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Advances in the study of autoimmunity, together with the emerging field of ecoimmunology, have shown the way forward in considering the immune system as part of an integrative approach to understanding how vertebrates respond to environmental stress (Denver et al. 2009; Martin et al. 2014; Avrameas 2016). This holistic viewpoint should be particularly important when studying individual animals living on the fringes of their geographic range and subjected to relatively harsh external stimuli. Data and circumstantial evidence complied from experiments and fieldwork in physiological ecology involving the common snapping turtle, Chelydra serpentina, suggest an understanding of this typically freshwater animal's response to high salinity habitats should include incidental immune system effects.

Salinity Relations of the Common Snapping Turtle. — Laboratory experiments conducted from 1987 through 1990 concerning the effect of water salinity on the growth of hatchling snapping turtles indicate that, despite the existence of a saltmarsh ecotype (Ernst and Lovich 2009), these animals are unable to hypoosmoregulate in salinities more concentrated than their internal osmotic concentration, about one-third that of seawater (100% seawater = 35 parts per thousand [ppt] = 1000 milliosmoles [mOsm]). Freshwater turtles typically have a blood osmotic concentration equal to or slightly less than that of human blood, about 290 mOsm (Dessauer 1970). The data (Table 1) indicate a marked reduction in the number of animals able to grow at salinities above 12 ppt (approximately 340 mOsm) compared with lower salinities. Simply put, they will progressively dehydrate when subjected to hyperosmotic salinities (Dunson 1984, 1986). Field data from the same time period reinforces this conclusion. The highest blood plasma osmotic concentration recorded for 14, apparently healthy, adult and subadult snapping turtles captured in a brackish tidal creek was 340 mOsm (mean = 307.6 ± 20.0 mOsm SD). The creek could be characterized as having a steep horizontal salinity gradient (0–25 ppt over 750 m). Six individuals captured at a nearby freshwater site had a significantly lower plasma osmotic concentration of 278.3 ± 20.2 mOsm SD. Additional field data, including trap capture rates and enclosure experiments, offer additional support that the turtles are unable to osmoregulate when forced to remain in relatively saline water found at capture sites (Kinneary 1993, 1996). These animals are apparently able to exploit high salinity habitats through a suite of behavioral adaptations including feeding, drinking, and movement patterns that limit the uptake and exposure to osmotically stressful water (Dunson 1986; Dunson and Mazzotti 1989; Kinneary 1992, 1993, 2008).

Table 1. Growth of hatchling snapping turtles in various salinities at 28°C. The turtles were fed 3 times per week. Photoperiod equals 12L:12D. One hundred percent seawater (SW) = 35 ppt; FW = freshwater; ppt = parts per thousand.^a

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The concern in this report is with the relatively few experimental animals (12 of 99; Table 1) that were not only able to maintain mass but continued to consume food and grow at a salinities (13 and 14 ppt) that were clearly stressful for all of their experimental cohorts. In a recent experiment, a group (n = 12) of hatchling snapping turtles were kept and fed in ostensibly hyperosmotic water (14 ± 0.6 ppt SD at 26°C ± 1.9°C SD). The experiment was discontinued after 2 wks (except for 2 individuals) when the animals as a group stopped eating and began to lose a substantial amount of body mass (–10.8% initial wet mass ± 11.2 SD) (Table 2). Again, as in previous high salinity experiments, 2 physiologically significant events occurred. A small number of individuals continued to eat and grow despite the osmoregulatory stress (one individual gained 5.5% and the other 20.2% of their initial wet mass through 4 wks). Second, as in past high salinity experiments, several of the turtles developed white, plaque-like cutaneous lesions overlying inflamed skin patches (quantitative data concerning the skin condition were not collected). This condition had also been noted, during previous fieldwork, in 6 of 11 clearly stressed larger individuals kept in field enclosures at high salinity capture sites or in an outdoor aquarium, but not in 8 turtles kept as freshwater controls (Kinneary 1993, 1996). The most parsimonious explanation is that the growth outliers are in fact isoosmotic with the higher salinities of their environment. The skin issue may be related with both factors involving the immune system.

