Transcriptional, translational and systemic alterations during the time course of osmoregulatory acclimation in two palaemonid shrimps from distinct osmotic niches

Abstract

Palaemonid shrimps exhibit numerous adaptive strategies, both in their life cycles and in biochemical, physiological, morphological and behavioral characteristics that reflect the wide variety of habitats in which they occur, including species that are of particular interest when analyzing adaptive osmoregulatory strategies. The present investigation evaluates the short- (hours) and long-term (days) time courses of responses of two palaemonid shrimps from separate yet overlapping osmotic niches, Palaemon northropi (marine) and Macrobrachium acanthurus (diadromous, fresh water), to differential salinity challenges at distinct levels of structural organization: (i) transcriptional, analyzing quantitative expression of gill mRNAs that encode for subunits of the Na⁺/K⁺-ATPase and V(H⁺)-ATPase ion transporters; (ii) translational, examining the kinetic behavior of gill Na⁺/K⁺-ATPase specific activity; and (iii) systemic, accompanying consequent adjustment of hemolymph osmolality. Palaemon northropi is an excellent hyper-hypo-osmoregulator in dilute and concentrated seawater, respectively. Macrobrachium acanthurus is a strong hyper-regulator in fresh water and hypo-regulates hemolymph osmolality and particularly [Cl⁻] in brackish water. Hemolymph hyper-regulation in fresh water (Macrobrachium acanthurus) and dilute seawater (Palaemon northropi) is underlain by augmented expression of both the gill Na⁺/K⁺-ATPase and V(H⁺)-ATPase. In contrast, in neither species is hypo-regulation sustained by changes in Na⁺/K⁺-ATPase mRNA expression levels, but rather by regulating enzyme specific activity. The integrated time course of Na⁺/K⁺- and V(H⁺)-ATPase expression and Na⁺/K⁺-ATPase activity in the gills of these palaemonid shrimps during acclimation to different salinities reveals versatility in their levels of regulation, and in the roles of these ion transporting pumps in sustaining processes of hyper- and hypo-osmotic and chloride regulation.

Introduction

The evolutionary history of the Crustacea reveals their ample adaptive radiation, an exploit that has enabled the occupation of many different osmotic niches, brought about by differential capabilities for anisosmotic extracellular and isosmotic intracellular regulation (Mantel and Farmer, 1983, Péqueux, 1995, Freire et al., 2008, Charmantier et al., 2009, Henry et al., 2012, McNamara et al., 2015). The successful colonization of dilute or concentrated media derives principally from the emergence of efficient mechanisms of ion uptake and/or secretion (Péqueux, 1995, Kirschner, 2004, Freire et al., 2008, Faleiros et al., 2010, Henry et al., 2012, McNamara and Faria, 2012), and from a reduction in epithelial permeability to passive ion and water movements (Smith, 1970, Péqueux, 1995, Rasmussen and Andersen, 1996, Rainbow and Black, 2001, Freire et al., 2008).

The morphological and functional responses of crustaceans challenged by markedly different saline environments involve adaptations manifest mainly in the epithelial cell layers that constitute the gills and branchial chamber (Bouricha et al., 1994, Freire and McNamara, 1995, McNamara and Lima, 1997, Lignot and Charmantier, 2001, Luquet et al., 2002, Luquet et al., 2005, Henry et al., 2012), integument (Ahearn et al., 1999), intestine (McNamara et al., 2005) and antennal glands (McNamara and Torres, 1999, McNamara et al., 2015). These epithelia regulate the transit of ions and molecules across the interfaces between the external medium and the extracellular fluid or hemolymph. Such adaptations are also manifest at different levels of biological organization (Faleiros et al., 2010) from the molecular (Towle et al., 1997, Towle et al., 2001, Weihrauch et al., 2001, Weihrauch et al., 2004, Luquet et al., 2005, Tsai and Lin, 2007, Jillette et al., 2011, Leone et al., 2015), cellular (Riestenpatt et al., 1996, Wilder et al., 2000, Belli et al., 2009) and tissue levels (Freire et al., 1995, Luquet et al., 1997, Ahearn et al., 1999) to the systemic and organismal levels (McNamara et al., 1983, Wilder et al., 1998, Freire et al., 2003, Augusto et al., 2009), many of which can be modulated by neurosecretory activity (Kamemoto, 1991, McNamara et al., 1991, Freire and McNamara, 1992, Santos and McNamara, 1996, Lucu and Towle, 2003).

