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Hemoglobin Polymerization in Red Blood Cells of Marine Fishes: A Case of Adaptive Phenotypic Plasticity?

Introduction

Hemoglobins (Hbs) for transport of [O.sub.2] in the blood remain the most intensively studied proteins for their function and structural variations. Globins or globin-like genes have been described in all kingdoms, including Archeobacteria, Protozoa, and Plantae (Weber and Vinogradov, 2001), and probably in all tissues of higher animals (Burmester et al., 2002; Trent and Hargrove, 2002). Despite the enormous diversity in their primary and quaternary structures (amino acid sequences and aggregation states), globin proteins exhibit a characteristic tertiary structure (the "globin fold"), suggesting a common ancestry. The ancestral globin gene likely evolved 670-1500 million years ago, when [O.sub.2] started to accumulate in the atmosphere (Hardison, 2012). This suggests that the protein's original function was variable, even plastic, allowing the protein to scavenge toxic [O.sub.2], CO, and NO gases (Hardison, 1996, 1999). As environments changed over evolutionary time, so did opportunities for Hb phenotypic plasticity.

Human sickle cell disease is an example of Hb plasticity. It results from a single mutation in which valine replaces a polar glutamic acid on the surface of the protein, resulting in the mutant hemoglobin HbS (Perutz and Mitchinson, 1950; Hofrichter et at, 1973). The structure of HbS reduces solubility in the deoxygenated state (Perutz and Mitchison, 1950; Ingram, 1957), and HbS polymers cause severe red blood cell (RBC) deformations. The most common is the sickle shape (Eaton and Hofrtichter, 1987). Human sickled RBCs are fragile and less deformable, and they result in anemia and blocked vessels, often causing extreme pain to the patient. In humans, the sickle cell trait (the heterozygous form) reduces transmission of the malarial parasite Plasmodium falciparum by impeding its transport in the body. But because polymerized RBCs have such low oxygen affinity, oxygen transport is also severely impaired.

Despite extensive work on the mechanisms that cause sickle cell trait and/or disease (the homozygous form) in humans, few detailed studies exist for other vertebrates, and there appear to be no documented studies in invertebrates. Polymerization of Hb and RBC sickling in fish has been reported since 1865 (cited in Perutz and Mitchinson, 1950), in deer (Taylor, 1983), in sheep (Butcher and Hawkey, 1979), in cats (Altman et al, 1972), and in some reptiles (Simpson et al, 1982; Mattei et al, 1985). Yet few reasons have been advanced for the persistence of Hb polymerization in populations other than humans, because no selective advantage seemed obvious.

Recently, Koldkjaer et al. (2013) proposed that Hb polymerization might serve as an innate, anti-parasitic immune response in fishes and maybe in all vertebrates. Empirical data to support this adaptive role for Hb are still lacking, yet it is clear that there is inherent phenotypic plasticity in fish Hb (Verde et al., 2002, 2006). Expressed as Hbs with different magnitudes of Root and Bohr effects, this phenotypic plasticity probably contributes to the capacity of fishes to colonize a rich diversity of aquatic habitats, from the deep oceans to alpine lakes (Sick, 1961).

In fishes, little detailed information about the environmental triggers for Hb polymerization was available until Harosi et al. (1998) investigated the phenomenon in two Gadiformes, haddock (Melanogrammus aeglefinus (Linnaeus, 1758)) and Atlantic cod (Gadus morhua Linnaeus, 1758), and in a closely related Batrachoidiform, the oyster toadfish (Opscmus tau (Linnaeus, 1766)). Harosi et al. (1998) provided evidence from Hb spectral signatures that Hb polymerization in fishes occurred only as deoxyhemoglobin (deoxyHb), similar to human HbS. Using scanning microspectrometry and polarized light microscopy, Harosi et al. (1998) established that RBC polymer morphology was related to oxygenation state, spectral absorbance, linear dichroism, and linear birefringence. Further, preliminary work from RBCs in G. morhua showed that Hb that was purified, separated, and concentrated to the level present in fish RBCs (16 mmol [L.sup.-1]) was the protein responsible for formation of Hb polymers when exposed to hypoxia (Hunt von Herbing et al, 2002). Thus, low [O.sub.2] was found to be a key environmental catalyst for Hb polymerization. In this way, Hbs that polymerize in fishes responded to conditions resembling those under which human HbS polymerizes to cause sickle cell disease.

Almost 10 years after Harosi et al. (1998), Koldkjaer and Berenbrink (2007) found that exhaustive exercise (captured on hook and line) caused the polymerization of >95% of RBCs in the gadiform whiting (Merlangius merlangus (Linnaeus, 1758)), characterized by angular and granular morphologies consistent with RBC sickling. Further, RBC sickling was accompanied by severe blood acidosis (pH ~7.3-7.5) from capture stress, as well as a release of stress hormones such as adrenaline, noradrenaline, and Cortisol, which have far-reaching effects on RBC acid-base chemistry. Koldkjaer and Berenbrink (2007) concluded, therefore, that low pH was critical for the formation of Hb polymers and that it must work synergistically with low [O.sub.2]. However, in some species it should be noted that the Root effect could generate high [O.sub.2] levels within a specific tissue and that low [O.sub.2] levels and low pH may not always be coincidental.

