Salt transport by the gill Na⁺-K⁺-2Cl^-symporter in palaemonid shrimps: exploring physiological, molecular and evolutionary landscapes

Abstract

Palaemonid shrimps include species from distinct osmotic niches that hyper-regulate hemolymph osmolality and ionic concentrations in dilute media but hypo-regulate in saline media. Their gill epithelia express ion transporters like the Na+-K+-2Cl- symporter (NKCC) thought to play a role in salt secretion. Using a palaemonid series from niches including marine tide pools through estuaries (Palaemon) to coastal and continental fresh waters (Macrobrachium), we established their critical upper salinity limits (UL50) and examined their short-(24 h) and long-term (120 h) hypo-regulatory abilities at salinities corresponding to 80% of the UL50’s (80%UL50). We tested for phylogenetic correlations between gill NKCC gene and protein expression and hemolymph Cl- hypo-regulatory capability, and evaluated whether niche salinity might have driven gill NKCC expression. The Palaemon species from saline habitats showed the highest UL50’s and greatest hypo-regulatory capabilities compared to the Macrobrachium species among which UL50’s were higher in the diadromous than in the hololimnetic species. While basal gill NKCC mRNA transcription rates differed among species, expressions were unaffected by exposure time or salinity, suggesting post-transcriptional regulation of protein synthesis. Unexpectedly, hemolymph Cl-hyper-regulatory capability correlated with gill NKCC gene expression, while gill NKCC protein synthesis was associated with hyper-regulation of hemolymph osmolality at the 80%UL50’s of almost all Macrobrachium species, suggesting a role for the gill NKCC symporter in salt uptake. The evolutionary history of osmoregulation in these palaemonid shrimps suggests that, while some molecular and systemic mechanisms have accompanied cladogenetic events during radiation into different osmotic niches, others may be driven by salinity.

Strategies of invertebrate osmoregulation: an evolutionary blueprint for transmuting into fresh water from the sea

“…Several strategies seem to have arisen independently and may be useful indicators of adaptation or of evolutionary processes in progress. The elevated salinity tolerances of some euryhaline Malacostraca suggest a more recent transmutation into fresh water from their ancestral marine environment compared to stenohaline freshwater taxa, and thus these may still be adapting (Augusto et al, 2007(Augusto et al, , 2009Faria et al, 2011;Maraschi et al, 2021). The retention of unexpected salinity tolerance in very old hololimnetic dwellers like the Anomura and Brachyura is remarkable; these rely on isosmoticity rather than on hypoosmotic regulation when confronted by experimental salt challenge (Faria et al, 2011;Mantovani and McNamara, 2021) a characteristic of some marine species.…”

“…Many species of diadromous freshwater Crustacea hypo-regulate their hemolymph osmolality and chloride concentrations when confronted by salinities above isosmotic (Maraschi et al 2021). This capability enables migration between distinct halo-habitats and the exploitation of richer estuarine waters or the ancestral marine environment, suggesting an evolutionary tradeoff against the genetic and energetic costs of retaining a hyper-/hypo-regulatory apparatus.…”

Evolutionary trade-offs in osmotic and ionic regulation and the expression of gill ion transporter genes in high latitude, Neotropical cold clime crabs from the ‘end of the world’

“…The species' critical limits were calculated using Probit analyses that adjust percentage survival to a linear regression model (Finney, 1971;Thurman, 2002Thurman, , 2003Maraschi et al 2021). The LL 50 and UL 50 values were 4 and 63 ‰S for A. albatrossis, and 12 and 50 ‰S for H. planatus, respectively.…”

“…To accompany the time courses of osmotic and ionic regulation and gill gene expression during hypo-or hyper-osmotic challenge, salinities corresponding to 80% of the LL 50 (80% LL 50 ) and UL 50 (80% UL 50 ) values (i. e., 5 and 50 ‰S for A. albatrossis, and 15 and 40 ‰S for H. planatus) were used, respectively. Thus, each species was challenged with equivalent, severe and symmetrical, but non-lethal salinities, enabling direct comparison of their responses (Mantovani and McNamara, 2021;Maraschi et al, 2021). Groups of 7 crabs each were exposed directly for 0 [=30 ‰S, control], 6, 24 or 120 h at these salinities, as given above.…”

“…The branchiostegites were removed from the freshly killed crabs and the three posterior gill pairs were dissected and set aside in TRIzol reagent (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) for total RNA extraction. The molecular methods followed those set out in Mantovani and McNamara (2021) and Maraschi et al (2021). Briefly, total RNA was extracted from the pooled 3 posterior gill pairs (≈50 mg) of each crab at each salinity and time combination in TRIzol (1: 10 w/v) under RNAse-free conditions.…”