Identification and characterisation of hemocyanin of the fish louse Argulus (Crustacea: Branchiura).

Hemocyanin transports oxygen in the hemolymph of many arthropod species. Within the crustaceans, this copper-containing protein was thought to be restricted to Malacostraca, while other crustacean classes were assumed to employ hemoglobin or lack any respiratory protein. Only recently it has become evident that hemocyanins also occur in Remipedia and Ostracoda. Here we report for the first time the identification and characterisation of hemocyanin in the fish louse Argulus, which belongs to the class of Branchiura. This finding indicates that hemocyanin was the principal oxygen carrier in the stem lineage of the pancrustaceans, but has been lost independently multiple times in crustacean taxa. We obtained the full-length cDNA sequences of two hemocyanin subunits of Argulus foliaceus by a combination of RT-PCR, RACE and Illumina sequencing of the transcriptome. In addition, one full-length and one partial cDNA sequence were derived from the transcriptome data of Argulus siamensis. Western blot analysis confirmed the presence of at least two hemocyanin subunits in A. foliaceus, which are expressed at the mRNA level at a 1:3.5 ratio. The addition to the branchiuran hemocyanin subunits to a multiple sequence alignment of arthropod, hemocyanins improved the phylogenetic resolution within the pancrustacean hemocyanins. Malacostracan, ostracod and branchiuran hemocyanins are distinct from the hexapod and remipede hemocyanins, reinforcing the hypothesis of a close relationship of Remipedia and Hexapoda. Notably, the ostracod hemocyanins are paraphyletic with respect to the branchiuran hemocyanins, indicating ancient divergence and differential loss of distinct subunit types.