Waterborne amino acids: uptake and functional roles in aquatic animals Free

Derived through the breakdown of terrestrial organic material that is subsequently washed into an aquatic system, or generated in situ, dissolved organic matter (DOM) is a ubiquitous component of freshwater and marine environments (Steinberg, 2002; Ogawa and Tanoue, 2003). In addition to its ubiquity, DOM is also characterized by its heterogeneity. Indeed, the DOM profile of any given water body will be unique, shaped by the nature of the DOM sources and the physicochemical properties of the aquatic environment (Rosenstock and Simon, 2001; Ogawa and Tanoue, 2003). Ultimately, DOM composition will vary in molecular size distribution, chemical constitution and concentration. For example, DOM may be present as complex multi-chained carbon molecules that are freely dissolved or associated with dissolved inorganics, or as simple free amino acids (AAs) (Ogawa and Tanoue, 2003; Lu et al., 2014). In terms of the latter, total dissolved AA concentrations in the open ocean are in the nano- to low micromolar range (approximately 50 nmol l⁻¹ to 2.5 µmol l⁻¹; Jorgenson, 1982; Lu et al., 2014; Zhang et al., 2016), whereas interstitial water AA concentrations in coastal basins can range from 2 to 60 µmol l⁻¹ (Jorgensen et al., 1981; Burdige and Martens, 1990). Collectively, dissolved free AAs can represent up to 50% of fixed nitrogen in the marine environment (Gilbert and Bronk, 1994).

The diversity of DOM extends to its roles in aquatic ecosystems. It is an important contributor to the global carbon cycle (Letscher and Moore, 2015; see Glossary), provides protection against ultraviolet light (Scully, 1994) and modifies the mobility and toxicity of aquatic metal toxicants such as copper and cadmium (Playle et al., 1993). However, much less is known regarding the direct effects of DOM on aquatic biota. Notably, DOM has been shown to increase immunostimulation, disease recovery and offspring production in aquatic organisms (Steinberg et al., 2003; Lieke et al., 2021). Also, a significant body of work has demonstrated nutritional roles for DOM, particularly the AA fraction (see Glossary). In some aquatic settings, elevated concentrations of dissolved AAs create the opportunity for uptake of these nutrients directly from the water across the integument (i.e. the external surfaces of an animal including the skin and gills) (Wright, 1982). This possibility was first proposed by Pütter (1909) and since then, there have been numerous studies that have indicated, to varying degrees, the capacity of aquatic biota to remove DOM from the water column [see reviews by Krogh (1931) and Stephens (1968) for excellent overviews of the early research in this field]. In fact, the uptake of waterborne AAs has been exhibited in the following phyla: Porifera, Cnidaria, Platyhelminthes, Brachiopoda, Mollusca, Nemertea, Annelida, Echinodermata, Arthropoda and even Chordata (West et al., 1977; Stephens, 1968; Glover et al., 2011; Katayama et al., 2016; Blewett and Goss, 2017). Although waterborne AA absorption is prevalent among marine species, knowledge of these processes in freshwater is limited, possibly because of reduced opportunities for uptake (i.e. limited settings with elevated dissolved AAs) or owing to the physiological limitations (see Box 1) of freshwater animals (Glover et al., 2013). Once absorbed, many potential roles exist for dissolved AAs, including serving as a supplementary nutrient source, osmoregulation, cytoprotection, shell production and molting (Preston and Stevens, 1982; Glover et al., 2011, 2016, 2017; Blewett and Goss, 2017). The capacity of some animals to absorb dissolved AAs may provide a competitive advantage that contributes to the success of these species.

The aim of this Commentary is to outline the mechanisms by which waterborne AAs are absorbed by aquatic animals and to highlight the functional roles that absorbed AAs may perform, placing particular emphasis on marine invertebrates. We identify knowledge gaps and draw connections between known uptake pathways and functionalities (nutrient uptake, osmoregulation, molting and shell creation). We also examine the prospect of AA uptake in freshwater organisms, which has previously been largely understudied.

Epithelial AA transporters have been extensively studied in mammalian models. Owing to the lack of mechanistic characterization of transporter function in aquatic biota, the paradigms developed in mammals have generally been extrapolated to aquatic animals, as is discussed below (see ‘Amino acid utilization’). In mammals, AA transporters can be broadly classified into three groups: uniporters, antiporters and cotransporters (Fig. 1) (Broer, 2002).

