) Isoelectric focusing. In Laboratory Techniques in Biochemistry and Moh, cular Biology (Edited by Work T. S. and Work E.). North-Holland, Oxford. Wheeler A. (1978) Key to the Fishes of Northern Europe. Fredrick Warne, London. Wheeler A., DeGroot S. J. and Nijhssen H. (1974) The occurrence of a second species of Lophius in Northern European waters. J. Mar. Biol. Ass. U.K. 54, 619 623.
0305-0491/86 $3.00 + 0.00 © 1986 Pergamon Press Ltd
Printed in Great Britain
COMPARATIVE EXAMINATION OF THE SOLUBLE MUSCLE PROTEINS OF TWO SPECIES OF ANGLER-FISH (LOPHIIDAE): LOPHIUS P I S C A T O R I U S (L.) A N D LOPHIUS BUDEGASSA (SPINOLA) W. W. CROZIER Department of Agriculture for Northern Ireland, Fisheries Research Laboratory, 38 Castleroe Road, Coleraine, Co. Londonderry, Northern Ireland (Received 17 July 1985) Abstract--l. A comparative examination of sarcoplasmic proteins of the two nominal European species
of angler-fish, Lophius piscatorius and L. budegassa was carried out using isoelectric focusing techniques. 2. Two protein bands differing in isoelectric point proved diagnostic for L. budegassa (pI 4.40 and pI 5.75) while a third characterized L. piscatorius (pI 4.65). 3. These species-specific protein profiles provide a method of species discrimination independent of morphological criteria. 4. Within-species heterogeneity of banding pattern suggested the presence of polymorphic gene loci of potential use in studies of population structure.
INTRODUCTION
Although the systematics of the family Lophiidae have been extensively studied (Regan, 1903; Caruso, 1981, 1983 and references therein) until recently the relative taxonomic status of even the commoner species of angler-fish remained contended (Le Danois, 1974). This taxonomic uncertainty resulted from the unreliability of morphometric measurements (due to the flaccid condition of these fish) and overlapping of meristic counts (Wheeler et al., 1974; Caruso, 1983). Two species within the genus Lophius are presently described for the North Atlantic (L. piscatorius in northern European waters and L. americanus in North American waters), though formerly only a single species L. piscatorius was recognised (Berrill, 1929). However, most confusion centred on the taxonomic distinction of L. piseatorius and L. budegassa Spinola. Although the latter has its geographical distribution centred in the Mediterranean (Le Danois, 1974; Wheeler, 1978) its range extends well into northern European waters (Wheeler et al., 1974; Fitzmaurice, 1976; Crozier, 1985) thus occurring sympatrically with L. piscatorius. Authors have suggested that only a single species is present throughout the entire European range of Lophius (Lozano y Rey, 1960), or that a morphologically intermediate form occurs in areas of range overlap (Du Buit and Lam Hoai Thong, 1971), though the most recent examination of the problem supports the existence of two species (Caruso, 1983). At present, management of the European fishery for angler-fish is based on a precautionary total allowable catch (1984 T.A.C. 40,000 tonnes), set using historical catch levels, no biological data being available for a scientific stock assessment. This T.A.C. is managed on a joint species basis, L. piscatorius and L. budegassa not being separated in official catch statistics (1984 report of the E.E.C. 371
Scientific and Technical Committee for Fisheries). Pectoral fin ray counts (Guillou and Njock, 1978) and the morphology of the illicium (Caruso, 1983) have been used to achieve separation, but these are difficult to evaluate in small individuals and cannot be used for routine separation as will be required for effective assessment of commercial exploitation of these species. Peritoneum colouration (black in L. budegassa, white in L. piscatorius) appears to provide the most adequate distinction between the species (Guillou and Njock, 1978) but its validity as a taxonomic feature is untested. Furthermore, in several European countries much of the angler-fish catch is landed as tails only (Dupouy, 1984), thus morphological characteristics cannot assist in the evaluation of the relative contributions of either species to the European fishery. The utility of electrophoresis of proteins in determining purely genetic affinities among and within species of plants and animals is well documented (for review see Ferguson, 1980). The difficulty encountered in separation of environmentally induced from genetically controlled variation which inevitably complicates the use of morphological features in taxonomy is avoided by biochemical examination of protein molecules which are directly coded by the genome. Moreover, characterization of protein types can be achieved using small amounts of material and from individuals of any age, often without damage to the specimen which may be required for parallel morphological examination. Analytical isoelectric focusing methods achieve separation of proteins solely on the basis of their net electric charge, electrophoresis being complete when the proteins have reached their isoelectric points (pIs). This method which separates and concentrates proteins characteristically different in pI is capable of resolving proteins differing by 0.0025 units in their pI values (Allen et al., 1974). Thus absolute positions of bands on
372
W.W. CROZIER
protein profiles obtained using isoelectric focusing are reproducible, as distinct from conventional electrophoresis where relative mobilities are timedependent (Jamieson and Turner, 1980). In many instances divergence in the process of speciation has resulted in the accumulation of distinctive genes which allow unequivocal separation of taxonomic units (e.g. McGlade et al., 1983) and enables construction of taxonomic keys which reflect purely genetic characteristics (Mork et at., 1983). The present paper reports comparative electropboretic examination of skeletal muscle proteins of L. piscatorius and L. budegassa in an attempt to verify the presently used methods of species identification, and to provide a method of species discrimination in cases where morphological criteria cannot be applied.
