Evolutionary shifts in body fluid regulation
The saline to freshwater transition represents a formidable barrier that most invertebrate species have been unable to penetrate (Hutchinson, 1957). Yet, the copepod E. affinis has been able to breach this boundary multiple times independently, within decades in the wild (Lee, 1999; Lee et al., 2003, 2011) and a few generations in laboratory selection experiments (Lee et al., 2011). Such invasions impose serious challenges for ionic and osmotic regulation, in terms of acquiring ions from dilute solutions against steep concentration gradients. Previous studies have found evolutionary increases in freshwater tolerance and in ion uptake activity following freshwater invasions by E. affinis (Lee et al., 2003, 2007, 2011). In this study, contrasts between ancestral saline and derived freshwater populations suggest that increases in body fluid regulation could evolve very rapidly following contemporary habitat shifts (Fig. 4; Table 1).
This study revealed the evolution of increased body fluid regulation at low salinities, in terms of increased hemolymph osmolality in freshwater populations of E. affinis relative to their saline progenitors (Fig. 4; Table 1). This evolutionary increase conformed to Hypothesis A, of greater hyperregulation associated with freshwater adaptation (Fig. 2). This pattern was consistent with the increased activity of ion uptake enzyme V-type H+ ATPase observed in freshwater populations under freshwater conditions (Fig. 1) (Lee et al., 2011). Moreover, the parallel evolutionary shifts in hemolymph osmolality that we observed in the two independently derived freshwater populations (Fig. 4a and b) were concordant with the parallel evolutionary increases in V-type H+ ATPase activity and expression found in those same freshwater populations (Lee et al., 2011).
The key question is why the freshwater populations would undergo an evolutionary increase in ionic regulation (in fresh water) relative to the saline populations. In freshwater environments, the need for ion absorption increases due to elevated diffusional loss of ions from animals in fresh water. Additionally, ion uptake becomes more energetically costly as the fluid becomes more dilute. Moreover, the evolutionary increase in hemolymph osmolality in the freshwater-adapted populations (Fig. 4) would require an even greater energetic cost associated with the increase in ionic regulation in fresh water. Elevated hemolymph osmolality might be critical for survival under freshwater conditions, given that saline populations of E. affinis cannot be sustained in fresh water across generations without imposing strong selection. Maintaining elevated hemolymph osmolality in fresh water might be important for preserving enzymatic function and easing the burden of cell volume regulation.
The transition from saline to freshwater habitats requires a switch in physiological strategy for maintaining hemolymph concentration, from one of relative conformation (of osmotic pressure and ionic concentration) in saline environments to that of increased osmo- and iono-regulation in freshwater habitats. Hemolymph is the extracellular body fluid that forms the buffer between the variable external environment and the cell. The cell maintains a relatively constant ionic composition across taxa and environments, whereas osmotic concentration (osmolality) of the cell varies to a greater degree and is kept close to that of the hemolymph in order to maintain near-constant cell volume (Péqueux, 1995; Willmer et al., 2004; Charmantier et al., 2009). On the other hand, both the ionic and osmotic concentrations of hemolymph may vary greatly according to the environment, especially for osmoconformers (Willmer et al., 2004). Most taxa in the sea are osmoconformers (e.g. most marine invertebrates) and tend to have hemolymph osmolalities that match that of the environment (i.e. they are isosmotic). In contrast, osmoregulators (e.g. most vertebrates) tend to show much more constant hemolymph or blood osmolalities across a broad range of salinities, maintaining gradients with the environment (Willmer et al., 2004). Invertebrate species that live in fluctuating salinities tend to be hyper–hypo-osmoregulators (e.g. many estuarine crustaceans); that is, they are isosmotic across intermediate salinities (ca. 15–30 PSU), but then hyperregulate at lower salinities (hemolymph osmolality > ambient water) and hyporegulate at higher salinities (hemolymph osmolality < ambient water). In general, freshwater species face serious challenges in very dilute environments of maintaining hyperosmotic body fluid concentrations against steep concentration gradients with the environment. Such gradients are maintained by mechanisms such as increased ion uptake, reduced ion efflux, or excretion of dilute urine (Péqueux, 1995; Patrick et al., 2001; Willmer et al., 2004; Augusto et al., 2007; Charmantier et al., 2009; Lee et al., 2011).