Uniporters transport AAs across epithelia without the need for coupled Na⁺ transport (Gauthier-Coles et al., 2021). For example, members of the rBAT and mCAT family of uniporters are Na⁺-independent AA transporters with similar structural characteristics to the well-studied glucose transporter family (GLUTs; Kakuda and MacLeod, 1994). While CAT-1 and similar isoforms (CAT-2A/B, CAT-3 and CAT-4) offer a unilateral transport of the cationic AAs arginine, lysine and histidine, true uniporter pathways are not considered to be major contributors to epithelial (i.e. gut) AA uptake in mammals. This is because unilateral transport results in a change in the electrochemical gradient (Broer, 2002) and therefore may be disruptive to other epithelial transport processes. Thus, it is suggested that uniporters are a mechanism for increasing or decreasing membrane potential by facilitating the influx and efflux of cationic AAs (Broer, 2002).

Antiporters are involved in cellular AA exchange. In these scenarios, AAs that are abundant in the transporting integument or cells (e.g. glutamine or glutamate) are moved out of a cell down a concentration gradient, providing the energy to absorb a less plentiful essential AA such as leucine (Broer, 2002). In many cases, this involves the exchange of a non-essential AA for an essential AA, such as in the case of LAT1/2 and y⁺LAT1/2, which transport neutrally charged AAs out of the cell and import neutral and cationic AAs, respectively (Gauthier-Coles et al., 2021). Antiporters, therefore, effectively trade one AA for another with no change in total AA concentration (Broer, 2002).

Cotransporters move AAs against a concentration gradient by using the favorable electrochemical gradient that exists inside of cells for the absorption of Na⁺ (Preston and Stevens, 1982). This gradient is achieved through the actions of the basolateral Na⁺ pump, which actively extrudes three intracellular Na⁺ ions in exchange for two extracellular K⁺ ions. The stoichiometry of AA cotransporters varies, with anywhere from 1 to 3 Na⁺ ions accompanying the uptake of the AA. In some cases, other ions are also co-transported, either instead of or in concert with Na⁺. For example, PAT1 is a H⁺-dependent, Na⁺-independent AA cotransporter found in mammalian cells (Anderson et al., 2009).

As noted above, the uptake of AAs directly across the integument of aquatic animals has been interpreted largely in the context of AA transporters in mammalian systems, in part because such uptake requires any given epithelial transporter to work against a chemical gradient. For example, marine animals generally maintain AAs at concentrations 10³–10⁶ times that of their external environment (Clark, 1968; Stevens and Preston, 1980). In marine decapod crustaceans, for instance, intracellular free AA concentrations have been measured in the range of 184 to 530 mmol kg⁻¹ (see table 2 in McNamara et al., 2004) – orders of magnitude greater than the concentrations of AAs in ambient water. For comparison, the mammalian tissue AA pool has a concentration that is 5- to 20-fold higher inside the cell than in the interstitial fluids (Preston and Stevens, 1982). Thus, while the general principles of AA transport in mammals are likely to apply to AA transport in aquatic biota, fundamental differences may exist. Nevertheless, researchers have used knowledge of the structure and function of AA transporters in mammals, combined with experimental work on AA transport characteristics in aquatic animals, to infer the transporters responsible for integumentary AA uptake.

To date, most mechanistic evidence for integumentary AA uptake in aquatic animals supports a role for AA cotransporters. For example, Wright (1987) showed that the transport of the AAs alanine and taurine across the gill of Mytilus californianus was Na⁺ dependent and inhibited by harmaline, a known inhibitor of Na⁺ cotransport (Wright, 1987). On the basis of uptake characteristics in waters of distinct Na⁺ concentration, integumentary AA cotransporters are also believed to exist in aquatic annelids, mollusks, echinoderms and arthropods (Table 1) (Preston and Stevens, 1982; Blewett and Goss, 2017). As an example of the latter, isolated perfused gill studies have observed that waterborne AAs can be absorbed across the branchial epithelia (see Glossary) of the green shore crab Carcinus maenas (Blewett and Goss, 2017). This finding countered previous hypotheses that arthropods could not access dissolved AAs because of the impermeability of their exoskeletons. Notably, the previous hypothesis overlooked the continuous supply of environmental water to the branchial chamber surrounding the crustacean gill, and the function of the branchial epithelia as a possible transport surface. In C. maenas, a Na⁺-dependent cotransporter absorbs l-leucine with a branchial Michaelis-Menten affinity (K_m; AA concentration required to achieve half saturation of uptake in kinetic analysis; see Glossary) of ∼1.7 µmol l⁻¹, which is well within recorded environmental concentrations (Blewett and Goss, 2017).