MATERIALS AND METHODS
The angler-fish samples, comprising 147 L. piscatorius (10.9 129.0cm) and 31 L. budegassa (22.7 61.2cm) were obtained from catches taken by commercial fishing vessels in the north-eastern Atlantic and Irish Sea during 1984/1985 (Table 1). Classification of individual fish into species was carried out prior to and independently from the biochemical analysis. This was based on criteria used by Wheeler et al. (1974) and Fitzmaurice (1976), meristic counts, morphology of the illicium and peritoneum colour being examined (Crozier, 1985). Samples of white skeletal muscle were dissected from most fish within 12 hr of capture and stored individually frozen at -80~C, however some fish were stored at -20cC on landing, until transport to the laboratory. Prior to electrophoresis, extraction of soluble proteins was achieved by mechanical homogenization in an equal volume of ice cold 0.01 M Tris HCL, pH 7.0, followed by centrifugation at 3000g for 15min at 4'~C. Isoelectric focusing was carried out on the LKB Ultraphor apparatus using commercially available 1 mm thick polyacrylamide gels (Amopholine PAG plates). Electrophoretic conditions followed manufacturers recommendations. Wide range (pH 3.5 9.5) and narrow range (pH 4.0-6.5) gels were used as appropriate to identify and then closely resolve areas of interest. Isoelectric points of protein bands were obtained by comparing their equilibrium positions with those of reference sets of proteins having known isoelectric points (Pharmacia pI calibration markers). Following electrofocusing, proteins were fixed in 0.7 M trichloroacetic acid/0.16 M sulphosalicylicacid and visualized by staining with 0.1% Coomassie Brillant Blue R.
RESULTS
Separations of muscle extracts in isoelectric focusing gels of pH range 3.5-9.5 showed in excess of 50 bands (Fig. 1). While several of the major and many of the minor bands were common to all individuals, the samples fell into two discrete groups, separable on
the basis of presence or absence of major bands A and B respectively. On comparison of individual samples with the initial morphological classification these groups corresponded exactly to L. piscatorius and L. budegassa. Thus band A was common to all L. piscatorius examined but absent in L. budegassa, while band B was fixed in L. budegassa alone. No intermediate electrophoretic profiles were observed. In order to further examine these differences individuals were subjected to isoelectric focusing using narrow pH range gels (pH 4.0-6.5), thus achieving greater resolution of the anodal part of the pH gradient. The resultant protein profiles are illustrated in Fig. 2. As before bands A and B separate the two Lophius species, thus confirming their validity as species-specific genetic markers. In addition the increased resolution revealed a further band (C) present only in individuals of L. budegassa. Thus bands B and C biochemically define L. budegassa, while band A defines L. piscatorius. The isoelectric points of these bands were estimated as: pH 4.40 (band B); pH 4.65 (band A); pH 5.75 (band C). Side by side electrophoretic comparisons of samples from the various sampling locations did not reveal any consistent inter-regional differences in banding pattern within either species. However, differences among individuals in presence or absence of several bands were detected within both species (Figs 1 and 2). These heterogeneities were unrelated to area of capture, age or sex of individuals and suggest the segregation of several polymorphic gene loci within L. piscatorius and L. budegassa. Given the acknowledged danger of artefactual heterogeneity on IEF profiles of samples subject to differing storage/extraction procedures (Righetti and Drysdale, 1976; King and Moffett, 1984) it is essential that non-genetic variation be identified prior to concluding that any heterogeneity has a genetic basis. Because of the differing sampling procedures necessary in the present investigation control experiments were carried out to determine whether time period between capture and sampling, and length of frozen storage altered protein profiles. The only detectable heterogeneity among samples subject to differing treatment was a slight shift towards the low pH end of the gel (more electropositive) of the entire banding pattern following prolonged (10 months) frozen storage. The agreement of the biochemical with the morphological distinction between the two species supports the validity of the morphological features used for the initial identification. In this respect the colour of the peritoneum provides the most convenient and indeed the most accurate method of separation, no intermediate colouration being observed among the 178 individuals examined.
