The hemoglobins of Anguilla anguilla (L.) ontogenetic variations

Comp.

Biochrm

Physrol., 1977,Vol. %A, pp.173IO 176.Pergamon Press.Printedin Great Brian

THE HEMOGLOBINS OF ANGUILLA ANGUZLLA ONTOGENETIC VARIATIONS* MARTINORIZZOTTI,ANTONIOCOMPARINIAND

(L.)

EMANUELE RODINC~

Institute of Animal Biology, University of Padua, Padua, Italy and Institute of Marine Biology, C.N.R.. Venice, Italy (Received

8 December

1976)

Abstract-l. The electrophoretic pattern of the hemoglobin of adult European eels shows two bands. The pattern of the elvers is composed of nine bands, divided into three groups with three bands in each. This pattern undergoes gradual modifications till it changes, after a few months of aquarium rearing, into the two band pattern typical of the adult eels. 2. The ontogenetic variation of the hemoglobin pattern of the eel, a catadromous fish, was postulated by some Authors to be functionally similar to that of an anadromous salmon. Our results do not support this hypothesis, while being in agreement with the habits and physiological requirements of elvers and eels.

The blood of the eel species so far examined shows two types of Hbt components clearly distinct by physical, chemical and functional properties. One of the distinctive properties is the isoelectric point (i.p.) and therefore the two types of components are easily separable by electrophoresis or ionic exchange chromatography of the hemolysate (Christomanos, 1974) into anodic (i.p. around 6) and cathodic ones (i.p:around 8). Yamaguchi et al. (1962) suggest, on the basis of their functional studies on the Hbs of Anguilla juponicu (Japanese eel), that the anodic components show the characteristics of the Hbs of marine fishes in general, i.e. they exhibit low oxygen affinity and strong Bohr effect, while in the cathodic components the oxygen affinity is high and the Bohr effect is absent or reverse, as usually observed in freshwater fishes. More recently, functional studies carried out on Anguillu juponicu (Hamada et al., 1964; Yoshioka et al., 1968; Okazaki et al., 1974), Anguillu rostrutu (American eel) (Poluhowich, 1972; Gillen & Riggs, 1973) and Anguillu unguillu (European eel) (Weber et al., 1976) demonstrate that the Hb systems of all these species are similar to that of A. juponicu. Eels are catadromous fish. Young individuals appear at the river mouths at the elver stage, reach an advanced phase of development in freshwater habitats, remaining there for some years as “yellow eels”, and then swim back to the sea for reproduction as “silver eels”. It seems then that the elver stage, during which there is a rapid transit from the sea to freshwater, is suitable for an indirect verification of the different functions attributed to the anodic and cathodic Hb components. In the present research we *This research has been supported by the Italian National Research Council (C.N.R.) through funds granted to the Institute of Marine Biology, C.N.R., Venice, and in part with contract N. 71.00237.04.115.4856 assigned to one of the Authors (E. Rodin6). t Hb(+hemoglobin(s).

have determined the quantitative ratio between the anodic and cathodic components during the transit of the elvers from the sea to freshwater, and compared it with the same ratio in later stages of development. We give also a description of the electrophoretic pattern of the Hbs in elvers and its ontogenetic variation. The only previous observation in elvers was made by Chieffi et al. (1960). These Authors, using paper and starch gel electrophoresis, reported the presence of only one Hb fraction in the elvers they analysed. MATERIALSAND

The adult eels were examined after a maintenance period in freshwater or brackish water; they came generally from the eel ponds near Chioggia or Comacchio, south of the Lagoon of Venice. The elvers came from the Tyrrhenian coast of Italy (Pisa, Castiglione della Pescaia, Orbetello. Naples). All the elvers were captured in January or February, kept in a freshwater aquarium and fed mainly with lyophilized Tub&x Blood was collected and treated as follows: individuals were anesthetised with MS 222 (Sandoz-Switzerland). their state of development determined (Grassi, 1914), anh total length and weight recorded. The caudal peduncle was then severed and the blood was collected in 20-30 vol of isotonic citrate-glucose solution. Red cells were washed and resuspended in this medium in which they kept well for several days at 0°C provided they were resuspended 2-3 times a day. Just prior to hemolysis the red cells were washed in neutralised isotonic NaCl. Washed red cells were hemolysed with an equal volume of neutralised S.lO-“M EDTA (Antonini & Brunori. 19711. , One droo mr of Ccl., was added to the fresh hemolysate and the sample centrifuged at high speed. All procedures were carried out at 4°C. The electrophoretic runs were generally made on strips of gelatinized cellulose acetate Chemegel (Chemetron Chimica, Milano. Italy) using a Tris-glycine buffer (7 g + 11 g/l respectively) at pH 8.8. The runs required 20-30min with a voltage gradient of 18OV. Amidoblack was generally used to stain the strips but the results were often checked with 0-Dianisidine (Owen et al., 1958). The colour intensity of the electrophoretic bands was evaluated by reading the Chemegel strips by reflection using the Chromoscan MK. II (Joyce Loebl,