To investigate the extent of Hb polymerization in fish RBCs, we greatly expanded the number of fish species tested. We documented the presence or absence of the Hb trait in vitro in the whole blood of 47 species over 10 years, from 3 major geographic areas (east and west coasts of the Atlantic Ocean and the Gulf of Mexico) and in vivo for one species, G. morhua. Although we focused on marine fishes, Hb polymerization was also tested in two freshwater and several euryhaline species. In addition, for 7 species in which Hb polymerization was known to occur in vitro, the percent of Hb polymerized RBCs (%Hb polymerized RBCs) over 24 h and polymer morphology were examined for interspecific differences. Finally, we investigated the effects of decreasing pH (7.99 to 6.96) on the severity of Hb polymerization, as well as the recovery (de-polymerization) from in vitro sickling by an increase in extracellular pH in two boreal species, G. morhua and O. tau.

Materials and Methods

Animals

Whole blood from 47 species was collected from fish caught by hook and line and trawl over a period of 10 years (2000-2010) at 6 locations: (1) Memorial University Marine Science Center, Newfoundland, Canada (2000-2003) (trawl); (2) St. Andrews Biological Station, New Brunswick, Canada (2000-2003) (trawl); (3) Marine Biological Laboratory, Woods Hole, Massachusetts (2000-2003) (trawl); (4) Trondheim Biological Institute, Trondheim, Norway (2002) (hook and line); (5) Mount Desert Island Biological Laboratory (MDIBL), Salisbury Cove, Maine (2009-2010) (hook and line and small bait traps); and (6) University of Austin Marine Science Institute, Port Aransas, Texas (2009) (netted from aquaculture tanks). The number of samples per species ranged from N = 3 to N > 10, dependent on species abundance at each location (see Tables 1, 2 for specific numbers). Fish were sacrificed with an overdose of tricaine methanesulfonate (MS-222, 250 g [L.sup.-1]). Blood (0.5-1 mL) sampled from the caudal vein using a heparinized syringe was immediately stored on ice prior to observation for Hb polymerization. All animal use procedures were approved by Institutional Animal Care and Use Committees (IACUC) at the University of Maine (IACUC nos. A97-02-02 [1997-2000], A2000-0204 [2000-2003], and A2003-05-02 [2003-2006]) and University of North Texas (IACUC nos. 0715 [2007-2010] and 11002 [2010-2013]). Permission to collect was also aided by animal care and welfare certifications held by Dr. John Mattel at St. Andrews Biological Station and Dr. Jarle Mork at the Trondheim Biological Institute in Trondheim, Norway.

Sample preparation and observation

Observations of blood samples from all 47 species were conducted at room temperature (20-23 [degrees]C). Each sample was mixed at a ratio of 10:1 with buffer (marine teleost Ringer's solution) (in mmol [L.sup.-1]): NaCl 140, KCl 2.5, [CaCl.sub.2] 1.5, [MgSO.sub.4] 1, [NaH.sub.2][PO.sub.4] 0.5, [NaHCO.sub.3] 0.5, and HEPES 10, adjusted to pH 7.97 with NaOH and air saturated at 20 [degrees]C. Subsamples (10 [micro]L) for microscopy were placed on a slide under a no. 15 coverslip and sealed around the edge with clear nail polish. We then let metabolically active fish RBCs deplete the [O.sub.2]. The effectiveness of this method was variable and dependent on species, but it closely followed the methods that one of us (IHvH) used in Harosi et al. (1998) to monitor Hb polymerization. Light microscopic observations and images of the whole mounts of the RBCs for each species were qualitatively recorded for 0, 12, and 24 h after extraction, and 10 slides were prepared and examined for each blood sample.

In addition to qualitative observation of all 47 species, quantitative estimates of %Hb polymerized RBCs were conducted for 7 species found to exhibit Hb polymerization: Boreogadus saida (Lepechin, 1774); Lycodes reticulatus Reinhardt, 1835; Gadus morhua Linnaeus, 1758; Urophycis chuss (Walbaum, 1792); Merluccius bilinearis (Mitchill, 1814); Pollachius pollachius (Linnaeus, 1758); and Notacanthus chemnitzii Bloch, 1788. Methods for preparing the slides were the same as for the qualitative observations above. For a subsample from each of the 7 species, 250-300 RBCs were counted at 0, 12, and 24 h for each replicate (N = 3), totaling between 700 and 800 RBCs per species. Extra whole blood was stored in an equal volume of iso-osmotic 1% glutaraldehyde for future counts and/or for transmission electron microscopy (TEM). All fixed samples were stored at 4 [degrees]C. Samples were counted on a Zeiss Axio Observer Z1 inverted microscope (Oberkochen, Germany) equipped with an Axiocam MRm cooled B/W Firewire camera (Zeiss), coupled with Axiovision image analysis software (Zeiss).

