Species-characteristic and subspecies-characteristic haemoglobins in some tilapiinefishes and a comparative study on theirglobin chainsT.M. Falk1,* E.K. Abban,2 W. Villwock1 and L. Renwrantz1

1Zoologisches Institut und Zoologisches Museum, Universitat Hamburg, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany2Institute of Aquatic Biology, PO Box 38, Accra, Ghana

Haemoglobins of ten tilapia species of the genera Oreochromis, Sarotherodon and Tilapia,three subspecies of the Nile tilapia, O. niloticus, and an artificial hybrid cross were analysedby isoelectric focusing aimed at characterizing the taxonomic value of these oxygen-bindingmolecules. Heterogeneous haemoglobin phenotypes were observed in haemolysate samplesof all the fishes, and species-characteristic as well as subspecies-characteristic patterns couldbe identified. F1 hybrids were distinguishable from pure parental specimens. Globin chainstudies by AU- and AUT-PAGE systems confirmed the heterogeneity and species specificityobserved by isoelectric focusing of the tetrameric molecules. A total of eight differenta-chains and eight different b-chains were detected by AU-PAGE and species-characteristicglobin chain variants were shown to occur in almost all species. Globin chain profiles ofhybrid specimens were characterized by the presence of all parental globin chain variants.

KEYWORDS: Genetic variation, Globin chains, Haemoglobins, Species differentiation, Tilapia(Oreochromis, Sarotherodon, Tilapia)

INTRODUCTION

Comparative studies on the identification and characterization of biochemicalgenetic markers that may be utilized in fisheries and aquaculture have increasedsignificantly over the last two decades. In tilapias, as in the majority of other fishgroups, the most common biochemical approach used for the characterization andidentification of species, natural populations and aquacultural strains has focusedon allozyme electrophoresis (McAndrew and Majumdar, 1983; Macaranas et al.,1986, 1995; Seyoum, 1990; Sodsuk and McAndrew, 1991; Pouyoud and Agnese, 1995).In addition, polyacrylamide gel electrophoresis (PAGE), isoelectric focusing (IEF),and immunological assays have been successfully applied to identify geneticallycontrolled, non-enzymatic species characteristics, for example, in tilapia plasmasamples (Avtalion, 1982; Oberst et al., 1992, 1993), tilapia muscle extracts (Oberst etal., 1993, 1996; Focant et al., 1994) and on erythrocyte membranes (Oberst et al.,

* Author to whom correspondence should be addressed (e-mail: tmfalk@pin-net.de).

0967–6120 © 1998 Chapman & Hall

Aquaculture International 6, 133–145 (1998)

1988, 1989, 1996). More recently, molecular genetic techniques have been in-troduced in this area of tilapia research (Seyoum and Kornfield, 1992; Bardakci andSkibinski, 1994; Naish et al., 1995; Dinesh et al., 1996), and particularly micro-satellites, a class of variable numbers of tandem repeats (VNTR) loci of nuclearDNA, have been shown to be a promising tool for population genetic analyses(Wright and Bentzen, 1995).

However, only a few biochemical genetic studies on haemoglobin molecules,although known to be taxonomically highly informative within several fish groups(Fyhn et al., 1979; Perez and Rylander, 1985; Val et al., 1987; Arefjev and Karnauchov,1989), are available for tilapias (Chen and Tsuyuki, 1970; Hines et al., 1971; Oberst etal., 1989; Macaranas et al., 1996). To the best of our knowledge, no data have beenpresented on tilapia globin chain characteristics or tilapia globin chain sequences.Thus, the objective of the present investigation was aimed at characterizing thetaxonomic value of haemoglobin and globin chain analyses within tilapiine fishes.Natural populations and strains of ten different tilapia species from three generawere studied.

MATERIALS AND METHODS

Most specimens of tilapias were obtained from natural populations of Ghanaianrivers or lakes. Table 1 lists the origin of tilapia samples used, their abbreviationcode and the numbers investigated. A minor group of tilapia specimens were kept inaquaria of the Hamburg Institute.

Blood sampling and haemolysate preparation

Blood sampling and preparation of erythrocytes were performed as described byOberst et al. (1988). Haemolysates were prepared by lysing one volume oferythrocyte pellet in four volumes of precooled (4 °C) distilled water. Subsequently,cell organelles and membranes were removed by two centrifugation steps (30 min at2200 g and 15 min at 30 000 g, 4–5 °C). The supernatant was mixed (1:1) with 80%PBS buffered glycerol (pH 7.4). Samples (30–35 mg Hb ml–1) were used immediatelyor stored at –20 °C.