Based on our results and those of Roddie et al. (1984), saline populations of E. affinis displayed patterns of hemolymph osmolality typical of hyper–hypo-osmoregulating estuarine species (Fig. 4; Table 1) (Roddie et al., 1984). The saline (brackish) populations in this study were close to isosmotic in the 15–25 PSU salinity range and became increasingly hyperosmotic below 15 PSU, with a minimum hemolymph osmolality of about 200 mOsm kg−1 (Fig. 4; Table 1), similar to results of Roddie et al. (1984). While we did not measure hemolymph osmolalities above 25 PSU, we would expect regulation to be hyposmotic above this salinity, as found by Roddie et al. (1984).
Few studies have examined evolutionary shifts in osmoregulation across a habitat cline. One study did find evolutionary differences in hemolymph osmolality between anadromous and freshwater landlocked populations of the South American shrimp Macrobrachium amazonicum (Charmantier & Anger, 2011). While the two populations showed similar hemolymph osmolalities at low salinities across most life history stages, the freshwater population showed the loss of ability to hyporegulate at higher salinities (Charmantier & Anger, 2011). Most previous comparative studies did not uncover evolutionary differences in hemolymph regulation, as they tended to assay animals directly from the field or acclimate adults to common treatment salinities for a few days to a few weeks prior to measurement assays (Bayly, 1969; Brand & Bayly, 1971; Schubart & Diesel, 1999; Freire et al., 2003; Augusto et al., 2007, 2009; Thurman et al., 2010). As such, these studies would not have removed effects of developmental acclimation to native salinities. Rearing conditions during development could have profound effects on adult physiology (Huey & Berrigan, 1996; Huey et al., 1999; Lee & Petersen, 2003). In addition, measurements of hemolymph of animals collected from the wild, or acclimated for short periods, would likely be confounded by pre-existing ion stores, which might dissipate very slowly over time (Beadle & Shaw, 1950; Lockwood, 1959; Scheide & Dietz, 1982; Wilcox & Dietz, 1995; Lin et al., 2002).
While this study focused on osmoregulation in adults, it would be worth investigating the evolutionary shifts in body fluid regulation of the larval (naupliar) stages (although this would be difficult due to small size). Previous results revealed that the naupliar stages of E. affinis are much more susceptible to mortality under low ionic conditions than adults (Lee et al., 2003, 2007, 2011). In addition, hemolymph osmolality has been found to vary among life history stages in some crustaceans (Guerin & Stickle, 1997; Charmantier, 1998; Charmantier & Anger, 2011). Given the small size of copepod larval stages (∼300 μm diameter), and larger surface area relative to volume, osmotic and ionic regulation of hemolymph might be much more difficult and energetically costly to maintain in fresh water at the early life history stages.
The evolution of increased body fluid regulation would entail increases in energetic costs in freshwater habitats. A higher ionic or osmotic gradient with the environment at lower salinities would require greater rates of ion uptake and/or reduced ion efflux (e.g. lower integument permeability). Higher energetic costs of osmoregulation are apparent from the increase in oxygen consumption of copepods at salinities above and below the isosmotic range, where they are hyper- or hypo-osmoregulating (Lance, 1965; Gyllenberg & Lundquist, 1978, 1979). Adequate food consumption might be critical to fuel the energetic costs associated with increased ionic and osmotic regulation. The increased energetic requirements might explain why invaders from brackishwater environments tend to invade freshwater habitats with elevated food availability or harbouring specific types of algae (van den Brink et al., 1993; Vanderploeg et al., 1996; Lauringson et al., 2007).
Increases in body fluid regulation, and associated increases in energetic costs, are problems that face organisms as they become farther removed from the sea (Withers, 1992; Willmer et al., 2004). This study is the first to reveal evolutionary shifts in body fluid regulation across a habitat cline, following rapid transitions from saline into freshwater habitats. Overall, our results are consistent with the evolution of increased physiological regulation accompanying transitions into stressful habitats.