Ambient salinity is an abiotic challenge intensively investigated in decapods, often rooted in their migratory behavior among different osmotic niches. In many palaemonid and penaeid shrimps, for example, such migrations are essential prerequisites for spawning, larval development and successful completion of the life cycle (McNamara et al., 1983, Jalihal et al., 1993, Pereira and Garcia, 1995, Alekhnovich and Kulesh, 2001). Osmoregulatory ability in the palaemonids in particular reveals distinct patterns ranging from excellent hyper-/hypo-osmotic and ionic regulation in intertidal species like Palaemon affinis (Kirkpatrick and Jones, 1985), P. pandaliformis (Freire et al., 2003) and P. northropi (Freire et al., 2003, Augusto et al., 2009; this study), and in some migratory diadromous species such as Macrobrachium olfersii (Freire et al., 2003), M. acanthurus (Signoret and Brailovsky, 2004; this study), and hololimnetic, continental species like M. potiuna (Freire et al., 2003). Other diadromous/brackish-water species exhibit modest osmoregulatory ability, for example M. equidens (Denne, 1968), M. rosenbergii (Sandifer et al., 1975), M. carcinus (Moreira et al., 1988, Signoret and Brailovsky, 2004), M. amazonicum (Augusto et al., 2007a, Faleiros et al., 2010), and the hololimnetic M. brasiliense (Faria et al., 2011). Such patterns of osmoregulatory ability likely underlie the distribution of the different species into distinct environments, reflecting their abilities to occupy diverse osmotic niches (McNamara et al., 2015).

Multidisciplinary approaches to osmoregulation in the Crustacea are incipient although innovative molecular biological methodologies have been employed to evaluate physiological mechanisms residing in the epithelial cell layers that form an active interface between the extracellular fluid and the aquatic environment. Such studies have attempted to elucidate the molecular basis of salt uptake and salt secretion in shrimps and crabs, identifying and characterizing the expression of candidate genes encoding for important ion transporters in gill cell membranes such as the Na⁺/H⁺ exchanger (Towle et al., 1997, Boudour-Boucheker et al., 2016) and Na⁺/K⁺/2Cl⁻ symporter (Luquet et al., 2005, Havird et al., 2013), and the Na⁺/K⁺-ATPase (Towle et al., 2001, Faleiros et al., 2010, Jillette et al., 2011, Havird et al., 2013, Leone et al., 2015, Maraschi et al., 2015, Pinto et al., 2016, Moshtaghi et al., 2016) and V(H⁺)-ATPase (Weihrauch et al., 2001, Weihrauch et al., 2004, Faleiros et al., 2010, Maraschi et al., 2015), using decapods acclimated to dilute or concentrated seawater as models.

The gill Na⁺/K⁺-ATPase has been considered the primary driving force for osmoregulation, playing an essential function mainly in salt uptake (Péqueux, 1995, Towle et al., 2001, Lucu and Towle, 2003). However, recent studies have demonstrated a role in both hyper- and hypo-osmotic regulation (Luquet et al., 2002). Na⁺/K⁺-ATPase activity in certain euryhaline crabs unable to survive in fresh water is significantly higher when in seawater than in dilute medium (Tsai and Lin, 2007), suggesting a role in hypo-osmoregulation. In contrast, in crabs that inhabit extremely dilute osmotic niches, Na⁺/K⁺-ATPase activity is unchanged on acclimation to fresh water or seawater, suggesting a complementary function in osmoregulation for other transporters such as the V(H⁺)-ATPase (Tsai and Lin, 2007).

In freshwater crabs and shrimps, in addition to the Na⁺/K⁺-ATPase, the V(H⁺)-ATPase plays a major role in osmotic regulation (Weihrauch et al., 2004, Faleiros et al., 2010, Firmino et al., 2011). Tsai and Lin (2007) have demonstrated apical and cytoplasmatic distributions of the V(H⁺)-ATPase in the gill ionocytes of crabs from different osmotic niches, proposing that the apical distribution reflects a mechanism typical of freshwater crustaceans (Faleiros et al., 2010), participating primarily in an osmoregulatory function. V(H⁺)-ATPases are also involved in acid-base equilibrium (Fehsenfeld and Weihrauch, 2017) and in ammonia‑nitrogen excretion (Weihrauch et al., 2017).

There is substantial evidence from immunocytochemical studies that the Na⁺/K⁺- and V(H⁺)-ATPases are present in the gill epithelia of palaemonid shrimps. The Na⁺/K⁺-ATPase has been demonstrated ultracytochemically in the gill septal cells of Macrobrachium olfersii (McNamara and Torres, 1999, and by immunofluorescence in M. amazonicum (Boudour-Boucheker et al., 2014) and M. acanthurus (Maraschi et al., 2015). The V(H⁺)-ATPase has been demonstrated by immunofluorescence in the gill pillar cells of M. amazonicum (Boudour-Boucheker et al., 2014) and M. acanthurus (Maraschi et al., 2015).