Isoelectric focusing (IEF) and haemoglobin detection

IEF separations were conducted on Servalyte precotes (pH range 3–10, Serva,Germany). Gels were prefocused (200–500 V; 5.2–6 mA/gel; 4 W/gel) at 6 °C.Haemolysate samples were applied to an application strip positioned 2.75 cm fromthe anode (pH 5.4–5.5) and the voltage was limited to 1700 V. Separations werefinished when a constant current of maximal 2 mA/gel was reached (after about3.5 h). Prior to use, haemolysate samples (20 mg Hb ml–1) were treated with2-mercaptoethanol (3%) for 1 h at 5 °C. Cyanide-stabilized haemoglobins wereprepared according to Perez and Maclean (1976).

Although IEF-separated haemoglobins could be identified by their red colour, gelswere incubated in 4-chloro-1-naphthol (Serva)/H2O2 (Merck, Germany), mainly tointensify the appearance of minor bands by the pseudoperoxidase activity ofhaemoglobins. According to Miribel and Arnaud (1988), the staining solution

134 T.M. Falk et al.

consisted of 60 ml methanol and 340 ml PBS (pH 7.4) containing 120 mg 4-chloro-1-naphthol and 1 ml 30% H2O2. The following pI marker proteins (Serva) were used:myoglobin whale: pI 8.3; myoglobin horse: pI 7.3 and 6.9; con-albumin: pI 5.9 andb-lactoglobulin: pI 5.34.

TABLE 1. Oreochromis, Sarotherodon and Tilapia species studied, their abbreviations codes,populations used and numbers investigated

Species/subspecies Abbreviation Sample location (* 5 Ghana)

No. ofindividualsinvestigated

Oreochromis andersonii OAn University of Stirling, Scotland 10

Oreochromis aureus OAu Lake Manzala, EgyptUniversity of Stirling, Scotland

510

Oreochromis n. niloticus ONi Southern part of Volta Lake*Densu River at Weija*Lake Manzala, EgyptUniversity of Stirling, Scotland

12143

23

Oreochromis n. sugutae ONs Suguta River, Kenya 3

Oreochromis n. eduardianus ONe Ruzizi River, Burundi 3

Sarotherodon galilaeus SGa Densu River at Weija*Black Volta at Busunu*Israel

847

Sarotherodon melanotheron SMe Densu River at Weija*Lower part of Volta Lake*

2012

Tilapia busumana TBu Lake Bosomtwi* 16

Tilapia dageti TDa Volta Lake at Kpandu* 14

Tilapia discolor TDi Lake Bosomtwi* 18

Tilapia guineensis TGu Volta lake at Battor*Densu River at Weija*Lower Volta at Sogakofe*Layo/Abidjan, Cote d’Ivoire

10878

Tilapia zillii TZi Black Volta at Busunu*Lower Volta at Battor*Densu River at Weija*Bouake, Cote d’IvoireSassandra, Cote d’Ivoire

410128

10

Oreochromis n.niloticus 3 Oreochromisandersonii

ONi 3 OAn University of Stirling, Scotland 20

135Haemoglobin and globin chain variations in tilapias

Globin chain electrophoresis

Essentially, acidic urea (AU-) PAGE and acidic urea Triton (AUT-) PAGE (12%acrylamide gels containing 8 M urea and 5% glacial acetic acid) was performedaccording to Alter et al. (1980). Haemolysate samples were diluted (1:10) with asolution consisting of 3.5 ml 12 M urea, 0.25 ml glacial acetic acid, 0.4 ml2-mercaptoethanol and 0.85 ml distilled water. After an incubation for 15 min at 5 °C,20% Methylene Green prepared in glycerol (300 ml 1% Methylene Green and 700 mlglycerol, Serva) was added. Volumes of 8 ml of each sample were applied to the gels.Gels were stained with Coomassie Brilliant Blue (Serva).

