Comparative analysis of three families of hygrophilid snails shows that the egg mass fluid protein haemocyanin-like 1 (Hcl-1) is unique to planorbids

The egg mass fluid (EMF) of the freshwater snail Biomphalaria glabrata (Hygrophila: Planorbidae) contains haemocyanin-like 1 (Hcl-1) protein, distinct from respiratory haemocyanins. The distribution of Hcl-1 was investigated among major families of Hygrophila, Physidae and Lymnaeidae, both of which employ respiratory haemocyanins, and Planorbidae, a group that evolved haemoglobin as a respiratory pigment. Immunoblotting detected c. 150 kDa protein (molecular weight of Hcl-1) cross-reactive with anti-keyhole limpet haemocyanin antiserum in the EMF of planorbids Bulinus globosus and Planorbella duryi (from a genus closely related to Biomphalaria), but not Physella acuta (Physidae) and Ladislavella elodes (Lymnaeidae). High throughput sequence data revealed Hcl-1 homologs from Bulinus globosus and Planorbella duryi, representative species that span the range of planorbid phylogeny, but not from Physella acuta (Physidae) and Lymnaea stagnalis (Lymnaeidae). A domain architecture comprising only three functional units (FUs) and predicted secondary structures within the C-terminal FU distinguish planorbid Hcl-1 protein from molluscan respiratory haemocyanins that are natively assembled as functional didecamers. Immunoblotting confirmed a monomeric configuration of native Hcl-1. Molecular clock analysis estimated divergence of Hcl-1 proteins from gastropod respiratory haemocyanins at 267 ± 143 Ma. It is hypothesized that Hcl proteins originated in the ancestor of the planorbid lineage when evolution of respiratory haemoglobin altered selective pressures for maintaining original function, facilitating mutation and refunctionalization of the ancestral respiratory haemocyanin in Planorbidae.