Abstract
Barnacles are sessile macro-invertebrates, found along rocky shores in coastal areas worldwide. The euryhaline bay barnacle Balanus improvisus (Darwin, 1854) (= Amphibalanus improvisus) can tolerate a wide range of salinities, but the molecular mechanisms underlying the osmoregulatory capacity of this truly brackish species are not well understood. Aquaporins are pore-forming integral membrane proteins that facilitate transport of water, small solutes and ions through cellular membranes, and that have been shown to be important for osmoregulation in many organisms. The knowledge of the function of aquaporins in crustaceans is, however, limited and nothing is known about them in barnacles. We here present the repertoire of aquaporins from a thecostracan crustacean, the barnacle B. improvisus, based on genome and transcriptome sequencing. Our analyses reveal that B. improvisus contains eight genes for aquaporins. Phylogenetic analysis showed that they represented members of the classical water aquaporins (Aqp1, Aqp2), the aquaglyceroporins (Glp1, Glp2), the unorthodox aquaporin (Aqp12) and the arthropod-specific big brain aquaporin (Bib). Interestingly, we also found two big brain-like proteins (BibL1 and BibL2) constituting a new group of aquaporins not yet described in arthropods. In addition, we found that the two water-specific aquaporins were expressed as C-terminal splice variants. Heterologous expression of some of the aquaporins followed by functional characterization showed that Aqp1 transported water and Glp2 water and glycerol, agreeing with the predictions of substrate specificity based on 3D modeling and phylogeny. To investigate a possible role for the B. improvisus aquaporins in osmoregulation, mRNA expression changes in adult barnacles were analysed after long-term acclimation to different salinities. The most pronounced expression difference was seen for AQP1 with a substantial (>100-fold) decrease in the mantle tissue in low salinity (3 PSU) compared to high salinity (33 PSU). Our study provides a base for future mechanistic studies on the role of aquaporins in osmoregulation.
Highlights
Aquaporins (AQPs) are pore-forming integral membrane proteins that mainly facilitate transport of water and small solutes through cellular membranes
Initial phylogenetic classification indicated that two of the eight B. improvisus aquaporins belong to the water specific AQPs within the classical aquaporin subfamily
The last two of the B. improvisus aquaporins are in a clade most closely related to big brain and were named Biblike1 (BibL1) and big brain aquaporin (Bib)-like2 (BibL2) (Fig 1)
Summary
Aquaporins (AQPs) are pore-forming integral membrane proteins that mainly facilitate transport of water and small solutes through cellular membranes. The superfamily of eukaryotic aquaporins is phylogenetically divided into four main subfamilies (referred to as "grades" [2]) based on their sequences: The classical aquaporins that mainly transport water, the aquaglyceroporins that transport glycerol and other small molecules, the aquaammoniaporins that transport ammonia and other small molecules, and the unorthodox aquaporins that are, at this stage, not well functionally characterized [2]. In addition to the water-transporting aquaporins, the classical aquaporins includes two arthropod specific subfamilies, big brain (BIB) and Eglp, for which water is not the main substrate [2]. BIB does not transport water [5] but has been reported to take part in ion conductance and cell-adhesion [6, 7]. Sequence-function relationship between the various classes is in some cases quite complex. This evolutionary complexity makes it essential to experimentally determine the functionality of newly identified aquaporins, especially for groups of organisms that are not well studied
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