RESULTS

IEF analysis of tilapia haemoglobins

Haemoglobin (Hb) phenotypes of all species investigated are shown in Fig. 1a–d.The present results, obtained by thin layer isoelectric focusing (IEF) on pH gradientsfrom pH 3 to pH 10, demonstrated a high degree of heterogeneity within tilapiahaemoglobin molecules. Estimated isoelectric points (pIs) of prominent compo-nents ranged between pH 5.88 and pH 8.06. On average, 24 different haemoglobinswere detected per species (OAn: 22; OAu: 22; ONi: 26; SGa: 21; SMe: 22; TBu: 25; TDa:26; TDi: 25; TGu: 26; TZi: 22), indicating the occurrence of multiple globin chainvariants involved in the composition of different tetrameric haemoglobin types.Moreover, variations of haemoglobin types among tilapia species have beenobserved, resulting in the identification of species-characteristic haemoglobinpatterns. Intraspecific variation appears to be consistent within the samplesobtained for each species. Only within OAu specimens did intraspecific variationoccur. One individual obtained from the University of Stirling (Fig. 1a, lane C) hasbeen characterized by the lack of four prominent haemoglobins compared with thepatterns observed for all other OAu specimens studied.

Differentiation among species, hybrids and subspecies

Considering the pI ranges of tilapia haemoglobins, genera-characteristic differenceshave been detected. As a common feature of the species of the genus Tilapia (Fig.1c), their most alkaline haemoglobins reached pI values up to pH 7.78, whereasthose of Oreochromis and Sarotherodon spp. reached pI values of pH 8.06 and pH7.96, respectively (Fig. 1a). Within the three genera, species-characteristic haemo-globin patterns could be identified for almost all the species studied (Fig. 1a–c), onlyTDa and TGu remained indistinguishable. Characteristic pI values of tilapiahaemoglobins are indicated in Fig. 1a–d. Furthermore, ONi ´ OAn hybrid specimenscould be distinguished from their parents by composite patterns composed ofindividual parental haemoglobins within the pI range from pH 6.04 to 7.08 (Fig. 1b).However, some haemoglobins present in one or even both parental speciesremained absent in all hybrid specimens studied.

Comparative Hb-IEF separations of three subspecies of the Nile tilapia are shownin Fig. 1d. For ONi and ONe identical patterns were obtained. However, strikingdifferences have been detected with respect to the intensity of individual haemoglo-bins at pIs of pH 6.88, 6.4 and 6.24. Haemoglobins of ONs differed from these

136 T.M. Falk et al.

findings. This subspecies was mainly characterized by the presence of five differenthaemoglobins within the acidic pI range from pH 6.4 to pH 6.5.

Variations in the pretreatment of haemoglobin samples did not influence resultsobtained by isoelectric focusing. Thus, identical haemoglobin patterns wereobtained using mercaptoethanol-treated oxyhaemoglobins or cyanide-stabilizedhaemoglobin samples (results not illustrated).

Globin chain analysis

To support the hypothesis that the heterogeneity of tilapia haemoglobins inindividual species is based on the variability of the composition of these tetramericmolecules, globin chain analyses were performed. Results obtained indicated thattilapia globin chains are heterogeneous within both the a-type (cathodal group) andthe b-type (anodal group) chains. A total of eight different a-chains and eightdifferent b-chains were identified. With regard to taxonomic issues, Fig. 2a–b showsthat all species of the genera Oreochromis and Sarotherodon including TDi could bedistinguished using the AU-PAGE system. For example, a2ONi is specific to ONi, a2OAnto OAn, a2SMe to SMe, and a2SGa to SGa. Moreover, some species are characterized bytheir combinations of chains: a1OAu/a2OAu is specific to OAu and a1TDi/a2TDi to TDi.Artificial F1-hybrid specimens (ONi ´ OAn) could be distinguished from the appro-priate pure species by the expression of all parental globin chain variants, whereasthe ‘odd type’ specimen of OAu (Fig. 1a, lane C) did not exhibit the b4OAu-globinchain present in all other globin chain samples of this species (not illustrated).

However, the majority of the species of the genus Tilapia were nearly indis-tinguishable using the AU-PAGE results (Fig. 2b). Therefore, globin chain samples ofthese species were analysed by AUT-PAGE (Fig. 3). Based on this separationtechnique, species-characteristic b-globin chain variants could be identified for TBuand TZi, whereas TDa and TGu remained indistinguishable.

The identity of the three major b-globin chain variants (b2, b4, b5) of the Niletilapia was confirmed by isolation and partial N-terminal amino acid sequenceanalysis (positions 1–40). The N-termini of both a-globin chains (a1, a2) of ONi wereblocked, a characteristic feature for a-globin chain variants of teleost fishes (Hilseand Braunitzer, 1968; Bossa et al., 1976; D’Avino et al., 1990).

