Analysis of aquaporins from the euryhaline barnacle Balanus improvisus reveals differential expression in response to changes in salinity

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.

Transcriptional dynamics following freezing stress reveal selection for mechanisms of freeze tolerance at the poleward range margin in the cold water intertidal barnacleSemibalanus balanoides

“…In addition, we found very low Tajima’s D values in aquaporins that were upregulated following freezing (Table 4), suggesting that selective sweeps had taken place on the sequence of these putative aquaporins. Follow-up with functional studies, particularly employing the frog oocyte expression system, would provide interesting evidence and further structural information in this poorly understood system (Lind et al, 2017).…”

The complex barnacle perfume: identification of waterborne pheromone homologues inBalanus improvisusand their differential expression during settlement

“…Six sequences that constituted full or partial fragments of WSP-like sequences were found, five of which were cloned and confirmed by Sanger sequencing. The sixth sequence was identified from a single molecule PacBio RNA dataset obtained from an adult of B. improvisus (Lind et al 2017). The sequences identified were named according to the level of identity to the published B. amphitrite WSP, eg the Bi_WSP clone was 74% identical and 91% similar at the protein level to the B. amphitrite WSP (Table 1), while the WSP-like 1-5 sequences had a gradually lower (in the range of 39%-70% identity and 71%-88% similarity) level of identity/similarity that followed the gene numbering.…”

“…Bi_WSPlike 1 was not cloned. Instead, a full-length sequence corresponding to Bi_WSP-like 1 was identified from an adult single-molecule PacBio RNA dataset (Lind et al 2017), where the corresponding consensus contig covering the ORF was supported by more than 30 PacBio reads. The sequences have been deposited in GenBank with accession numbers MK275628-MK275633.…”

The Barnacle <em>Balanus improvisus</em> as a Marine Model - Culturing and Gene Expression

“…One species, the bay barnacle Balanus (Amphibalanus) improvises ( B. improvisus ), can tolerate almost freshwater and Charles Darwin described this species from a small stream in the estuary of Rio de la Plata in Uruguay 1 . The extreme tolerance tolow salinity makes B. improvisus a particularly relevant model for the studies of osmoregulatory mechanisms 2 3 . This barnacle prefers brackish conditions but is capable of living in waters with salinities from around 1.6 psu to as high as 40 psu 4 .…”

“…There is also a draft genome and several transcriptomes available, and these resources have been used for the cloning of several genes ( e.g. , genes of importance in osmoregulation) 2 3 .…”