Table 2. Raw data for growth of hatchling snapping turtles offered commercial pet food 5 times per week in 14 ppt salinity (400 mOsm). Photoperiod equals 12L:12D.

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The Immune System of Vertebrates. — While much of the immunological literature focuses on the mammalian system, there is little doubt the many constitutive elements of the mammalian innate and adaptive immune response have been refitted from phylogenetic predecessors, not only cutting across the various vertebrate lineages but having foundation in invertebrate defense systems (Schluter et al. 2005; Lee 2006; Litman et al. 2010; Zimmerman et al. 2010; Ghorai and Priyam 2018; Müller et al. 2018). The total amount of cells and biomolecules associated with this vastly complex network is both staggering and variable, being dependent to an extent on both biotic and abiotic stimuli. Advances in the study of immunity have made clear that, incidental to its primary function, the immune system has complex homeostatic functions that must be considered if we are to have a more complete understanding of vertebrate ecology and evolution (Graber 1983; Bona 1988; Marchalonis and Schluter 1994; Lee 2006; Graham et al. 2010; Mahendra et al. 2013; Avrameas 2016; Avrameas et al. 2018). In turtles it has been demonstrated that, in vitro, basophils from Chelydra serpentina will release histamine when triggered by an antigen, and in the sea turtle Caretta caretta, lymphocyte proliferation was positively correlated with an environmental contaminant, indicating modulation of the immune system by environmental parameters (Zimmerman et al. 2010).

Autoimmunity. — Although the vertebrate classes possess different kinds of immunoglobulins, they are phylogenetically related and appear to have arisen from a more ancient system of morphoregulatory molecules, probably a cell adhesion system ancestral to both the immune and nervous systems (Edelman 1987, 1989). An evolutionary viewpoint helps frame an explanation for the bidirectional interactions between the immune, nervous, and endocrine systems (Clatworthy et al. 1994; Denver et al. 2009; Adamo 2014; Avrameas 2016).

Included in this ubiquitous vertebrate immune response are natural antibodies or autoantibodies (NAbs). It is well established that the sera of healthy individuals from different animal species contain an enormous number of NAbs that are polyreactive and recognize almost all self and environmental antigens (Avrameas 2016). NAbs appear to function at the intersection of the innate and adaptive immune system response (Zimmerman et al. 2010; Sandmeier and Tracy 2014; Zimmerman 2018). In addition to fish, birds, and mammals (Schluter et al. 2005; Lee 2006; Zimmerman et al. 2010), NAbs have been identified in reptiles, including snakes, turtles, and a crocodilian (Zimmerman et al. 2010; Sandmeier and Tracy 2014; Stromsland and Zimmerman 2017; Ghorai and Priyam 2018).

It is now widely accepted that NAbs can exhibit different biological functions due to their actions on biomolecules, receptors, and other cellular structures. In turtles, there is evidence polyreactive NAbs are secreted without antigen stimulation (Stromsland and Zimmerman 2017; Zimmerman 2018). Like the nervous system, the immune system plays an integrative role with continuous communication channels between the organism's exterior and interior. The system adjusts its homeostatic activity accordingly by taking account of stimuli that are constantly received and changed (Avrameas et al. 2007; Avrameas 2016). That auto-polyreactivity or autoimmunity has a well-defined physiological role and has been preserved during the evolutionary process can no longer be ignored (Avrameas et al. 2018). And, although the term autoimmunity is generally used in a maladaptive or immunopathological context (Rose and Mackay 2014), there is mounting evidence this property can have overall adaptive consequences depending on the environmental milieu. In mammals, NAbs have been found to mimic the function of hormones (Bottazzo et al. 1986; Núñez Miguel et al. 2009; McLachian and Rapoport 2013) and exhibit catalytic activity (Mahendra et al. 2013). Evidence suggests that chronic activation of the immune system by the external milieu of an organism may lead to expansion of NAbs in genetically prone individuals (Avrameas et al. 2018).