DISCUSSION

The present investigation on haemoglobins and globin chains of closely relatedspecies of the genera Oreochromis, Sarotherodon and Tilapia indicates that theseoxygen-binding molecules are taxonomically highly informative in character.Species-characteristic haemoglobin components have been identified for eight outof ten different tilapia species studied. Only TDa and TGu seem to possess identicalcompositions of haemoglobins and globin chains. These findings are supported bycomparable publications on the haemoglobin heterogeneity and species specificityof these molecules in freshwater and marine teleost fishes including tilapias (Chenand Tsuyuki, 1970; Hines et al., 1971; Perez and Maclean, 1976; Fyhn et al., 1979;Perez and Rylander, 1985; Val et al., 1987; Arefjev and Karnauchov, 1989; Oberstet al., 1989; Macaranas et al., 1996). In almost all the studies, species-characteristic

137Haemoglobin and globin chain variations in tilapias

(a) (b)

FIG. 1. (a) IEF separation of tilapia haemoglobins, A, O. n. niloticus; B, O. aureus; C, ‘odd type’ specimen of O. aureus, D, O. n.niloticus; E, S. galilaeus; F, S. melanotheron; G, S. galilaeus. Numbers represent pI values. Stain, 4-chloro-1-naphthol/H2O2. (b) Hb-IEFprofiles of O. andersonii (A, E), O. n. niloticus 3 O. andersonii hybrid specimens (B, D) and O. n. niloticus (C). Numbers represent pIvalues. Stain, 4-chloro-1-naphthol/H2O2.

138T.M

. Falk et al.

(c) (d)

FIG. 1. (c) Hb-IEF profiles of substrate-spawning tilapias. A, E, T. discolor; B, T. zillii; C, T. guineensis; D, T. busumana. Numbersrepresent pI values. Stain, 4-chloro-1-naphthol/H2O2. (d) Hb-IEF profiles of three subspecies of Nile tilapia. A, O. n. sugutae; B, O. n.eduardianus; C, O. n. niloticus. Numbers indicate pI values. Stain, 4-chloro-1-naphthol/H2O2.

139H

aemoglobin and globin chain variations in tilapias

differences have been observed within monomorphic as well as polymorphichaemoglobin phenotypes.

Despite these taxonomically important observations, differences regarding thetotal number of haemoglobins in individual species ranging from 7 to 17 compo-nents have been reported in tilapias (Chen and Tsuyuki, 1970; Hines et al., 1971;Oberst et al., 1989; Macaranas et al., 1996) and are probably attributed to differentsample preparation and separation techniques used. We observed an averagenumber of 24 haemoglobin components per species with a maximum of 26components for ONi and TGu by thin layer isoelectric focusing. Technical artefactslike aggregation or dissociation phenomena which may influence the multiplicity ofhaemoglobins observed by IEF separations could almost be excluded, becausemolecular weight (MW) determinations by gel filtration chromatography (Biogel P-

(a) (b)

FIG. 2. (a and b) Separation of tilapia globin chains by AU-PAGE and diagrammatic presentationof chain variants listed and numbered in order of their electrophoretic mobility. Minor chainvariants are put in parentheses and are also indicated in the Figure (a) A, O. n. niloticus; B, O.aureus; C, O. andersonii; D, S. melanotheron; E, S. galilaeus; F, T. zillii. (b) A, T. guineensis; B. T.zillii; C, T. discolor; D, T. busumana; E, T. dageti.

140 T.M. Falk et al.

200 column; 70 ´ 1.6 cm) at different pH values (pH 5.0/7.3/9.1) revealed constantMWs of about 69.6 kDa for the haemoglobin molecules of ONi, SGa and TZi (resultsnot shown). Oxygenation state variations within the haem groups which also maycause an increased variability of apparent tilapia haemoglobins were studied bycomparative IEF separations of mercaptoethanol-treated and cyanide-stabilizedsamples, and identical patterns have been obtained after both pretreatments oftilapia haemoglobins.

It is important to note that polymorphic haemoglobin or globin chain phenotypeshave not been detected regularly within the tilapia species and subspeciesinvestigated. However, it cannot be ruled out that the ‘odd type’ specimen of OAumay represent such an exception. Hines et al. (1971) also identified two differenthaemoglobin phenotypes within OAu specimens.

