Salinity-stimulated changes in expression and activity of two carbonic anhydrase isoforms in the blue crabCallinectes sapidus

Abstract

SUMMARY Two isoforms of the enzyme carbonic anhydrase (CA) in the blue crab gill,CasCAg and CasCAc, were identified, sequenced, and found to match the membrane-associated and cytoplasmic isoforms, respectively. The membrane-associated isoform is present in much higher levels of mRNA expression in both anterior and posterior gills in crabs acclimated to high salinity (35 p.p.t.), but expression of the cytoplasmic isoform in the posterior gill undergoes a significantly greater degree of up-regulation after exposure to low salinity (15 p.p.t.). CasCAc has the largest scope of induction (100-fold) reported for any transport-related protein in the gill,and this may be necessary to overcome diffusion limitations between gill cytoplasm and the apical boundary layer. Furthermore, the timing of the changes in expression of CasCAc corresponds to the timing of the induction of protein-specific CA activity and CA protein concentration. No changes in CA mRNA expression or activity occur in the anterior gills. The pattern of up-regulation of expression of mRNA of the α-subunit of the Na+/K+-ATPase is similar to that for CasCAc, and both precede the establishment of the new acclimated physiological state of the crab in low salinity. A putative `housekeeping' gene, arginine kinase, also showed about a threefold increase in expression in response to low salinity,but only in the posterior gills. These results suggest that for studies of expression in crustacean gill tissue, a control tissue, such as the anterior gill, be used until an adequate control gene is identified.

Osmoregulation in the Hawaiian anchialine shrimpHalocaridina rubra(Crustacea: Atyidae): expression of ion transporters, mitochondria-rich cell proliferation, and hemolymph osmolality during salinity transfers

“…at 32‰) and at 3, 8, 24 and 48 h and 7 days post-transfer. Based on those results and previous studies (Luquet et al, 2005;Serrano et al, 2007;Havird et al, 2013), shrimp transferred to 2‰ and 45‰ were sampled at 24 h and 7 days post-transfer. Shrimp were anesthetized on ice and gill undercarriages (see above) dissected into tubes containing ice-cold denaturing solution from the RNAgents Total RNA Isolation System (Promega, Madison, WI, USA).…”

“…In euryhaline crustaceans, expression of these genes usually increases dramatically in the osmoregulatory gills during comparable salinity transfers (reviewed by Havird et al, 2013). For example, NKA expression in C. granulatus increased 25-to 55-fold after transfer from SW to 45‰ and 2‰ (Luquet et al, 2005), with similar results for Scylla paramamosain (Chung and Lin, 2006), Pachygrapsus marmoratus (Jayasundara et al, 2007), C. sapidus (Serrano et al, 2007), C. maenas (Serrano and Henry, 2008;Jillette et al, 2011), Macrobrachium amazonicum (Faleiros et al, 2010) and Litopenaeus vannamei (Wang et al, 2012). Although utilized in fewer studies, CAc (but not CAg) (see Serrano and Henry, 2008), NKCC, HAT and AK (e.g.…”

“…For example, Chasmagnathus granulatus (subsequently renamed Neohelice granulate) (Sakai et al, 2006) increased NKA expression 33-to 55-fold (Luquet et al, 2005) when transferred from 30‰ to 2‰. Similarly, cytoplasmic CA (CAc; the osmoregulatory isoform of CA) underwent a 100-fold increase in expression during high to low salinity transfers in Callinectes sapidus and Carcinus maenas (Serrano et al, 2007;Serrano and Henry, 2008). Analogous patterns have been reported for additional osmoregulatory genes, including the Na…”

“…Although it is unclear whether this hypothesized chronic upregulation of osmoregulatory processes is an adaptation to anchialine habitats, such a strategy may allow H. rubra to cope with the rapid and continuous salinity fluctuations they encounter, and it will be interesting to see whether this pattern of elevated osmoregulatory processes is characteristic of euryhaline atyids or anchialine crustaceans in general. Finally, it is unlikely that using the gill undercarriage instead of individual gills skewed the expression results, as the genes under investigation do not significantly change in non-osmoregulatory tissues during salinity transfers (see Henry and Cameron, 1982a;Henry, 2001;Serrano et al, 2007;Henry et al, 2012).…”

“…To compare H. rubra mRNA expression of genes previously identified as playing roles in crustacean osmoregulation during salinity transfer (Luquet et al, 2005;Serrano et al, 2007;Havird et al, 2013), specific primers were designed for H. rubra targeting six genes: Na + /K + -ATPase α-subunit (NKA), the cytoplasmic, osmoregulatory isoform of carbonic anhydrase (CAc), the membrane-associated, respiratory isoform of CA (CAg), the Na + /K + /2Cl -co-transporter (NKCC), the H + -ATPase (HAT) and arginine kinase (AK). These primers were specifically designed for H. rubra using transcriptomes generated from the East Hawaii and Windward Oahu genetic lineages (EP and HILO) (see Craft et al, 2008) (Fig.…”