Transmission electron microscopy

For TEM, blood samples from a total of 12 species (6 species that showed Hb polymerization and 6 species in which Hb polymerization was not observed) were placed in 4% iso-osmotic glutaraldehyde at 4 [degrees]C immediately after sampling (Tables 1, 2). Fixed RBC samples were washed 3 times in pH 7.97 saline, embedded in 2% agarose, and stained for 1 h in 1 % [O.sub.2][O.sub.4]. The embedded cells were washed in 30% ethanol for 10 min and incubated for 1 h in 0.5% uranyl acetate, followed by 10 min in each of 30%, 60%, 70%, 80%, 90%, and 100% ethanol and 2 washes in 100% acetone to dehydrate the samples. Samples were incubated in acetone:resin for 30 min in a 1:1 ratio and pure resin, then placed in molds for 24-48 h in a 60 [degrees]C oven. Sections were cut on an ultramicrotome and stained for 5 min each with 5% uranyl acetate and 2% Reynold's lead citrate. Sections were examined for the presence of the Hb matrix within the RBCs on a Tecnai transmission electron microscope (FEI Tecnai-12, Hillsboro, Oregon) equipped with an Olympus camera and Olympus Soft Imaging Solutions software (Shinjuku, Tokyo).

In vivo hemoglobin polymerization under hypoxia

Juvenile cod G. morhua (N = 6, mean standard length, 15 [+ or -] 0.5 cm) obtained from the University of Maine Aquaculture Research Center, Bangor, were evaluated in a one-time experiment (on June 13, 2002) for the occurrence of Hb polymerization in vivo. Two treatments were set up: (1) (control) 3 juvenile cod in a temperature-controlled (15 [+ or -] 1 [degrees]C) 10-L tank containing air-saturated (21% oxygen) seawater (control), and (2) (experimental hypoxia, 10% oxygen) 3 juveniles in a tank containing seawater bubbled with 100% nitrogen to generate hypoxia. The experiment consisted of 3 tanks per treatment, with 3 fish per tank (N = 9). Oxygen concentration decreased gradually to 10% air saturation, considered the lethal oxygen threshold for G. morhua (Plante et al, 1998). After 1 h for controls, or in the case of the experimental hypoxic treatment when death occurred, fish were euthanized with MS-222 (250 mg [L.sup.-1]), fixed in 4% glutaraldehyde, and prepared for TEM (see Transmission Electron Microscopy, above) to look for the presence of Hb polymers in the vessels.

Effects of pH on hemoglobin polymerization and recovery

Experiments on the effects of pH on Hb polymerization and RBC sickling were carried out on two species, G. morhua and Opsanus tau, between June and July 2008 at MDIBL, Salisbury Cove, Maine. Juvenile G. morhua (7.81 [+ or -] 0.04 g, N = 15) were purchased from the Center for Cooperative Aquaculture Research (CCAR, University of Maine), Franklin, Maine. Opsanus tau adults (330 [+ or -] 51.9 g, N = 8) were collected using shallow baited traps in Salisbury Cove, Maine. Both species were housed separately outside at ambient temperature (16.5 [+ or -] 0.5 [degrees]C) in a flow-through seawater holding system. All fish were fed ad libitum daily with squid. Four complete trials were carried out for G. morhua and 6 trials for O. tau.

Prior to blood sampling, fish were euthanized in MS-222 (250 mg [L.sup.-1]), then 0.5-1.0 mL of blood was immediately extracted using a 22.5-gauge heparinized syringe needle via the caudal vein. If fish were too small (which was the case in juvenile G. morhua) and if enough blood could not be extracted for experiments, fish were euthanized in MS-222 (250 mg [L.sup.-1]), and the body was severed between the second and third dorsal fins. Blood was collected with heparinized pipette tips and transferred to microcentrifuge tubes held on ice. Whole blood (500 [micro]L) was also collected from 4 adult Atlantic halibut (Hippoglossus hippoglossus (Linnaeus, 1758)), held at CCAR and used as controls for experiments, because Atlantic halibut Hb does not polymerize (LHvH, pers. obs.).

All collected whole blood was kept on ice, before centrifugation (4 [degrees]C; 2500 rpm for 4 min x 3 times). After centrifugation, the plasma was removed and replaced with 10 volumes of isotonic saline. The composition of the saline was (in mmol [L.sup.-1]): 160 NaCl, 3 KC1, 1.5 [MgCl.sub.2], 1.5 [CaCl.sub.2], 5 D-glucose, and 20 HEPES, adjusted to pH 7.99, osmolality 339 mOsm [kg.sup.-1] at 16.5 [+ or -] 0.5 [degrees]C. Samples were stored overnight at 4 [degrees]C, with an air bubble, to allow for oxygenation and to reduce sedimentation.

After incubation, samples were centrifuged, washed twice, and re-suspended in pH 7.99 saline solution, resulting in 20%-25% final hematocrit. Three 50-[micro]L aliquots were removed and placed in 1.5-mL microcentrifuge tubes containing 200 [micro]L of experimental saline at 6 pH levels (6.96, 7.2, 7.4, 7.6, 7.8, and 7.99 [pH control]). All samples were incubated in a water bath at 16.5 [+ or -] 0.5 [degrees]C and manually turned every 10 min to prevent sedimentation. At the end of the 45-min period of incubation, all samples not used in recovery experiments were fixed with 2% glutaraldehyde solution in a 1:1 ratio for future cell counts and observations. For the pH experiments, a total of 700-900 RBCs were counted (250-300 RBCs from each of the 3 replicates) for each pH level.

