Research Note

The Tamaraw (Bubalus (B.) mindorensis) Hemoglobin Phenotype and Comparison among the Asian Buffaloes Based on Isoelectric Focusing

Chester D SOLIS, Yoshi KAWAMOTO*, Kazuaki TANAKA, Joseph S MASANGKAY**, Kei-ichiro MAEDA and Takao NAMIKAWA

School of Agricultural Sciences, Nagoya University, Chikusa-ku, Nagoya-shi 464-01 * Primate Research Institute , Kyoto University, Inuyama-shi 484 ** College of Veterinary Medicine , University of the Philippines at Los Banos, Laguna, 4031, the Philippines

(Received June 16, 1995)

Abstract Hemoglobins (Hb) from seven captive tamaraws were examined and compared to that of water buffaloes (Bubalus bubalis): five river type and eleven swamp type; and two species of anoas: two lowland anoas (Bubalus (Anoa) depressicornis H. Smith, 1827) and eight mountain anoas (B. (A.) quarlesi Ouwens, 1910) using ultrathin polyacrylamide gel isoelectric focusing (PAGIF). The Hb phenotype of the tamaraws displayed a monomorphic single band, compared to two bands (major and minor bands) in water buffaloes and one to three allelic bands found in polymorphic anoa individuals. We designate this band HbTamaraw,having the highest isoelectric point (pI) focused at the same level as the minor band. Dissociation of globin chains by urea-PAGIF showed that HbTamaraw consists of a single type of α and β globins, whose α focused at

the same pI level as the α minor band of water buffalo, as it is also true between the single α band of the anoa and the α major band of water buffalo focused at a lower pI. The β band of the

tamaraw shared the same pI level to the β hands of both the water buffaloes and lowland anoas.

The HbTamaraw differs by having a single α component from that of the water buffaloes, and an a band with a higher pI level from that of the anoas. Anim. Sci. Technol. (Jpn.) 66(12):1014-1018, 1995 Key words: Bubalus mindorensis, tamaraw, water buffalo, hemoglobin, isoelectric focusing

The tamaraw (Bubalus (B.) mindorensis Mindoro, the Philippines. Various nomencla- Heude, 1888)6,7) is an endangered wild buffalo tures including Bubalus arnee mindorensis9) and which can only be found in the island of Anoa mindorensis5) have been given to this rare

タ マ ラ オ(Bubalus (B.) mindorensis)ヘ モ グ ロ ビ ン の 等 電 点 電 気 泳 動 法 に よ る 表 現 型 と ア ジ ア ス イ ギ ュ ウ の そ れ と の 比 較:Chester D Solis・ 川 本 芳*・ 田 中 和 明 ・Joseph S Masangkay**・ 前 多 敬 一 郎 ・並 河 鷹 夫(名 古 屋 大 学 農 学 部,名 古 屋 市 千 種 区646-01,*京 都 大 学 霊 長 類 研 究 所,犬 山 市484,**フ ィ リ ピ ン 大 学 獣 医 学 部,ラ グ ナ 県4031)

Anim. Sci. Technol. (Jpn.) 66(12):1014-1018 1014 1995 Isoelectrophoretic Hemoglobin Phenotype of the Tamaraw species as incurred by the insufficient knowl- 30W constant power, maximum voltage of edge to its basic biological character. Up to 1,500V for 60-90 min. Globin chains were the present, its precise taxonomic classification analyzed by an ultrathin urea-PAGIF gel remains elusive, yet crucial for its active con- which contains 8M urea. Focusing was servation. Presently it is classified under the carried out at the same constant power and Asian buffalo group10) which include the Anoa voltage for 120 min. The same staining proce- of Sulawesi in Indonesia and the water buffalo, dure was employed. however the exact divergence process of this Results group is yet unclear. Based on few characters and at morphological level, they have been Figure 1 A shows the tetrameric Hb pheno- placed under the genus Bubalus, where the type patterns of the Asian buffaloes by ultra- tamaraw and the water buffalo fall under the thin PAGIF at 5-8 pH gradient. The tamaraw subgenus Bubalus, while the two species of Hb displayed uniformly single major band anoas fall under subgenus Anoa6). At molecu- focused at an approximate pI level 7.2-7.3. We lar level, several studies on the hemoglobin of designate this single band as the HbTamaraw in buffaloes have been made using various elec- the present paper. In comparison with the trophoretic methods2,4,8,11,14,15). In this paper, other Asian buffaloes, the water buffaloes we describe the analysis of tamaraw hemoglo- showed two bands, a minor band having the bin phenotype for the first time and compare it same pI with the HbTamaraw,and a major band with the other Asian buffaloes using the more (pI=7.0-7.1) with lower pI together with one to resolutive ultrathin polyacrylamide gel iso- three different band types in anoas (pI=6.8- electric focusing (PAGIF) to increase knowl- 7.1). edge on its genetic character and confer signifi- Dissociation of the Hb molecules using ultra- cant genetic insights in understanding the di- thin urea-PAGIF revealed two constituent vergence process among these Asian buffaloes. globin chains in tamaraw, corresponding to single α globin with high pI and single β globin Materials and Methods with low pI (Fig. 1B). The tamaraw α globin