Transport of AA across the branchial membrane has also been identified in marine bivalves and the hagfish (Stewart, 1978; Wright, 1985; Glover et al., 2011). For example, at least 4 separate transport pathways for multiple AAs have been described in marine bivalve gills (Stewart, 1978; Wright, 1985). Two of these pathways transport neutral AAs (glycine, alanine, leucine and serine), while one transports lysine and the other acts as a proline transporter (Wright, 1988). Hagfish present evidence of an allosteric pathway (see Glossary) for glycine and alanine with uptake characterized as having a relatively low affinity of between 11 and 17 µmol l⁻¹ (Glover et al., 2011), a value that is well above the open ocean environmental concentrations that can approach 1 to 3 µmol l⁻¹ (Wright, 1985). This relatively low affinity is probably due to their feeding and burrowing habits. Hagfishes burrow into decaying carcasses and AA-rich mud, which are likely to contain extremely high concentrations of free dissolved AAs (Burdige and Martens, 1990; Glover et al., 2011). In addition to branchial uptake, hagfish also possess the capability of taking up free AAs (alanine and glycine) through their epidermis using at least two possible distinct pathways, which are believed to be composed of System A (SLC38A; see Glossary) and System asc (SLC7A; see Glossary) transporters based on the transport characteristics (Fig. 2) (Glover et al., 2011).

A further example of a characterized AA transport pathway in a marine organism comes from cephalopods (Eguileor et al., 2000). Cuttlefish (Sepia officinalis) possess epithelial cells on their arms that have microvilli and high mitochondrial activity that are characteristic of the absorptive cells in the gut epithelium. Using in vitro isolated arms and brush-border membrane vesicle techniques, Eguileor et al. (2000) provided evidence for both Na⁺-dependent and Na⁺-independent AA uptake pathways (Fig. 2). For example, while the uptake of proline was reduced significantly in the absence of Na⁺, cephalopod arms exposed to 0.5 µmol l⁻¹ radiolabeled leucine continued transporting, even in the absence of environmental Na⁺ (Eguileor et al., 2000). Analysis of AA uptake in membrane vesicle preparations (see Glossary) confirmed the Na⁺ dependence of proline uptake, but also showed that leucine uptake was cation dependent, relying on both Na⁺ and other cations to drive the absorptive process (Eguileor et al., 2000).

Many marine species exhibit multiple AA transport pathways in their integument. However, the vast majority of AA transport pathways have yet to be identified at a molecular level in marine invertebrates. One exception to this is a study by Applebaum and colleagues (2013), who conducted a molecular assessment of AA transporters in Antarctic echinoderms. Through RNA extraction, RT-PCR and nucleotide sequencing, 11 specific AA cotransporters resembling the SLC6 AA transport family were identified in the epithelium and tube feet of sea stars and sea urchins. These 11 AA cotransporters were found to be expressed throughout all stages of development and in a variety of tissues, ranging from the embryo body wall, adult tube feet, and throughout the digestive tract (Applebaum et al., 2013). Notably, the described AA cotransporters were similar to the AA cotransporters described in mammalian systems. Having an assortment of transporters with overlapping specificities and affinities likely provides an organism with the capacity to transport AAs under a wide range of environmental conditions and physiological states (i.e. increased transporter efficiency throughout developmental stages to facilitate growth; Applebaum et al., 2013).

In general, however, little is known about the use and expression of AA transporters in marine invertebrates. The pathways that are defined are largely restricted to those associated with cotransporters (Applebaum et al., 2013), leaving the molecular identification of uniporters and antiporters largely absent in marine biota. However, it can be argued that their prevalence in mammalian cells, and the described conservation of cotransporter families within marine invertebrates, means that additional investigation should uncover roles for AA antiporters and uniporters in marine invertebrates.

Historically, waterborne AAs have been considered as a primary (i.e. supplying over 50% of the nutritional requirements for bodily function) or secondary nutrient source. However, as outlined below, more recent studies have shown various other roles for dissolved AAs including hypoxia tolerance, shell formation and osmoregulation.