Recovery from in vitro Hb polymer formation by an increase in extracellular pH was studied in air-equilibrated RBCs at 16.5 [+ or -]0.5 [degrees]C. After 45 min, 3 50-[micro]L subsamples of RBCs suspended in low-saline pH (6.96) were taken and fixed for analysis. The remainder of the sample was centrifuged (10 s at ~9000 x g, following Koldkjaer and Berenbrink 2007), and the plasma was replaced with high-saline pH (7.99). The resuspended cells were incubated as before, and 3 50-[micro]L sub-samples were removed at 5, 15, 30, and 45 min and were fixed in 2% glutaraldehyde for future counts and observations. Recovery experiments (N = 3) were carried out for both species per time point.

Statistics

To investigate the effect of time on %Hb polymerized RBCs, one-way analysis of variance (ANOVA) was used to determine the extent of polymerization (0%-100%) for 7 species over a 24-h period. To investigate pH on Hb polymerization, a two-way ANOVA compared the effects of pH and time on %Hb polymerized RBCs between two species: G. morhua and O. tau. To determine the rate of recovery (depolymerization) from low pH (6.96) to high pH (7.99) of RBCs, a two-way ANOVA was conducted across species over time. All data were represented as means [+ or -] standard deviations. Statistical significance was accepted at P < 0.05. All analyses were conducted in SAS, version 9.4 (SAS Institute, 2015).

Results

In vitro hemoglobin polymerization

Of 47 fish species sampled in vitro, Hb polymerization was observed in 15 species spanning 9 families and 5 orders. These included species within the orders Gadiformes (N = 7) and Batrachoidiformes (N = 2) (Table 1). In the gadiform Gadus morhua and the batrachoidiform Opsanus tau, RBCs morphed from clear ovoid cells (Fig. 1a, c) into sickled cells containing Hb polymers within the first few minutes under test conditions (Fig. 1b, d). In contrast, 33 species in 23 families and 11 orders did not exhibit Hb polymerization or RBC sickling to any degree (Table 2).

In some orders sampled in the present study, expression of the Hb polymerizing trait varied among species but not within families, while other orders showed differences within families. In the Scorpaeniformes, one species in Sebastidae, Sebastesfasciatus, exhibited Hb polymerization, while four species in three other families, Cottunculus microps family Psychrolutidae, Myoxocephalus octodecemspinosus family Cottidae, Myoxocephalus scorpius family Cottidae, and Hemitripterus americanus family Hemitripteridae, did not exhibit the trait (Tables 1, 2). In contrast, in the large order Perciformes, the expression of the Hb polymerizing trait varied among species within the same family. Tautoga ontis and Lycodes reticulatus, in Labridae and Zoarcidae, respectively, exhibited Hb polymerization (Table 1), while Ctenolabrus rupestris and Zoarces americanus, in the same two families, did not exhibit the trait (Table 2).

To determine the extent (percent) and relative rate of Hb polymerization among species, %Hb polymerized RBCs in vitro were monitored over a 24-h period for Boreogadus saida, L. reticulatus, G. morhua, Urophycis chuss, Merluccius bilinearis, Pollachius pollachius, and Notacanthus chemnitzii (Fig. 2). For all 7 species, observations at 24 h showed that a significantly (P < 0.05) higher percentage of RBCs contained Hb polymers than at the start (0 h) (Fig. 2). For 2 of the 7 species, L. reticulatus and N. chemnitzii, no Hb polymerized RBCs were present in the first hour of observation, compared to at 24 h (P < 0.05). Yet for the 4 gadiform species, >20% Hb polymerized RBCs occurred at 0 h versus 100% at 24 h (P < 0.05), with the exception of P. pollachius, in which 100% Hb polymerization occurred at 0 and 24 h (Fig. 2).

Using transmission electron microscopy to characterize hemoglobin polymerization in different species

Transmission electron microscopy images confirmed the presence of intracellular Hb polymers in six species in three families (Gadidae, Merlucciidae, and Phycidae) in the order Gadiformes (Fig. 3). Prominent bundles were clearly visible throughout the cytosol and in the nucleoplasm. In one gadiform species, saithe (Pollachius virens), hemolysis was acute, and intact cellular membranes were not visible (Fig. 4a). Transmission electron microscopy images showed only membrane remnants (Fig. 4b). In contrast, TEM images of six species in which RBCs did not exhibit Hb polymerization showed the cytosol and nucleoplasm as uniform and free of any intracellular polymers (Fig. 5).

In vivo hemoglobin polymerization under hypoxia

Transmission electron micrographs showed bundles of distinct Hb polymers in an artery of a juvenile G. morhua exposed to reduced oxygen concentrations (Fig. 6a-d). Cross sections of the central dorsal artery of a juvenile G. morhua also showed several RBCs with Hb polymers in the cytosol (Fig. 6a, b), and they appeared granular and rough (Fig. 6c, d). In contrast, no Hb polymerization was observed in cross sections from control fish (juvenile G. morhua exposed to bubbled air in seawater) (Fig. 6e). The cytosol and nucleoplasm of RBCs from control fish appeared uniform in the TEM images, indicated by the typical smooth appearance of non-polymerized Hb (Fig. 6e).