Table 1. Samples of Lophius examined in the present investigation Date
Location
22.6.84 June 1984 5.7.84 18.10.84 15.11.84 9.5.85
West Scotland Irish Sea Irish Sea South-west Ireland Irish Sea West Ireland
A p p r o x i m a t e position 56"20'N 53"20' 54'20' 51"20' 53"45' 54'~30 '
7 50'W 550' 455' 11"30' 530' 9"25 '
L. piscatorius 17 21 50
L. budegassa 19
I1 48 11 147
1 31
Electrofocusing of Lophius muscle proteins
373
pH 3.5
C-
Origi
pH 9.5 b
p
p
p
p
b
p
b
p
b
p
p
b
b
Fig. 1. General protein patterns of skeletal muscle extracts of two Lophius species separated by isoelectric focusing in a pH gradient 3.5-9.5. Samples: (a) Lophius piscatorius; (b) L. budegassa.
~-~
pH 4.0
Origi
pH 6.5 p
p
p
b
p
b
p
b
p
p
b
p
p
p
Fig. 2. General protein profiles of Lophius spp. separated by isoelectric focusing in a pH gradient 4.0-6.5. Labelling as Fig. 1. Band C is indicated for individuals of L. budegassa.
DISCUSSION The advantage of using biochemical characteristics to identify species is that they can provide a genetic base, allowing verification on organismal differences. This approach has been applied successfully to .aid
resolution of taxonomic difficulties in many organisms including tapeworms (Bylund and Djupsund, 1977), nematodes (Fleming and Marks, 1982) and fishes, including eels (Jamieson and Turner, 1980), coregonids (Ferguson et al., 1978) and redfish (McGlade et al., 1983). Further applications utilize
374
W.W. CROZIER
specificity of proteins at the species level to identify fillets of marine fish (Lundstrom, 1977) and to determine the species composition of pelagic samples of fish eggs (Mork et al., 1983). The results reported here have direct applicability to the future scientific assessment of the increasingly important European fishery for Lophius spp., as biochemical examination of muscle proteins will offer a convenient and reliable method for determining the relative contributions of L. piscatorius and L. budegassa to total landings of angler-fish. Lundstrom (1981) using agarose and polyacrylamide gels examined skeletal muscle extracts of Lophius americanus by isoelectric focusing. Although homologies between particular protein bands cannot be established without direct side by side electrophoretic comparison, examination of the illustrations given in Lundstrom (1981) suggests that while L. americanus display many bands in c o m m o n with L. piscatorius and L. budegassa they possess distinctive (and possibly species-specific) bands in the pH range 3.5-4.5. Lundstrom (1981) reported reproducible differences among individuals in respect of banding pattern (ten putative genotypes being identified) suggestive o f the presence of polymorphic gene loci in this species. The present research permits for the first time definitive separation of L. piscatorius and L. budegassa at the biochemical level. The species-specific protein profiles described here can be viewed as evidence that these two lophiid angler-fishes have diverged sufficiently from their c o m m o n ancestor to have accumulated distinctive genes. A comparative examination of the genetic expression of a range of enzymatic proteins presently being carried out by the author may well reveal further evidence of genetic divergence between these species, gene frequency data allowing calculation of genetic divergence and estimation of approximate time of divergence (Nei, 1975). The suspected presence of polymorphic gene loci in both European angler-fish species warrants further investigation, as these would potentially serve as genetic markers in an examination of population structure. Acknowledgements--The author wishes to thank Mr L. Girvan and Dr R. Grainger for assistance with obtaining samples of Lophius. Dr D. King made useful comments on a preliminary draft of the manuscript. The provision of a research grant by the Jeffreys Association Limited is gratefully acknowledged. REFERENCES
Allen R. C,, Harley R, A. and Talamo R. C. (1974) A new method for determination of alpha-l-antitrypsin phenotypes using isoelectric focusing on polyacrylamide gel slabs. Amer. J. clin. Pathol. 62, 732 739. Berrill N. J. (1929) The validity of Lophius americanus Val. as a species distinct from L. piscatorius Linn., with notes on the rate of development. Contrih. Can. Biol. Fish. 12, 145-151. Bylund G. and Djupsund B. M. (1977) Protein profiles as an aid to taxonomy in the genus Diphyllobothrium. Z. Parasitenk. 51, 241-247. Caruso J. M. (1981) The systematics and distribution of the tophiid angler-fishes : I. A revision of the genus Lophiodes with the description of two new species. Copeia 3, 522-549.
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