IEF analyses of haemoglobins are also considered to be a promising taxonomictool even below the species level of tilapias. Subspecies-discriminative haemoglobinprofiles have been identified among populations of the Nile tilapia. Especially ONsdisplayed characteristic haemoglobin components within the acidic pH range (pH6.4–6.5), whereas ONe and ONi revealed identical haemoglobin patterns. Theseresults are almost in line with haemoglobin data of two subspecies of the Nile tilapiastudied by Macaranas et al. (1996). The ‘Kenya strain’, subspecies O. n. vulcani fromLake Turkana, could be distinguished from all other ‘strains’ (subspecies O. n.niloticus) by a reduced genetic variability of haemoglobin types and the absence of‘band 3’. In addition, polymorphic haemoglobin phenotypes have been described

FIG. 3. Globin chain profilesof Tilapia species obtained byAUT-PAGE and diagrammaticpresentation of chain variantslisted and numbered in orderof their electrophoreticmobility. A, E, T. busumana;B, T. zillii; C, T. guineensis; D,T. dageti.

141Haemoglobin and globin chain variations in tilapias

for farmed (‘Israel strain’, ‘Singapore strain’, ‘Taiwan strain’, ‘Thailand strain’) andwild (‘Egypt strain’, ‘Ghana strain’, ‘Senegal strain’) populations of O. n. niloticus,whereas O. mossambicus and O. aureus specimens displayed monomorphic haemo-globin patterns (Macaranas et al., 1996). These variations of haemoglobin types,particularly within the Philippine farmed ‘strains’ of O. n. niloticus mentioned above,may probably be explained by introgressive hybridization events, a hypothesissupported by allozyme data (Macaranas et al., 1986, 1995). However, both studiescoincidentally confirmed the taxonomic value of haemoglobin analyses at and belowthe species level of tilapias and more expanded research programmes would berequired to evaluate the natural degree of genetic variability within the globin chainclusters of the Nile tilapia, in that about seven subspecies have been described(Trewavas, 1983).

Electrophoretic studies on globin chains of various fish species have corrobo-rated the tetrameric structure of haemoglobin molecules in fish, consisting of twodifferent types of polypeptide chains (a-like and b-like) and have established theoccurrence of multiple a- and b-globin chain variants (Tsuyuki and Ronald, 1971;Perez and Maclean, 1976; Mied and Powers, 1978; Masala et al., 1992; Ohkubo et al.,1993). To determine the total number of globin chains involved in the process ofhaemoglobin synthesis within the tilapiine species studied here, globin chainanalyses using AU- and AUT-PAGE systems were applied. Overall, a total of eightdifferent a-globins and eight different b-chain variants have been detected by AU-PAGE, and species-characteristic globin chain variants were identified for all themouthbrooding species (Oreochromis and Sarotherodon spp.) and TDi. Furthermore,defined F1-hybrids (ONi ´ OAn) of both crossing directions could be more easilydistinguished from the parental species compared with haemoglobin IEF analysis inthat all parental a- and b-globin chain variants were present within electrophoreticglobin chain profiles of hybrid specimens. Based on these genetic variationsobserved within both globin chain types of tilapias, the multiplicity and species/subspecies specificity of tilapia haemoglobins could be explained by the occurrenceof tetrameric chain combinations differing in their subunit compositions.

A slightly modified separation technique, the AUT-PAGE, was used to identifycharacteristic globin chain variants among the species of the genus Tilapia whichwere indistinguishable on the basis of the AU-PAGE results, and species-discrim-inative b-globin chain variants could be identified for TZi and TBu. However, TGuand TDa revealed identical globin chain patterns, although it has been shown thatthe AUT-PAGE system allows a separation of globin chain variants that differ inneutral-to-neutral amino acid replacements only (Alter et al., 1980; Di Luccia et al.,1991). Thus, it is interesting to note that the single major a-globin chain of theTilapia species studied (AU-PAGE results) could be resolved into four differentcomponents common to all species (AUT-PAGE results).

CONCLUSIONS

1. Haemoglobin and globin chain studies are considered to be taxonomicallyhighly informative in tilapias.

2. It is indicated that natural or artificial interspecific hybridization events couldbe recognized and verified using haemoglobin and globin chain data.

142 T.M. Falk et al.

3. There is evidence that variations of haemoglobin types could occur amongnatural populations of tilapias.

ACKNOWLEDGEMENTS

The present work has been accomplished during a collaborative project funded byBMZ/GTZ, Germany, and involved the Institute of Aquatic Biology, Ghana, theUniversity of Hamburg, Germany, and the International Center for Living AquaticResources Management, the Philippines.

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145Haemoglobin and globin chain variations in tilapias