Effects of pH on hemoglobin polymerization

Experiments on the effects of pH on Hb polymerization in RBCs of G. morhua and O. tau showed similar patterns in the degree of polymerization but somewhat different RBC morphologies (Fig. 7). At pH 7.99, a typical value for normoxic resting animals at 15-16 [degrees]C (Butler et al, 1989; Perry et al, 1991 ;Ultsch and Jackson, 1996; Larsen etal, 1997; Knudsen and Jensen, 1998; Koldkjaer and Berenbrink, 2007), about 90% of RBCs were normal in both G. morhua and O. tau (~97%). When pH dropped to 7.6, 46.0% of all RBCs in G. morhua and 63% in O. tau contained numerous intercellular bars and had distorted cell membranes. At pH 6.96, 100% of RBCs in G. morhua and 90% of RBCs in O. tau showed severe polymerization.

While in both species %Hb polymerized RBCs increased with decreasing pH ([F.sub.1, 57] = 246.67, P < 0.001), there was no significant difference between species ([F.sub.1, 57] = 0.64, P> 0.05; Fig. 7). In addition, the interaction between species and pH was not significant ([F.sub.1, 56] = 1.17, P> 0.05). For recovery experiments (RBCs at pH 6.96, then exposed to pH 7.99), %Hb recovered RBCs increased significantly with time ([F.sub.1, 21] = 22.6, P < 0.001; Fig. 8), but rates of recovery did not significantly differ between species ([F.sub.1, 21] = 0.19, P > 0.05; Fig. 8).

Discussion

In vitro Hb polymerization in fish RBCs resulting from exposure to low oxygen and low pH occurred in 32% of the 47 species surveyed, representing 15 species, 10 families, and 5 orders (Table 1). This significantly expands our knowledge of the wide distribution of this unusual trait. While we cannot confirm that polymerization was induced by hypoxia, because oxygen concentrations were not measured directly on the microscope slides, similar intracellular bars in vitro have been identified as Hb polymers or aggregates of deoxyHb in a previous study (Harosi et al, 1998). While we accept that the intracellular bars within RBCs consisted of polymerized deoxyHb, we refer to the RBCs that contained them as Hb polymerized RBCs, thus differentiating cells that contain intracellular Hb polymers from sickled RBCs (cells in which membrane deformation results from polymerization). This terminology differs from that used in Harosi et al. (1998), Koldkjaer and Berenbrink (2007), and Koldkjaer et al. (2013), in which all RBCs containing Hb polymers (whether the cell membranes were distorted or remained smooth) were referred to as sickled cells. This distinction between polymerized RBCs and sickled RBCs in the present study was useful in estimating the extent of polymerization (0%-100%) and its variation among species (Fig. 2).

The supra-ordinal taxon Paracanthopterygii includes Gadiformes (e.g., cods, hakes, rattails, and their allies), with about 11-14 families, 75 genera, and >500 species. Of the 15 species that tested positive for the Hb polymerizing trait, many were in the order Gadiformes (7 species) or were related to Gadiformes. Two species in Batrachoidiformes, Opsanus tau and Opsanus beta, also exhibited the Hb polymerizing trait. Batrachoidiformes is a sister order thought to have diverged 20 million years ago from Gadiformes (Carroll, 1988; Helfman et al, 1997). Also, the zoarcid Arctic eelpout, Lycodes reticulatus, exhibited clear Hb polymerization in its RBCs. Zoarcids were once taxonomically grouped with Gadiformes but now belong to Perciformes (Nelson, 2006).

Gadiformes fish inhabit waters in every ocean of the world and at one time supplied more than 25% of the world fish harvest (FAO, 2004), prior to the cod fishery crash in the early 1990s. They are widely distributed, from deep-sea benthic habitats to coastal waters in higher latitudes (Nelson, 2006). They are also found in tropical seas and are among the most diverse group of fishes inhabiting deep waters, where they can be the dominant species in biomass (Myers et al, 2001). Within Gadiformes, the family Gadidae is well known because it contains many economically and ecologically important fishes such as Atlantic cod (Gadus morhua), silver hake (Merluccius bilinearis), and haddock (Melanogrammus aeglefinus) (Nelson, 2006). Past studies identified other gadids as also exhibiting Hb polymerization, including the following: poor cod, Gadus minutus (re-classified, Trisopterus minutus (Linnaeus, 1758)); whiting, Merlangius merlangus (Linnaeus, 1758); pouting cod, Gadus luscus (re-classified, Trisopterus luscus (Linnaeus, 1758)); and pollock, Pollachius pollachius (Linnaeus, 1758) (Yoffey, 1929); as well as the subspecies Baltic cod (G. morhua callarias Linnaeus, 1758) (Thomas, 1971). In total, almost 50% (i.e., 10 of 21 species) within Gadidae exhibited the trait, suggesting that Hb polymerization may be a phylogenetic marker for the family and possibly for the order Gadiformes. Therefore, the prevalence of the Hb polymerizing trait in such an important fish group is admittedly a puzzle, as is its effect on the group's health and survival.