Blood samples were collected in EDTA tubes (Tα) band together with the α minor (IIα) band from seven captive tamaraws from Mindoro, of the water buffaloes were observed to have the Philippines, sixteen water buffaloes (five same pI at the highest level among all the river type and eleven swamp type) from Indo- globin bands. Anoas also showed single α nesia and the Philippines, and ten anoas (two globin (Aα) band with a different pI from that of IIα but the same as the α major (Ia) band of the lowland anoas and eight mountain anoas) kept at Indonesian zoos. Hemolysates and globin water buffaloes, The β globin of the tamaraw, components were prepared according to a pre- however apparently showed the same pI with vious method3) with slight modification. those of the rest of the buffaloes examined, Ultrathin polyacrylamide gel isoelectric focus- except for some mountain anoas, e. g. MA-2, ing (PAGIF) was carried out in a 200μm-thick MA-5 and MA-6, which show polymorphic β gel at 5-8 pH gradient, then stained with alleles (Fig. 1B). Coomassie Brilliant Blue R-250 after fixation Discussion with 10% trichloroacetic acid according to the manufacturer's instruction (Pharmacia LKB Bi- The resolving power of the methods used in otechnology, Uppsala, Sweden). Focusing the present study clearly revealed the elec- was performed on an isoelectric focusing (IEF) trophoretic phenotype of the tamaraw Hb, flat bed apparatus with focusing condition at designated as HbTamaraw, which we assume to

1015 SOLIS, KAWAMOTO, TANAKA, MASANGKAY, MAEDA and NAMIKAWA

Fig.1A. Comparative electrophoretic Hb phenotype (with diagram) among the representative Asian buffaloes (T=tamaraw, LA=lowland anoa, MA=mountain anoa, IS=swamp type from Indonesia, PS=swamp type from the Philippines, IR= river type from Indonesia and PR=river type from the Philippines of water buffaloes) focused on the ultrathin PAGIF at 5-8 pH gradient. Bands not identified are represented by broken lines.

Fig 1B. Comparative electrophoretic anaiysis (with diagram) of the Hbα (Tα= tamaraw α, Aα=anoa α, Iα=α major and IIα=α minor of water buffalo)and β globin constituents among the Asian buffaloes (T=tamaraw, LA=lowland anoa, MA=mountain anoa, IS=swamp type from Indonesia, PS=swamp type from the Philippines, IR=river type from Indonesia and PR=river type from the Philippines of water buffaloes) by ultrathin urea-PAGIF at 5-8 pH gradient. Bands not identified are represented by broken lines.

1016 Isoelectrophoretic Hemoglobin Phenotype of the Tamaraw consist a unique single molecular form of phism. This may indicate low degree of ge- tetrameric α2 β2. The separation method fur- netic variability in the tamaraw population. ther allowed us to ascertain the presence of one The maintenance of genetic variability in a type of α and β dissociated globin chains. The small population is important in reducing appearance of minor bands (not identified) sim- homozygosity and inbreeding depression1). ilar to those observed in other studies4,11,15) is a Thus such findings relating to its population phenomenon recognized to be widespread and structure may prove useful to confer consider- common in such technique of very high resolu- able insights aiming for the effective breeding tion which frequently resolves proteins into and management of captive colonies and con- many more bands. Consequently, as com- servation of endangered species. pared with other techniques, the present Very few studies have inferred some basic results remain to be reliable which were more biological characteristics on this animal. definely demonstrated consistent with the Based on measurement of external characters, previous studies elsewhere using conventional the tamaraw exhibited values in between the electrophoresis and isoelectric focus- water buffaloes and the anoas; while based on ing4,8,11,14,15). karyotypes, only the tamaraw and the swamp The HbTamarawdisplayed a phenotype pattern type buffalo showed proximity by sharing the evidently distinct despite its close relationship same fundamental number (FN=58) among the to the other buffaloes examined, as revealed by bubaline species13). Likewise, behavioral pat- the methods used. Notable differences ob- tern of the captive tamaraws indicated that served among these Asian buffaloes lie upon they demonstrate similarity with the water their α globin chains. The water buffaloes buffaloes in several behavioral parameters have two kinds of α globins (Iα and IIα), while observed12). the tamaraws consist of only one kind with The tamaraw exhibits uniqueness in its Hb similar pI with that of IIα. The anoas also phenotype among the Asian buffalo group as focused a single α band, however with a pat- evidenced by its distinct α globin band. tern having similar pI with the Iα band. The Employing isoelectric focusing to analyze Hb two α globins in water buffaloes are known to phenotype as a genetic marker can be useful in be under the control of two structural loci as a the differentiation of Asian buffalo species as result of duplication of α globin locus14). The well as potentially to demonstrate the degree IIα differs from Iα as previously described , by of genetic variability in their populations. three amino acid substitution, and having However detailed primary structural level higher positive net charge and greater cathodic analysis of its Hb, as well as analyses extend- character4). Such α globin fashion among ing to other genetic markers may provide fur- these Asian buffaloes poses some interesting ther understanding on the evolutionary rela- inquiries on their evolutionary relationship. tionship of these globin genes and to the gene-