The nutritional contributions of waterborne AA uptake in aquatic species have been studied in many systems. For example, echinoderms have been noted to absorb and utilize AAs at extremely high rates (Preston and Stevens, 1982). In the case of the sand dollar (Clypeaster subdepressus), when external AA concentrations reach over 35 µmol l⁻¹, 100% of their oxidative requirements (i.e. the energy required to maintain bodily functions) can be obtained through waterborne AA uptake (Preston and Stevens, 1982). Although AA concentrations of 35 µmol l⁻¹ are seldom recorded in the open ocean, many tidal mudflats and coastal substrate porewaters (see Glossary) may reach these levels (Jorgensen et al., 1981; Preston and Stevens, 1982; Burdige and Martens, 1990).

Although some examples show the use of free AAs as a primary source of nutrients, they are most widely considered to be a secondary nutrient source, as has been consistently demonstrated in marine bivalves (Manahan et al., 1983; Preston and Stevens, 1982). Species such as Mytilus californianus, Mya arenaria, Modiolus modiolus, Mytilus edulis and Rangia cuneata exhibit the uptake of up to 16 different AAs, which accounts for up to 37% of their oxidative requirements (Anderson and Bedford, 1973; Bamford and Campbell, 1976; Manahan et al., 1983; Preston and Stevens, 1982). In echinoderms, the brittle star (Microphiopholis gracillima) shows increased rates of AA uptake when deprived of other food sources, whereas the feather star (Cenometra bella) increases AA transport rates between two and 14 days after evisceration of digestive tissue, with incorporation of waterborne AA's shown in regenerated tissues (Smith et al., 1981; Clements et al., 1993).

These echinoderm examples exemplify the variable usage of absorbed AAs in times of stress and increased nutrient demand. Indeed, secondary nutrient transport pathways are most likely to contribute to energy resources in times when food availability is low (i.e. molting, spatial seclusion or high competition) (Jorgensen and Kristensen, 1980; Glover et al., 2016; Blewett and Goss, 2017). To investigate the relative importance of waterborne AA uptake, Glover et al. (2016) examined the uptake of l-lysine, l-alanine and l-phenylalanine in hagfish that were fasted or immersed in a blended fish slurry. The relative lack of change to the uptake rate in epidermal AA transport pathways indicated that waterborne AA uptake likely served as a constitutive nutrient uptake pathway and was not upregulated to take advantage of elevated environmental AA concentrations associated with their immersive feeding behavior (Glover et al., 2016). Thus, the uptake of dissolved AA likely serves as a primary source of AAs in periods of fasting, which can last upwards of 9 months in hagfish (Tamburri and Barry, 1999).

Another case where waterborne AAs may contribute specifically towards energy use is in developing animals. The ability for the early life-stages of some species to take up AAs at a higher rate than adults is well described (Meyer and Manahan, 2009). For example, developing sea urchins (Strongylocentrotus purpuratus) exhibit an 11-fold increase in an AA transporter gene (Sp-AT2) over a 4-day period, resulting in a 6-fold increase in waterborne alanine transport capacity (Meyer and Manahan, 2009). Notably, throughout development, larval sea urchins also exhibit a 32-fold increase in internal alanine content – with alanine constituting roughly 8% of total protein composition – outlining the importance of dissolved AAs as a nutrient source during development (Pace and Manahan, 2006). This increased rate of AA transport and nutrient acquisition could allow for increased growth, thereby enhancing the survival rates of larval invertebrates.

While much of the research on invertebrate AA uptake has focused on the use of free AAs as a nutrient source, there are several other benefits that could be just as useful to organisms. One such use of free AAs is in hypoxia, where they may facilitate enhanced tolerance of low oxygen levels. The uptake of dissolved AAs bypasses some of the costs associated with absorption of AAs from food (e.g. mechanical and chemical digestion) and thus reduces energy expenditure under a scenario where such resources must be conserved. For example, polychaetes live in AA-rich benthic sediment environments with ambient AA concentrations in the high micromolar range (Costopulos et al., 1979). However, these habitats are also known to have extremely low oxygen levels and are often anoxic. In a study examining how anoxia affected AA uptake (Costopulos et al., 1979), experimental evidence suggests that accumulation of waterborne AAs incurred a lower metabolic cost than nutrient uptake through digestion. Thus, the use of dissolved AAs reduces the overall usage of metabolic energy stores and facilitates survival in an otherwise metabolically challenging environment.