Three other orders, Notacanthiformes, Perciformes, and Scorpaeniformes, contained species that expressed the Hb polymerizing trait in the present study (Table 1). Combining our results with reports over the past century (Yoffey, 1929; Dawson, 1932; Hansen and Wingstrand, 1960; Fago et al, 1993; Harosi et al, 1998; Koldkjaer and Berenbrink, 2007; Riccio et al, 2011; Koldkjaer et al., 2013) brings the total number that exhibit this trait to >50 species. Most of these are marine fishes, with the exception of one freshwater teleost, the Central and South American charcin, Hoplias malabarica (Reischl, 1976). The two freshwater species studied here, the zebrafish (Danio rerio) and the common white sucker (Catostomus commersonii), did not exhibit Hb polymerization. Therefore, it is impossible to judge whether this Hb trait is more common in marine versus freshwater environments.

While only a fraction of the more than 33,000 species of fishes currently verified (Erschmeyer, 2010) were tested for Hb polymerization, its occurrence in >50 species suggests that this unusual trait may have been conserved, at least in these taxa. Though possibly a conserved trait, the morphology and extent of polymerization vary among species (Figs. 2, 4). In the gadid G. morhua (this study) and its sister species M. merlangus (Koldkjaer and Berenbrink, 2007), RBC membranes rarely ruptured as a result of Hb polymerization, despite almost 100% of their RBCs containing intracellular Hb polymers and distorted cell membranes (e.g., sickled cells). Similarly, the batrachoids O. tau (this study) and O. beta (Koldkjaer et al, 2013) seemed unaffected by RBC sickling. Yet in one gadid, Pollachius virens, disruption of cell membranes and hemolysis was extreme (Fig. 4a, b). Thus, compared to other species, P. virens may be more sensitive to disruption of oxygen transport, perhaps associated with Hb polymerization, when exposed to prolonged hypoxia and/or low pH.

In addition to morphological differences, rates of Hb polymerization and/or severity of polymerization (i.e., the degree to which cell membranes were distorted or sickled) were also observed among species (Fig. 2). In G. morhua, Urophycis chuss, M. bilinearis, and P. pollachius, all (100% of) RBCs were polymerized in the first 24 h, while in Boreogadus saida, L. reticulatus, and Notacanthus chemnitzii, only about 20% of RBCs contained polymerized Hb over the same period. These morphological and species-specific rate differences in Hb polymerization may reflect the variable nature of fish Hbs and their molecular structure (polymorphisms), thought to be the primary cause of the adaptive capacity of Hb to respond to environmental change (Karpov and Novikov, 1980; Weber and Jensen, 1988; Verde et al, 2006; Anderson et al, 2009).

Results from the present study support previous results that Hb polymerization is strongly pH dependent. In our study >60% of G. morhua RBCs and >40% of O. tau RBCs contained Hb polymers at a pH of 7.6 (Fig. 7), while related species of M. merlangus and O. beta showed >50% of RBCs containing Hb polymers at the same pH (Koldkjaer and Berenbrin, 2007; Koldkjaer et al, 2013). While Hb polymerization increased as pH decreased (from 7.99 to 6.96) in all studies, polymerization was also readily reversible when pH reverted to normal levels (Fig. 8). In vitro it took only 2 min for 100% of the sickled RBCs to return to normal when pH increased from 7.03 to 7.97 (Koldkjaer and Berenbrink, 2007). In our study it took ~15 min for 50% of G. morhua and O. tau polymerized RBCs to recover to normal when pH increased from 6.96 to 7.99 (Fig. 8). In vivo it appeared to take about 24 h after being caught on hook and line for the percentage of sickled RBCs to decrease from 95% to 63% in M. merlangus (Koldkjaer and Berenbrink, 2007). Differences between in vivo and in vitro methods, such as the washing of cells to remove catecholamine effects, which involves substantial RBC volume changes, may be responsible for the variation in results among these studies. But it does seem that, along with polymerization, reversibility is common in fishes that exhibit the Hb polymerizing trait.

Reversibility of polymerization and its corresponding state changes (from liquid to crystal, see Harosi et al, 1998) suggest that Hb phenotypic plasticity may result from structural differences at the molecular level. In G. morhua, primary sequences of Hb A and B chains revealed substitutions of conserved amino acids when compared to other fishes (Tipping and Birley, 2001). Homology modeling of the B chains shows that three of these substitutions are in proximity to each other (e.g., B 56) and may result in new surface cysteines capable of forming disulphide bridges (Hunt von Herbing et al., 2002, 2008, 2009). These interactions established for Hbs (Tondo et al., 1974) are consistent with polymerized RBCs in G. morhua (Thomas, 1971; Harosi et al., 1998), as are two extra histidine residues on the external surface of the B-globin chain in G. morhua and B. saida (Verde et al, 2006). These structural anomalies may also be implicated in the in vitro formation of Hb polymers in the temperate marine goosefish, Lophius americanus, order Lophiiformes, family Lophidae (Borgese et al, 1988, 1992), and the red gurnard, Chelodonichthys kumu, order Scorpaeniformes, family Trigilidae (Fago et al, 1993). Whether these morphological variations observed in Hbs that polymerize lead to changes in oxygen transport and fish metabolism is still to be determined.