On the other hand, although the β globin bands tic character and phylogeny of the tamaraw of these buffaloes displayed similar pI, the and the rest of the Asian buffaloes. water buffaloes differ from the lowland anoa Acknowledgment by two amino acid residues, despite no charge difference observed in electrophoresis, thus We sincerely thank Dr. V. G. Momongan (IAS claiming a significant differentiation between -CA) , Dr. M.F. Manuel (Dean, CVM) and the the two species8). staff of CVM-ADDL at UPLB for the facilities It is also interesting to note that these and assistance; the officers and technicians of tamaraws showed the absence of Hb polymor- the Tamaraw Conservation Project at Mindoro

1017 SOLIS, KAWAMOTO, TANAKA, MASANGKAY, MAEDA and NAMIKAWA for the invaluable source of samples; and help 8) Kakoi H, Namikawa T, Takenaka O, Takenaka from Mr. H. Nagabukuro. This research is A, Amano T, Martojo H. Divergence between supported by grants from the Inui Memorial the anoas of Sulawesi and the Asiatic water buffaloes, inferred from their complete amino Trust for Research on Animal Science, and acid sequence of hemoglobin β chains. Z. zool. from the Fund for Global Environment Syst. Evolut. -forsch., 32:1-10. 1994. through the Nagoya Foundation of Animal Sci- 9) Klos H-G, Wunschmann A. The wild and do- ence (Dr. T. Tomita, director). mestic oxen. In: Grzimek's Animal Life Encylopedia. (Grzimek B ed. in chief) 331-398. References 13 (Mammals IV). Van Nostrand Reinholt Com- 1) Alderson GLH. A system to maximize the pany, New York, Cincinnati, Toronto, London, Melbourne. 1972. maintenance of genetic variability in small 10) Mahadevan P. Distribution, ecology and adap- populations. In: Genetic Conservation of Do- tation. In: World Animal Science. (Holmes mestic Livestock vol. II. (Alderson L, Bodo I JHG, Tulloh NM eds.) 1-12. C6 (Buffalo Produc- eds.) 18-29. CAB International. Wallingford, tion). Elsevier Science Publishers B. V., Am- UK. 1992. sterdam, The Netherlands. 1992. 2) Balani AS, Barnabas J. Polypeptide chains of 11) Masina P, Ianneli D, Iorio M, Ramunno L. He- buffalo hemoglobins. Nature, 205:1019-1021. moglobin polymorphism in Italian water 1965. buffalo (Bubalus bubalus (Arnee)). Anim. Blood 3) Chernoff AI, Pettit NM Jr. The amino acid Grps Biochem. Genet., 8:65-72. 1977. composition of hemoglobin. III. A qualitative 12) Momongan VG, Walde GI. The behavioral pat- method for identifying abnormalities of the tern of tamaraws (Bubalus mindorensis Heude) polypeptide chains of hemoglobin. Blood, 24: in captivity during the dry and wet seasons. A 750-756. 1964. study conducted under the Tamaraw Conser- 4) Di Luccia D, lannibelli L, Ferranti P, Iorio M, vation Program, University of the Philippines Annunziata M, Ferrara L. Water buffalo at Los Banos, Philippines, 1-22. 1993. (Bubalus bubalis) hemoglobins: An electro- 13) Namikawa T, Masangkay JS, Maeda K-I, phoretic and chromatographic study. Comp. Escalada R, Hirunagi K, Momongan VG. Exter- Biochem. Physiol., 94B:71-77. 1989. nal characters and karyotypes of the captive 5) Fischer H, Hohn H. Der karyotyp eines tamaraws Bubalus (B.) mindorensis, at the Gene weiblichen tamarau (Anoa mindorensis). Giess. Pool in the island of Mindoro, Philippines. J. Beitr. Erbpathol. Zucht. hyg., 6:173-177. 1976. Anim. Genet., 23:19-28. 1995. 6) Groves CP. Systematics of the anoa (Mamma- 14) Ranjekar PK, Barnabas J. Haemoglobin pheno- ia, Bovidae). Beaufortia (Series of Miscellane- l types in water buffalo (Bos bubalus) during ous Publications, Zoological Museums of the development. Comp. Biochem. Physiol., 28: University of Amsterdam, No. 223), 17:1-11. 1395-1401. 1969. 1969. 15) Schreiber A, Notzold G, Held M. Molecular and 7) Grubb P. Artiodactyla: Bovidae: bovinae. In: chromosomal evolution in anoas (Bovidae: Mammals Species of the World: A Taxonomic Bubalus spec.). Z. zool. Syst. Evolut. -forsch., and Geographic Reference. 2nd ed. (Wilson DE, 31:64-79. 1993. Reeder DAM eds.) 377-414. Smithsonian Insti- tutional Press. Washington and London. 1993.

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