Based on a diversity of physiological and biochemical findings obtained in a variety of crustaceans from fresh water and dilute media, McNamara and Faria (2012, Figure 4) have proposed a model for salt uptake and secretion in palaemonid shrimps that integrates findings for some of the ion transporters investigated to present. During hyper-osmoregulation in fresh water, salt uptake is thought to be powered by two main driving forces, the V(H⁺)-ATPase, located in the apical membranes of the pillar cell flanges (Boudour-Boucheker et al., 2014, Maraschi et al., 2015), and the Na⁺/K⁺-ATPase, found in the deep narrow invaginations of the septal cell membranes to which the pillar cells are coupled (McNamara and Torres, 1999). Na⁺ enters the cytosol of the pillar cell flanges through an apical Na⁺ channel against its concentration gradient, driven by the outside positive electrical potential created by H⁺ extrusion via the apical V(H⁺)-ATPase. Na⁺ crosses the pillar cell cytosol to the septal cells through basal Na⁺ channels or via a Na⁺/K⁺/2Cl⁻ symporter, leading to active Na⁺ transport to the hemolymph through the Na⁺/K⁺-ATPase. Chloride enters the pillar flange cytosol through an apical Cl⁻/HCO₃^– antiporter and may exit either directly to the hemolymph by Cl⁻ channels in the lower flange membranes, or via basal pillar cell Cl⁻ channels or through the Na⁺/K⁺/2Cl⁻ symporter to the septal cell invaginations and hemolymph, accompanying the Na⁺ gradient. The supply of H⁺ and HCO₃^– as counter ions derives from the hydration of metabolic CO₂ by cytosolic carbonic anhydrase (Henry et al., 2012).

During salt secretion in hyper-osmotic media, Cl⁻ is thought to be transported from the hemolymph to the septal cells by the Na⁺/K⁺/2Cl⁻ symporter, flowing to the perikarya of the pillar cells through basal Cl⁻ channels, and subsequently across the apical pillar flanges via apical Cl⁻ channels. Na⁺ recycles to the hemolymph through the Na⁺/K⁺-ATPase in the septal cell membrane invaginations, and K⁺ is recycled via K⁺ channels, producing an inside negative cytosolic potential that drives Cl⁻ efflux to the pillar cells, reaching their apical flanges and apical Cl⁻ channels. Such Cl⁻ efflux would create a negative electric potential in the subapical space below the cuticle that drives paracellular Na⁺ efflux between adjacent pillar cell flanges, leading to the secretion of a NaCl rich fluid (McNamara and Faria, 2012).

The monophyletic Palaemonidae (Pereira, 1997) constitute an appealing group in which to investigate response patterns to salinity variation. To evaluate whether osmoregulatory patterns in different palaemonid genera are effected by similar biochemical and molecular mechanisms, we chose two species of palaemonid shrimps representative of the genera Palemon and Macrobrachium that exhibit life cycles intimately linked to variation in environmental salinity. Palaemon northropi is a marine species that inhabits rocky tide pools (Freire et al., 2003), frequently confronted by reduced and elevated salinities, ranging from ≈ 3 to ≈ 33‰S or more, as a function of tides, precipitation and evaporation (Augusto et al., 2009). Macrobrachium acanthurus is a diadromous, freshwater shrimp encountered in freshwater streams that depends on brackish/coastal waters for its larval development (McNamara et al., 1983). Both species exhibit extended larval developmental sequences during which the zoeae are found in coastal waters.

Given the effect of salinity acclimation on the activity and expression of the Na⁺/K⁺- and V(H⁺)-ATPases (Weihrauch et al., 2004, Luquet et al., 2005, Faleiros et al., 2010, Firmino et al., 2011, Leone et al., 2015), their location in the gill epithelia, and their known roles in osmoregulatory mechanisms, we evaluate the short term (hours) and long-term (days) time courses of the hyper- and hypo-osmoregulatory acclimation responses in each species. We also examine at what level of regulation, i.e., transcriptional or post-translational, these ion transporters are affected during osmotic challenge corresponding to salt uptake and secretion. Thus, we investigate the responses of each species to salinity challenge at different levels of structural organization, i.e., (i) transcriptional, analyzing the quantitative expression of gill mRNAs that encode for subunits of the ion transporters Na⁺/K⁺-ATPase and V(H⁺)-ATPase; (ii) translational, examining the kinetic behavior of the gill Na⁺/K⁺-ATPase; and (iii) systemic, accompanying consequent adjustment of hemolymph osmotic and ionic concentrations.

Analysis of such responses in these two palaemonid species from very distinct yet overlapping osmotic niches should disclose the influence of osmotic niche on molecular, biochemical and physiological adjustments, and aid in comprehending the ion transport mechanisms that have enabled the penetration of the caridean shrimps into media of variable salinity and into fresh water.