In contrast, the plasticity of the physiology of this Hb trait is clear from our observations of different specific-specific rates of polymerization at pH levels considered to be low in fish blood (pH 7.3-7.5) (Holeton et al, 1983; Milligan and Wood, 1987). Blood acidosis, which often results from exhaustive exercise when fish are caught on hook and line, is often accompanied by elevated plasma adrenaline and noradrenaline levels (Primmett et al, 1986; Berenbrink and Bridges, 1994; Perry et al, 1996). To combat stress-induced low pH, many teleosts possess RBC membrane B-adrenerically activated Na/H exchangers (BNHEs), whose stimulation increases intracellular pH (Nikinmaa, 1982; Coussins and Richardson, 1985; Berenbrink and Bridges, 1994). In Hb polymerizing fishes, BNHEs may also protect against pH-induced sickling in vivo (Koldkjaer and Berenbrink, 2007). But even a modest decrease in intracellular pH (pHi) (which could occur if a Hb polymerizing fish were exposed to lower than normal external pH [pHe 7.7-8.1]) might be enough to reduce glycolysis and impair aerobic metabolism. Conflicting results exist, however, of the impact of environmental pC[O.sub.2] on fish metabolism, and they seem to depend upon the species tested (see references in Heuer and Grosell, 2014). For example, G. morhua, which exhibits Hb polymerization, experienced little impact on aerobic metabolism when exposed to pC[O.sub.2] levels up to 6000 [micro]atm. But two tropical reef fish species (untested for Hb polymerization) experienced increased--not decreased--maximum metabolic rate and reduction of aerobic scope when exposed to -900 [micro]atm (Rummer et al, 2013).

For the commercially important G. morhua, any metabolic compromises associated with Hb polymerization may have implications for its growth and survival. This species is now intensively farmed and faces overcrowding in aquaculture pens, where [O.sub.2] saturation often drops below 70% and where high ammonia levels cause low pH (Bjornsson and Olafsdottir, 2006). These conditions could lead to chronic increased levels of Hb polymerized RBCs and could potentially affect productivity. But it is difficult to predict the true impact of Hb polymerization until detailed structure-function analyses are conducted comparing species exhibiting polymerization with those with non-polymerizing Hbs. Only then may we begin to understand the reason why the Hb polymerizing trait persisted through evolution, as well as its potential affects on present-day fish distribution and abundance.

In summary, we suggest that in vitro and also perhaps in vivo hemoglobin polymerization and RBC sickling are much more widespread in fishes than once thought. From our study, as well as previous studies, we suggest that (1) Hb polymerization is present in a broad range of fish taxa; (2) this Hb trait was conserved in fishes that occupy a wide biogeographical and temperature range (the Arctic to the tropics) and a broad range of habitats (bathydemersal to pelagic) and environmental conditions (brackish to marine); and (3) Hb polymerization is pH dependent and reversible in most fishes. While this trait may once have been adaptive or neutral, in present-day fishes its function remains unclear. Understanding the function of Hb polymerization in fish RBCs may provide insights as to how selection favored the evolution and maintenance of this trait in some fish groups but not in others. The potential impacts of the Hb polymerizing trait in fishes may become more critical as large-scale environmental conditions (temperature, pH, oxygen concentration, and salinity) and their synergistic effects (Pan and Hunt von Herbing, 2017) change over this century and affect species survival in the oceans.

Acknowledgments

Thanks to Bob Cashon, Jarle Mork, John Martel, Tiffany Horton, and Jennifer Sanford, who helped with the data collection, as well as the staff at St. Andrews Biological Station, St. Andrews, New Brunswick; Mount Desert Island Biological Laboratory, Salisbury Cove, Maine; Trondheim Biological Institute, Trondheim, Norway; and University of Texas Marine Science Institute, Port Aransas, Texas. This work was supported by National Science Foundation OPP0118372 to GHvH. KS-S was also supported in part by the University of North Texas Ronald E. McNair Post-Baccalaureate Program. This paper is dedicated to the memory of Ferenz Harosi, who helped champion our original paper in 1998. Thanks also to T. C. F. Pan, who made valuable comments on the manuscript, as well as to two anonymous reviewers and the associate editor who provided excellent feedback and suggestions.

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I. HUNT VON HERBING(*) AND K. SCHROEDER-SPAGN ([dagger])

Department of Biological Sciences, University of North Texas, Denton, Texas 76203

Received 12 February 2018; Accepted 13 September 2018; Published online 14 December 2018.

(*) To whom correspondence should be addressed. Email: vonherbing@unt.edu.

([dagger]) Present address: Coastal and Marine System Sciences, Texas A&M University-Corpus Christi, 6300 Ocean Drive, Corpus Christi, Texas 78412.

Abbreviations: BNHE. B-adrenerically activated Na/H exchanger; deoxyHb, deoxyhemoglobin; Hb. hemoglobin; HbS, mutant hemoglobin (hemoglobin sickle); MS-222. tricaine methanesulfonate; RBC. red blood cell; TEM, transmission electron microscopy.