Arguably the most notable example of AA utilization in hypoxia tolerance is displayed in the hagfish. Bucking et al. (2011) showed that upon exposure to hypoxic conditions, hagfish exhibit increased utilization of specific AAs. Specifically, an increase in branchial glycine uptake was observed, resulting in enhanced glycine accumulation in the brain. In contrast, no changes in alanine transport and handling were observed (Bucking et al., 2011). These authors hypothesized that under hypoxic stress, the selective increase in glycine allows for metabolic depression via postsynaptic hyperpolarization (see Glossary) in the brain (Bucking et al., 2011). When bound, glycine-gated chloride channels within the cell membrane open, allowing the influx of Cl⁻ ions, increasing transmembrane voltage differences and reducing cell excitation and action potential firing (Gundersen et al., 2005). This decreases neural ATP utilization in cell-to-cell signaling and thereby facilitates the conservation of vital energy stores (Bucking et al., 2011). Notably, this is a similar strategy to that employed by some anoxia-tolerant vertebrates such as freshwater turtles (Nilsson and Lutz, 1991). The phenomenon of increased AA accumulation in hypoxia is not restricted to hagfish, as this has also been observed in the blue mussel (Mytilus edulis; Belivermis et al., 2020), suggesting that the uptake of dissolved AAs can play a specific role in hypoxia tolerance in aquatic biota.

While direct evidence for waterborne AA utilization in shell formation is lacking, it is worth considering that hard-shelled invertebrates utilize AAs to form the basis of their exoskeletons and shells. In mollusks, serine and glycine form a negatively charged organic matrix with glycoproteins, which acts as a mineralization starting point in the formation of a calcium carbonate shell (Weiner and Hood, 1975). Furthermore, the use of waterborne AAs could be extremely beneficial to molting species such as crustaceans as their shells contain 10 different AAs: methionine, arginine, threonine, tryptophan, histidine, isoleucine, lysine, leucine, valine and phenylalanine (Lage-Yusty et al., 2011). Crustaceans generally cease food consumption in their pre-molt stage (see Glossary), resuming normal consumption upon entering the intermolt stage (see Glossary) with a newly hardened shell (Wheatly, 1985). It is likely that during this period, when the shell is soft, the animal is especially vulnerable to predation and food-seeking activity is considered high risk. Therefore, utilizing an ambient nutrient source without having to move and feed has obvious advantages. Concentrations of free AAs in the whole body of crustaceans (Callinectes sapidus) change throughout molting stages, and of the 18 AAs measured, five (glutamic acid, methionine, aspartic acid, isoleucine and lysine) increased significantly following a molting event. This is counter to the expectation associated with fasting (Wheatly, 1985), thereby indicating potential uptake from the water. However, the impact of post-molt shell consumption has also not been considered, which might also contribute sources of AAs to the molting crab (Fig. 3). The idea of AA uptake during molts is particularly compelling because the barrier function of the exoskeleton (see Glossary) is diminished, making the uptake of AAs across the integument more likely (Glover et al., 2013). However, Williams et al. (2009) describe the onset of impermeability of crustacean shells within 1 h post molt, reducing the likelihood of a newly formed soft shell offering any impactful absorption surface. Further research is required to confirm these proposed theories.

