Alone among piscine taxa, the antarctic icefishes (family Channichthyidae, suborder Notothenioidei) have evolved compensatory adaptations that maintain normal metabolic functions in the absence of erythrocytes and the respiratory oxygen transporter hemoglobin. Although the uniquely "colorless" or "white" condition of the blood of icefishes has been recognized since the early 20th century, the status of globin genes in the icefish genomes has, surprisingly, remained unexplored. Using a-and f-globin cDNAs from the antarctic rockcod Notothenia coriiceps (family Nototheniidae, suborder Notothenioidei), we have probed the genomes of three white-blooded icefishes and four red-blooded notothenioid relatives (three antarctic, one temperate) for globinrelated DNA sequences. We detect specific, high-stringency hybridization of the a-globin probe to genomic DNAs of both white-and red-blooded species, whereas the 8-globin cDNA hybridizes only to the genomes of the red-blooded fishes. Our results suggest that icefishes retain inactive genomic remnants of a-globin genes but have lost, either through deletion or through rapid mutation, the gene that encodes p-globin. We propose that the hemoglobinless phenotype of extant icefishes is the result of deletion of the single adult ,3-globin locus prior to the diversification of the clade.In 1954 Ruud (1) published the first systematic analysis of the "white" blood of an antarctic icefish, Chaenocephalus aceratus. He reported that fresh blood was nearly transparent, contained leukocytes at 1% by volume, but lacked erythrocytes and the respiratory transport pigment hemoglobin. Furthermore, the oxygen-carrying capacity of C. aceratus blood was approximately 10% that of two red-blooded notothenioids. Subsequent investigations extended these observations to other icefish species and revealed that icefish blood contains small numbers of "erythrocyte-like" cells that, nevertheless, are devoid of hemoglobin (2, 3). Thus, limited to oxygen physically dissolved in their blood, the icefishes have evolved compensatory physiological and circulatory adaptations-e.g., modest suppression of metabolic rates, enhanced gas exchange by large, well-perfused gills and cutaneous respiration, and large increases in cardiac output and blood volume-that ensure adequate oxygenation of their tissues (4, 5).We have initiated studies to determine the status of globin genes in channichthyid genomes and to evaluate potential evolutionary mechanisms leading to the hemoglobinless phenotype. Icefishes evolved from the red-blooded Notothenioidei (6, 7), which, in contrast to temperate fishes, are characterized by a paucity of hemoglobin forms (7-10). Adults of the family Nototheniidae (antarctic rockcods) generally possess a major hemoglobin, Hb 1 (-95% of the total), and a second, minor hemoglobin, Hb 2, that differ in their a chains (al and a2, respectively) (11-13). The more phyletically derived harpagiferids and bathydraconids have a single hemoglobin. The trend toward reduced hemoglobin multiplicity in t...