173 C&P.5&/2*--u

METHODS

I74

MARTIN> RI/LOTTI. AI\;TOONIO COMPARN ANI) EMAIUU LI

ROINW

England) and by dlssolvmg the single bands in 3 ml of X0”. acetic acid and then measuring the absorbance with a spectrophotometer

at 6N nm.

RESULTS

The electrophoretic patterns were more complex than expected on the basis of the previous report by Chieffi et al. (1960). Up to nine electrophoretic bands were observed, comprising three distinct groups of components (see Fig. 1). These are referred to as the anodic (A), the intermediate (I) and the cathodic (C) groups. In those elvers which were examined a day after capture at the river mouth the cathodic group was entirely absent and the intermediate group was very weak. Elvers which had been reared for one month in freshwater possessed all three groups of Hbs. the anodic group being the most concentrated and the cathodic group the least concentrated. Within each group the central band (Al, I,. C2) was the most concentrated. After two or three months in the freshwater aquarium the nine band pattern established in the first month began to evolve gradually into the typical adult pattern. The intermediate group decreased in intensity and lost first the band I,. then the band I3 (after 6 or 7 months), finally disappearing entirely (Fig. 2). Within six months the anodic group also lost its minor bands (A,, A3), while the cathodic group lost the band C,, and the band C2 was much less intense. At the same time a relative increase in the concentration of the cathodic group was observed: in fact. around the tenth month of rearing, the latter was almost as concentrated as the anodic group. The typical adult pattern consists of a single band (Az) in the anodic group together with a single band (C,) in the cathodic group (without taking into account a few other components present in very small amounts). These ontogenetic variations of the Hb pattern seem to be correlated only with the time of maintenance m freshwater. No correlation has been found with the stages described by Grassi, nor with other parameters such as length or weight. Among the samples of newly captured elvers in stages 3, 4 and 5 we occasionally found a few more pigmented individuals (stages 8 and 9) which showed the electrophoretic pattern of a more advanced freshwater stage; it is likely that these few individuals had. prior to capture, spent some time in freshwater near the river mouth. The nine band pattern has been found after one or two months of rearing, in all the developmental stages (from 4 to 9) present in the population. The electrophoretic pattern varies simultaneously with the rearing time in the entire aquarium population: after 6 months there were no individuals under stage 8. while there were already some above stage IO, yet all had the same Hb pattern. The Hb patterns of single elvers showed only a little individual variability with regard to intensity and time of disdppearance of the bands. On the other hand. no difference has been noticed between specimens of different geographic origin. Lastly. we have always obtained the patterns described even when the electrophoretic medium. the buffer system or the staining method were changed.