Amino acids play a direct role in osmoregulation, acting as osmolytes within tissues (Abe, 2002; Shinji et al., 2012). The cellular uptake or loss of AAs such as alanine, glycine and taurine maintain cell volume by counteracting volumetric changes associated with the movement of ions and water resulting from changes in external salinity and thus ionic and osmotic gradients (Gilles, 1987; Kube et al., 2007; Burg and Ferraris, 2008). In essence, although inorganic ions are essential for biochemical processes, prolonged periods of high cellular inorganic ion concentrations can alter protein structure and function, disrupting further ion and nutrient transport processes (Burg and Ferraris, 2008). Where high levels of inorganic ions within cells can alter cellular protein function, free intracellular AAs stabilize protein function through maintaining cell osmolality (Burg and Ferraris, 2008). This role for dissolved AAs has been best demonstrated in mollusks. For example, in response to both hypo- and hyperosmotic conditions, the Asian hard clam (Meretrix lusoria) increases cellular expression of the taurine transporter (TAUT) in the gill and mantle, both of which are tissues with direct exposure to the external environment (Lin et al., 2016). Likewise, after 3 weeks of cyclic salinity exposure, the ribbed mussel (Geukensia demissa) exhibits a significant increase in the uptake of leucine and phenylalanine during periods of immersion at 60% saltwater (Neufeld and Wright, 1998). In marine mollusks, increased AA transporter abundance, increased rates of AA uptake and increased cellular AA levels signify the direct use of dissolved AAs in osmoregulatory strategies. In contrast to the findings above that link uptake of waterborne AAs to osmoregulation, other studies have failed to establish a relationship between these two processes. As a by-product of osmoregulation, nonessential AAs are also suggested to aid in mollusk freezing tolerance, reducing lethal temperatures by 2–3°C (Aarset, 1982). In the osmo-conforming hagfish, the effects of short-term changes in salinity on epidermal AA uptake, and the accumulation of waterborne amino acids in plasma, red blood cells and muscle tissue, were examined using radiolabeled AAs to distinguish environmentally sourced AAs from the existing internal AA pool (Glover et al., 2017). Although changes in AA accumulation rates in the plasma were noted – indicative of altered AA handling as a function of salinity – the nature of these changes was inconsistent with a role for waterborne AAs in osmoregulation. Similarly, no evidence was found for changes in AA accumulation rates in other tissues, which would have been expected if AAs played a role in cellular volume regulation. Likewise, in vitro skin uptake assays failed to provide support for the hypothesis of variable demand for AAs as osmolytes in altered salinity (Glover et al., 2017).

Therefore, although strong evidence exists for an osmoregulatory role of dissolved AAs in bivalve mollusks, there is little compelling evidence for such a role in other phyla. However, it must be noted that there has been limited investigation of this hypothesis outside of a few species.

Knowledge of the mechanisms for the transport of dissolved free AAs in marine environments can be vital to understanding the success of many invertebrate species, especially under conditions where food items may be scarce or subject to heavy competition. For example, the European green crab (Carcinus maenas) is considered one of the world's most successful marine invaders, and consequently, has become the most widely distributed crab in the intertidal region globally (Leignel et al., 2014). In combination with its extreme salinity tolerance (4–54 ppt; Leignel et al., 2014), the European green crab is also currently one of only four arthropod species known to take up waterborne AAs (Blewett and Goss, 2017; Griffin et al., 2023). This raises a question as to whether the ability to take up AAs offers some advantage in the intertidal zone. This advantage could be in terms of an additional source of nutrients for energy metabolism, or through aiding osmoregulation and/or hypoxic tolerance. Thus, the uptake, utilization and role of waterborne amino acids in arthropod species offers a field rich in further research potential, possibility outlining adaptive mechanisms that are currently unknown in marine invertebrates.

Many studies have shown assimilation of dissolved AAs from the environment; however, the transport pathways that achieve uptake remain largely understudied, and there is little information regarding their interactions within a constantly changing environment. These knowledge gaps are, in part, due to the relative lack of molecular characterization. Future research should focus on identifying transport pathways through molecular analyses to complement and facilitate the interpretation of transport studies. Identifying the structure, function, energetic requirements and transport capacity of specific AA transporters will also allow further interpretation of their uses in osmoregulation, hypoxia tolerance and the nutritional benefits, in a wide array of marine animals.

Recent studies have identified species capable of absorbing and utilizing dissolved AAs that were previously considered incapable of directly accessing waterborne nutrients (Blewett and Goss, 2017). It is safe to assume that many other species have a similar ability but remain unstudied. The uptake of free AAs offers both a main nutrient source and a supplemental nutrient source in marine biota and could prove to be extremely beneficial in times when food availability is otherwise low. The uses for absorbed AAs in osmoregulation and hypoxia tolerance have not yet been examined in the vast majority of marine animals. Although many species have been identified that are capable of AA uptake, many pathways, uses and adaptational benefits of this process remain unknown. In the future, delineation of the transport pathways and mechanisms driving AA transport should be considered vital because they offer additional connections between the physiological capabilities of marine animals and the environments they inhabit. It is also important to recognize the extreme complexity and diversity in transport capability, not only among individual AAs within a species, but also among closely related species. Outlining the vast diversity and comparability of different transport pathways may also unlock unknown connections between animals and their ability to survive in an ever-changing ecosystem.