Unlike mammals, some bony fish species have been reported to possess multiple forms of the complement component C3. To explore the structural and functional diversity of bony fish C3, we have isolated eight distinct cDNA clones encoding C3 from a single carp (Cypri-nus carpio). The eight sequences were grouped into five C3 types, designated C3-H1, C3-H2, C3-S, C3-Q1 and C3-Q2, each sharing 80-86 % amino acid sequence identity with the others. A striking amino acid substitution was noted at the position corresponding to the catalytic histidine, which is conserved in C3 from all the animals analyzed to date and provides the thioester with the ability to bind covalently to hydroxy groups on the target cells or to be hydrolyzed quickly; C3-S, C3-Q1 and C3-Q2 have serine, glutamine and glutamine residues, respectively, in place of the histidine which is conserved in C3-H1 and C3-H2. On the other hand, five distinct C3 forms, named C3-1 to C3-5, were purified from the serum of a single carp. N-terminal sequencing and covalent binding to [ 3 H]glycine identified C3-1 as the translated product of C3-S, while C3-2 was that of C3-H1, and C3-5 that of C3-H2. C3-1 showed a hemolytic activity threefold higher than that of C3-2, whereas C3-5 was inactive, suggesting that the thioester catalytic mechanism is not a necessary determinant for C3 activity and that C3 lacking the catalytic histidine plays a significant role in the complement system of carp and probably other bony fish.

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Unlike mammals, some bony fish species have been reported to possess multiple forms of the complement component C3. To explore the structural and functional diversity of bony fish C3, we have isolated eight distinct cDNA clones encoding C3 from a single carp (Cyprinus carpio). The eight sequences were grouped into five C3 types, designated C3‐H1, C3‐H2, C3‐S, C3‐Q1 and C3‐Q2, each sharing 80 – 86 % amino acid sequence identity with the others. A striking amino acid substitution was noted at the position corresponding to the catalytic histidine, which is conserved in C3 from all the animals analyzed to date and provides the thioester with the ability to bind covalently to hydroxy groups on the target cells or to be hydrolyzed quickly; C3‐S, C3‐Q1 and C3‐Q2 have serine, glutamine and glutamine residues, respectively, in place of the histidine which is conserved in C3‐H1 and C3‐H2. On the other hand, five distinct C3 forms, named C3‐1 to C3‐5, were purified from the serum of a single carp. N‐terminal sequencing and covalent binding to [3H]glycine identified C3‐1 as the translated product of C3‐S, while C3‐2 was that of C3‐H1, and C3‐5 that of C3‐H2. C3‐1 showed a hemolytic activity threefold higher than that of C3‐2, whereas C3‐5 was inactive, suggesting that the thioester catalytic mechanism is not a necessary determinant for C3 activity and that C3 lacking the catalytic histidine plays a significant role in the complement system of carp and probably other bony fish.

Unlike mammals, bony fish possess multiple genes encoding the complement component C3, a member of the alpha2-macroglobulin (alpha2M) protein family, presumably expanding the diversity of immune recognition. To examine whether the alpha2M gene has also duplicated and diverged in the bony fish lineage, cDNA cloning of alpha2M from a pseudotetraploid teleost, the common carp (Cyprinus carpio), was conducted and resulted in the isolation of three distinct alpha2M sequences from a single individual, indicating the presence of multiple alpha2M genes in this species. The deduced amino acid sequences contained a post-translational cleavage signal, predicting a C3-like two-chain structure, as in lamprey alpha2M. Two distinct alpha2M proteins were purified from carp serum; both proved to be Mr 380,000 dimers, the subunits of which are composed of disulfide-linked alpha chains (Mr 93,000) and beta chains (Mr 85,000), as reported for the alpha2M from plaice, another teleost species. The presence of an internal thioester in the alpha chain was demonstrated by its autolytic fragmentation and direct incorporation of [14C]methylamine. Interestingly, the three forms of carp alpha2M exhibited outstanding sequence diversity in the bait region which displays target sequences for various proteases, and in the C-terminal region of the alpha chain assigned as the receptor-binding domain, while an Asn residue at the position corresponding to the catalytic His in C3 was completely conserved in the carp alpha2Ms, as in most alpha2Ms of other animals. The possible functional significance of the sequence diversity is discussed.

A histological examination was made of the ontogenetic development of the digestive and immune systems of the larval and juvenile kelp grouper Epinephelus bruneus reared in the laboratory. The liver, gall bladder, pancreas and the demarcating region between the intestines and rectum were formed within 3 days post-hatch (dph). During the preflexion phase (within 16 dph), revolution of the intestine concluded, and pharyngeal teeth and the mucous cells of the esophagus were differentiated. In the transitional period to the juvenile stage (25 dph), the blind sac of the stomach, gastric glands and pyloric caeca began to form. From the viewpoint of the differentiation phase of the adult-type digestive system, the kelp grouper is similar to Heterosomata, hitherto reported. The primordial thymus, kidney and spleen were present at 12, 1 and 6 dph, and the small lymphocytes in these lymphoid organs appeared at 21, 30 and 33 dph, respectively. The developmental sequence of the lymphoid organs and the appearance ages of the lymphoid organs and small lymphocytes in the lymphoid organs in the kelp grouper are similar to those of other marine fish previously reported, except for the Pacific bluefin tuna Thunnus orientalis.