Molecular characterization and evolution of haemocyanin from the two freshwater shrimps Caridina multidentata (Stimpson, 1860) and Atyopsis moluccensis (De Haan, 1849)

Haemocyanin (Hc) is a copper-containing respiratory protein, floating freely dissolved in the hemolymph of many arthropod species. A typical haemocyanin is a hexamer or oligohexamer of six identical or similar subunits, with a molecular mass around 75 kDa each. In the crustaceans, the haemocyanins appear to be restricted to the remipedes and the malacostracans. We have investigated the haemocyanins of two freshwater shrimps, the Amano shrimp Caridina multidentata and the bamboo shrimp Atyopsis moluccensis. We obtained three full-length and one partial cDNA sequences of haemocyanin subunits from the Amano shrimp, which were assigned to the alpha- and gamma-types of decapod haemocyanin subunits. Three complete and two partial haemocyanin cDNA sequences were obtained from the bamboo shrimp, which represent subunit types alpha, beta and gamma. This is the first time that sequences of all three subunit types of the decapod haemocyanins were obtained from a single species. However, mass spectrometry analyses identified only alpha- and gamma-type subunits, suggesting that a beta-subunit is not a major component of the native haemocyanin of the bamboo shrimp. Phylogenetic and molecular clock analyses showed that malacostracan haemocyanins commenced to diversify into distinct subunit types already similar to 515 million years ago. beta-subunits diverged first, followed by alpha- and gamma-type subunits similar to 396 million years ago. The haemocyanins of phyllocarids and peracarids form distinct clades within the alpha/gamma-cluster. Within the Caridea, an early divergence of distinct alpha-type subunits occurred similar to 200 MYA. The tree of the gamma-subunits suggests a common clade of the Caridea (shrimps) and Penaeidae (prawns).