Our results show that at the time of their entry into continental freshwaters. the blood of elvers has a relatively higher proportion of anodic Hbs than that of adult eels (Fig. 2). The relative proportion of anodie Hbs decreases continuously during the first six months in freshwater. This would support the view of Yamaguchi et al. (1962) that the anodic Hb components of the adult eel are functionally similar to the Hbs of marine fishes. while the cathodic components are similar to the Hbs of freshwater fishes. Consequently it appears that environmental salinity influences gene activity in the blood forming tissues of the elvers, so causing the observed variation of the ratio between anodic and cathodic Hbs. On the other hand. a later increase of the anodic Hbs up to 65% is necessary for the young eels to reach the final adult pattern (see Fig. 2. dotted line). This latter observation is in good agreement with the data of Yamaguchi c’t ul. (1962) on the increase in concentration of the anodic Hbs from about 55 to 65”; in young eels. Yamaguchi c’t rrl. (1962) seem to suggest. on the basis of the functional meaning attributed to the two components. that this increase in anodic Hbs represents a preadaptation to the marine environment to which the animal must return for breeding purpose. Such a “prcadaptdtion” cannot be attributed to the effect of variable environmental salinity on gene activity, since it is attained long after the passage into freshwater has been completed. Furthermore, the final pattern lasts in freshwater for many years, which seems quite unlikely for a “preadaptation”. Yamaguchi it ul. (1962) compare the variation in the Japanese eel to that occurring in the Pacific anadromous salmon Oncorhyr~lnrs krtu (chum salmon) in which the anodic and cathodic Hbs are considered to resemble functionally those of the eel. But the chum salmon arranges its Hbs pattern for the breeding environment (freshwaters) very early; the variation occurs in the juvenile phase in the rivers during the migration to the sea. The comparison would be valid if the attainment of the adult pattern in the eel took place before its entry into the rivers. This variation in the chum salmon can be regarded as a general characteristic of .Salmormi&i and Clupeoidei (classification of Greenwood (‘t u/., 1966). In fact Wilkins (1970) noticed that these suborders differ from the other Teleosts (and from the eels too, as the present research demonstrates) because the ontogenetic variation of the Hbs “is a protracted process, occupying the greater part of the life cycle of the individual”. Moreover. the same variation seems to be present both in the anadromous species and in some freshwater species or forms of Salrnonoidci (Vanstone 1st ul., 1964; Koch r’t (I/.. 1966). Therefore it appears unwarranted to postulate preadaptation of a single Hb in the eel to the marine or to the freshwater environment. It seems more justified to propose that the whole Hb system (including interactions with other erythrocytlc components) adapts to definite environmental parameters. like salinity. oxygen tension and temperature. paying attention to habits and physiological requirements (see Brunori. 1975). In this context. and on the basis

The hemoglobins

c

I

3 2 I

3 2 I

of A. anguilla

A 321

(a) II

(b)

(b) I

(cl II

(d) I

(e)

(e)

:

Fig 1. Electrophoretic patterns of the hemoglobins of Anguih anguih in the course of development. C, I and A are the cathodic, intermediate and anodic components, respectively. a, elvers pattern at the river mouths; b, c, d, elvers patterns after about one, four and seven months of freshwater aquarium rearing; e, adult eel pattern; f, human hemoglobin A, for comparison.

175

REFERE;\I<‘ES

BKI UOKI M. (1’175) MoIccular adaptation I<) phys~ologtcai requirements: the hemoglobm system of trout. In C‘lrrrct~r Toprcs ilr Crllultrr Regularion. (Edited by HORE(‘ICE?. B. L. & STAIIT.WN. E. R-1. Vol. 9. pp. I 39. Academic Press. New York.

months

Fig. 3. Ontogenetic cathodic. intermedlate

of

acpuarium

rearing

variation of the relative and anodic hemoglobins Ull+iill&

amount of in Af~yuillu

of the available data. the predominance of the anodic Hbs in the elvers may be necessary for increased musculary activity during the eels ascent of the river. The Bohr effect of these Hbs increases the efficiency of tissue oxygenation in conditions of high (but not extreme) tissue acidity (Riggs. 1970). Nothing is known about the properties of the intermediate Hbs. The multiplicity of Hbs observed in the elvers may represent a primitive adaptation to the variable environmental conditions of the river mouth. The Hb system in yellow and silver eels, although more simple than that of the elvers. nevertheless allows regulation of swimbladder pressure (ascribable to the anodic Hb) and adequate tissue oxygenation in the hypoxic and euryhaline conditions. For external hypoxia. due to long periods spent resting in mud, the high oxygen affinity of cathodic Hb can help. We cannot exclude also an inner hypoxia due to strongly intensified activity during predation, for instance, and consquent hyperacidity, which can also be overcome by cathodic Hb, owing to its lack of any Bohr effect (Riggs, 1970). Finally there is considerable evidence for salt and organic phosphate effects (Gillen & Riggs, 1973 ; Okazaki et al., 1974; Weber rt al., 1976) which play an important role in compen~tjng against salinity (Poluhowich, 1972; Geoghegan & Poluhowich. 1974) and oxygen tension variations (Wood & Johansen, 1972). Ae~/t~w~~d~~rn~)~fs-

thank Prof. B. Battaglia for on the manuscript; Dr. V. Scali and Dr. M. Masetti of the University of Piss, Dr. G. RavaeTlan, Dr. F. Ghion of the Ichthylologic Center of Venetian Ponds (C.I.V.V.) for furnishing etvers and young eels; Mr. U. Arezzini and Mr. C. Friso for technical assistance. We especially acknowledge the kind help of Dr. N. P. Wilkins in providing several critical suggestions and a revision of the English in the manuscript.