DOI: 10.1086/700832
Table 1 Fish species in which hemoglobin (Hb) polymerization was
observed in red blood cells (RBCs)

Fish classification                  N
Order: Batrachoidiformes
  Family: Batrachoididae
    Atlantic toadfish,               10
    Opsanus tau
    (Linnaeus, 1766)
    Gulf toadfish, Opsanus           10
    beta (Goode & Bean, 1880)
Order: Gadiformes
  Family: Gadidae
    Atlantic cod, Gadus              30
    morhua Linnaeus, 1758
    Haddock, Melanogrammus           10
    aeglefinus (Linnaeus, 1758)
    Pollock, Pollachius pollachius   10
    (Linnaeus, 1758)
    Saithe. Pollachius virens        10
    (Linnaeus, 1758)
    Polar cod, Boreogadus saida       5
    (Lepechin, 1774)
  Family: Merlucciidae                5
    Silver hake, Merluccius
    bilinearis (Mitchill, 1814)
  Family: Phycidae                    5
    Red hake, Urophycis chuss         5
    (Walbaum, 1792)
Order: Notacanthiformes
  Family: Notacanthidae
    Spiny eel, Notacanthus            5
    chemnitzii Bloch. 1788
Order: Perciformes
  Family: Gobiidae
    Norwegian mudfish.                5

    Pomatoschistus microps
    (Kroyer, 1838)
  Family: Labridae
    Tautog, Tauloga onitis            5
    (Linnaeus, 1758)
  Family: Serranidae
    Speckled hind, Epinephelus        5
    drummondhayi Goode &
    Bean, 1878
  Family: Zoarcidae
    Arctic eelpout. Lycodes           5
    reticulatus Reinhardt, 1835
Order: Scorpaen!formes
  Family: Sebastidae
    Acadian redfish. Sebastes         5
    fasciatus Storer. 1854

Data are grouped alphabetically into orders, families, and species. N =
number of individuals tested of each species.

Table 2 Fish species in which hemoglobin (Hb) polymerization was not
observed in red blood cells (RBCs)

Fish classification                      N
Order: Atheriniformes
  Family: Menidiinae
    Atlantic silverside. Menidia         8
    menidia (Linnaeus, 1766)
Order: Carcharhiniformes
  Family: Triakidae
    Smooth dogfish. Mustelus             12
    canis (Mitchill. 1815)
Order: Clupeiformes
  Family: Clupeidae
    Atlantic herring. Clupea             24
    harengus harengus Linnaeus, 1758
    Blueback shad. Alosa aestivalis      6
    (Mitchill, 1814)
    Menhaden, Brevoortia gunteri         8
    Hildebrand, 1948
Order: Cyprinodontiformes
  Family: Fundulidae
    Killifish. Fundulus heteroclitus     12
    (Linnaeus, 1766)
    Sheepshead minnow, Cyprinodon        5
    variegatus variegatus
    Lacepede, 1803
  Family: Catostomidae
    Common white sucker, Calostomus      5
    commersonii (Lacepede, 1803)
  Family: Cyprinidae
    Zebrafish, Danio rerio               10
    (Hamilton, 1822)
Order Lophiiformes
  Family: Lophiidae
    Monkfish, Lophius piscalorius        3
    Linnaeus, 1758
  Family: Ephippidae
    Atlantic spadefish, Chaetodipterus   3
    faber (Broussonet, 1782)
  Family: Haemulidae
    Pigfish, Orthopristis chrysoptera    3
    (Linnaeus. 1766)
  Family: Labridae
    Norwegian labrid, Ctenolabrus        3
    rupestris (Linnaeus, 1758)
  Family Moronidae
    Striped bass. Morone saxatilis       3
    (Linnaeus, 1758)
Order: Perc!formes
  Family: Sciaenidae
    Atlantic croaker, Micropogonias      3
    undulatus (Linnaeus, 1766)
    Red drum, Sciaenops ocellatus        5
    (Linnaeus, 1766)
    Southern kingcroaker, Menticirrhus   3
    americanus (Linnaeus, 1758)
    Spot croaker, Leiostomus xanthurus   3
    Lacepede, 1802
    Weakfish, Cynoscion regalis          3
    (Bloch & Schneider, 1801)
  Family: Scombridae
    Mackerel, Scomber scombrus           5
    Linnaeus, 1758
  Family: Rachycentridae
    Cobia, Rachycentron canadum          3
    (Linnaeus, 1766)
  Family: Zoarcidae
    Ocean pout, Zoarces americanus       5
    (Bloch & Schneider, 1801)
Order: Pleuronectiformes
  Family: Pleuronectidae
    Winter flounder, Pleuronectes        10
    americanus Walbaum, 1792
  Family: Paralichthyidae
    Summer flounder, Paralichthys        5
    dentatus (Linnaeus, 1766)
    Southern flounder, Paralichthys      3
    lethostigma Jordon & Gilbert, 1884
  Family: Scophthalmidae
    Turbot, Scophthalmus maximus         3
    (Linnaeus, 1758)
Order: Rajiformes
  Family: Rajidae
    Little skate, Leucoraja erinacea     10
    (Mitchill, 1825)
Order: Scorpaeniformes
  Family: Psychrolutidae
    Polar sculpin, Cottunculus           3
    microps Collett, 1875
  Family: Cottidae
    Longhorn sculpin, Myoxocephalus       5
    octodecemspinosus
    (Mitchill, 1814)
    Shorthorn sculpin, Myoxocephalus      3
    scorpius (Linnaeus, 1758)
  Family: Hemitripteridae
    Sea raven, Hemitripterus americanus   5
    (Gmelin. 1789)
Order Squaliformes
  Family: Squalidae
    Dogfish, Squalus acanthias            5
    Linnaeus, 1758

Data are grouped alphabetically into orders, families, and species. N =
number of individuals tested of each species.
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