Immunity and Snapping Turtles. — In the snapping turtle case, the disruption of immunological homeostasis caused by a stressful osmotic environment, and manifested in only a few immunological phenotypes, could cause the cutaneous lesions observed in some individuals. Some skin conditions such as psoriasis and chronic cutaneous lesions in humans are known to be driven by interplay between the innate and adaptive immune systems. They have a genetic component, and it appears exposure to environmental agents play a role in their etiology (Boehncke 2015; Harden et al. 2015; Pollard 2015). It is of interest that environmental agents also play a role in Graves's disease in humans. It has been estimated that factors including infections, pollution, and iodine intake account for about 20% of predisposition to this thyroid disorder characterized by autoantibodies that result in thyroxine overproduction (Shukla et al. 2018).

It must be acknowledged we are sailing in turbulent waters here. With only gross appearance and no diagnostic sampling, it is impossible to make a definitive statement about the cause(s) of the skin lesions observed in turtles both in the field and the laboratory. Various factors including salinity-induced infections, autoimmunity, and inflammation may all be at play. The confounding effects of stress hormones on the immune system must also be taken into consideration (Adamo 2014). The point is, one causal factor does not necessarily preclude another, and any or all could result in an incidental and purely colligative effect of driving up internal osmotic concentration. An increase from 340 mOsm blood osmotic concentration—the upper normal limit as suggested by field data—to 400 mOsm concentration would make those few individual turtles isoosmotic at 14-ppt salinity and capable of growth at a higher salinity. The microevolutionary consequences of such an increase in internal osmotic concentration in multiple individuals on the fringes of a population's normal distribution are intriguing. In one case, the approximately 18% increase in internal osmotic concentration would equate to a gain of 100–150 m of physiologically compatible habitat along an approximately 750-m brackish tidal creek characterized by a steep salinity gradient (Kinneary 1993). Perturbations in internal osmotic concentration incidental to an immune response may answer questions left open in the literature. For example, immune system constituents may account for the unidentified substance amounting to about 20%–30% of the osmotic pressure in plasma samples of hatchling crocodiles Crocodylus acutus from relatively high salinity habitats in Florida Bay (Dunson 1982).

Summary. — The conclusion from field and laboratory data from multiple sources is that Chelydra serpentina, the common snapping turtle, cannot hypoosmoregulate and, with the exception of brief excursions, are restricted to habitats that are no more than isoosmotic, which is somewhere around 12 ppt (340 mOsm; 34% seawater). Advances in the study of autoimmunity and ecoimmunology suggest that the immune system of vertebrates plays a role in maintaining homeostasis, beyond fighting infectious agents, and may give clue to explaining why a small percentage of snapping turtle hatchlings can grow at relatively high salinities, at least up to 14 ppt (400 mOsm; 40% seawater). The most parsimonious explanation is that the constitutive components of the immune system, including NAbs, have an incidental colligative effect and raise internal osmotic concentration to higher levels. The cutaneous lesions noted in some individuals, both in the field and in the laboratory at higher salinities, further support there is an immune system component at play. It is acknowledged that the above hypothesizes causality when there may only be correlation. However, advances in serological assays employed in the field of ecoimmunology make the hypotheses presented here testable. These include bacteriocidal and enzyme-linked immunosorbent assays that measure natural antibody levels and quantify the functional effects of immunological proteins (Brock et al. 2014; Garnier and Graham 2014; Sandmeier et al. 2016; Stromsland and Zimmerman 2017). In addition, what limits species ranges is one of the most puzzling questions in evolutionary biology (Kirkpatrick and Barton 1997; Sexton et al. 2009; Futuyma and Kirkpatrick 2018). The complex homeostatic functioning of the immune response resulting in secondary effects on internal osmotic concentration may offer clues to the beginning stages in the evolution of a species geographic range in aquatic environments characterized by steep salinity gradients. There are still unanswered questions concerning the incipient stages leading to diadromy, in its various forms, in the approximately 250 species (out of over 21,000 species) of bony fish (McDowell 1988, 1997; McCormick 2009). It has been suggested that, in any case, the reason(s) may be subpopulation or even individual specific (McDowall 1988; Quinn and Brodeur 1991; Feder et al. 2000). As future biologists continue to view the immune system from a more holistic perspective, it will give foundation to a more complete understanding of vertebrate adaptation to varying physical milieus. Hopefully, this article will serve as a small step toward a novel and integrative level of understanding the natural history of the common snapping turtle.