reading and commenting

We

ClIKISrOMA\OS .A. ( 1974)Molekularr Btoiogic dcr HBmoglohine. III. A. DIG Fischh%moglohinc. Sci. press. greg. par,s,anos. Athens. Gt(X;)ff.CiAh W. D 81 PoL~~~Io~~~H J. .I. (1974) The major erythrocytic organic phosphates of the America1 eel. ,,lflyrrilllr ro~tratu. Camp. Hioch~m. Pll!siol. 49B. 28 I 790. GILLI-N R. G. & RIGGS A. (lY73) Structure and function of the isolated hemoglobiJ]s of the American eel. .4r1g.ui/lu rrlslrclftr. J hioi. Chc,rn. 248, 1961 1969. GKASSI B. (I9 13)Quel the si sa e qucl chc non SI sa intorno alla storia naturale dcll’Anguilla. K. Corn.. roll. if. 37. GRI.~NW~X)I~P. H.. ROSEN D. E.. W~~~T~MA?IS. H. & MVER G S. (1966) Phyletic studies of teleostean fishes with a provisional classification of living forms. Bull. 4m. Mus. ih’ut. Hist. 131, 339 35. HAMAI~A K., Sl-MrrA N:.. OKAZAKI T.. SII~K[;YA R.. SASAKADA S. & SATAKI. K. (19641 Hemoglobills from erythrocq tes 01‘ eel. .&y~ril(u /aportic,tr. J. Biochmm. 55. 154. IQ. K~~II H. J. A.. B~.RC;STR~~M E. & E~A\s J. C. (1966) A size correlated shift m the proportion of the haemoglohin components of the AtIan& Salmon (Sulmo .sutur L.) and of the Sea Trout (&lmo truit(l L.1. M&&l. I ‘laamsc~ Acud. Kl. U’c~r. 28. I 20. O~ZAKI T.. MISAWA J. Xr SI~I~KI~YAR. (1974) The effects of organic phosphates on the oxygen equilibria of two distinct hcmogfohins of :1x eel. .~~]~I~;~~~~ ,@poni~u. Biocltc~rtt. hiuphys. Rc’s. Comm~r~r. 56, IO3I 1037. Owl.\ S. A., SILRIKMAN H. 1. & Gor G (IY5X) Detection of haemoglobin, hacmoglobtn haptoglobin complexes and other substances with pcrorhsr activity after zone electrophoresis. &‘arurr. Lo&. 182, 1373. P01.r tfowIct1 J. J. ( IY71) Adaptive significance of eel multipie hemoglobins. Plr),.srol. Zool. 45. 7 15 222. Rims A (1970) Properties of fish llcmoglobins. In Fisfz Physiok~yy. (Edited by HOAK W. S. & -RANDALL D. J.), Vol. IV. pn. 709.-Z’;1. Academic Press. New York. VAUSTO~I ‘\;J. E.. ROBEKN F. & TSL:YLIKI H. (1964) Changes in the multiple llcrn~~glohill pattern of some Pacific salmon. genus Oitc,orifq’rlclrii.s. during the parrsmolt transformation. Cmt. J. Pll!xio/ Pharmacol. 42, hY7 701. WIBEK R. E., LYKK~:IK)I Ci. 6i JOHANS~.N K. (lY76) Physlological properties of ccl ~~crnoglobi~~: hypoxlc acclimation. phosphate effects and multiplicity. .I. ewp. Biol. 64, 75 XS. WILKIUS N. P. (1970) The subunit composition of the haemoglobin of the Atlantic salmon (S&no s&r L.I. &achim. hioplrp. Actu 214. 5243. WOOD S. C. & JOHASSEY K. f 1972) Adaotation to hvnoxia by increased HbO, aflimty and’ decrdased red ce;l’ATP concentration, Wumrt,. .Vs,w Biui. 237. 17X-279. YAMACXK.HI R., KO~HIYAMA Y.. HASHIMOTO K. 6t MATSULIKA F. (1962) Studies on multiple hemoglobins of eel. II. Oxygen dissociation curve and relative amounts of components F and S. &iii. Jup. Snc. SC;. Fish. 28, 192-200. YMIIIOKA M.. HAEAAI>A K.. OKAZAKI T.. KAJITA A. & Skrurciiva R. (1968) Hemoglobin from the erythrocytes of the ccl. Anytrillrr /~ponicu. II. Further studies on the properties of the two hcmoglohins. .I Bio&m. 63, 70 16.