The effects of temperature and salinity on the blood chemistry and survival of juvenile Atlantic tarpon Megalops atlanticus

Abstract

Atlantic tarpon Megalops atlanticus are highly migratory sportfish that support recreational fisheries throughout their range. In U.S. waters, juveniles can be found in coastal and estuarine habitats along the Gulf of Mexico and Atlantic seaboard, with temperature limiting their northern latitudinal distribution. Juveniles may overwinter in these areas during the first several years of life. Low temperatures are known to cause mortality in adults, but the challenges of temperature are less understood for juveniles. Furthermore, salinity, which can change dramatically in these habitats, may have a synergistic effect with temperature. To examine the physiological effects of temperature and salinity on juvenile tarpon, wild fish were acclimated to a range of conditions that potentially occur in the northern range of their estuarine habitats. The haematology of juvenile tarpon was examined in two salinity (≤2 and ≥30 ppt) and temperature (15 and 25°C) treatments, followed by a low temperature tolerance test. After two weeks in treatment conditions, blood samples were analyzed for hematocrit, pH, red blood cell concentration, haemoglobin content, and plasma osmolality. Increased plasma osmolality was observed in fish at low temperature (15°C compared to 25°C) and at high salinity (≥30 ppt compared to ≤2 ppt). Blood pH was increased at 15°C compared to 25°C, with the highest pH at 15°C and low salinity. Haemoglobin, hematocrit and red blood cell concentration were higher at 25°C than 15°C, with haemoglobin lowest at 15°C and low salinity. For the low temperature tolerance test, all fish were acclimated to 15°C for 2 weeks, then transferred to separate tanks where temperature was gradually decreased at 0.9 ± 0.1°C/hr until fish lost equilibrium. Fish at low salinity lost equilibrium more rapidly (1 ppt, 12.65 ± 0.46°C) than fish at high salinity (30 ppt, 11.26 ± 0.14°C). Results indicate juvenile tarpon are susceptible to low temperature, which is exacerbated by low salinity, findings useful in assessment of juvenile tarpon overwintering habitat.

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REGULAR PAPER
The effects of temperature and salinity on the blood chemistry
and survival of juvenile Atlantic tarpon Megalops atlanticus
Manuel E. Coffill-Rivera
1
| Yvanna D. Paez Mendez
1
| Logan Little
1
|
Patrick M. Graham
2,3
| James S. Franks
2
| Sandra Bibiana Correa
1
|
J. Wesley Neal
1
| Peter J. Allen
1
1
Department of Wildlife, Fisheries and
Aquaculture, Mississippi State University,
Starkville, Mississippi, USA
2
Gulf Coast Research Laboratory, Center for
Fisheries Research and Development, The
University of Southern Mississippi, Ocean
Springs, Mississippi, USA
3
Division of Coastal Sciences, School of Ocean
Science and Engineering, The University of
Southern Mississippi, Ocean Springs,
Mississippi, USA
Correspondence
Manuel E. Coffill-Rivera, Department of
Wildlife, Fisheries and Aquaculture, Mississippi
State University, Mississippi State, Mississippi
39762, USA.
Email: manuelcoffill@gmail.com and
peter.allen@msstate.edu
Funding information
Mississippi State University, Undergraduate
Research Scholars Program; Puerto Rico Sea
Grant, University of Puerto Rico, Grant/Award
Number: 2020-2021-007; USDA National
Institute of Food and Agriculture McIntire-
Stennis, Grant/Award Number: 1026075
Abstract
Atlantic tarpon Megalops atlanticus are highly migratory sportfish that support recrea-
tional fisheries throughout their range. In US waters, juveniles can be found in coastal
and estuarine habitats along the Gulf of Mexico and Atlantic seaboard, with tempera-
ture limiting their northern latitudinal distribution. Juveniles may overwinter in these
areas during the first several years of life. Low temperatures are known to cause mor-
tality in adults, but the challenges of temperature are less understood for juveniles.
Furthermore, salinity, which can change dramatically in these habitats, may have a
synergistic effect with temperature. To examine the physiological effects of tempera-
ture and salinity on juvenile tarpon, wild fish were acclimated to a range of conditions
that potentially occur in the northern range of their estuarine habitats. The haematol-
ogy of juvenile tarpon was examined in two salinity (≤2 and ≥30 ppt) and tempera-
ture (15 and 25C) treatments, followed by a low-temperature tolerance test. After
2 weeks in treatment conditions, blood samples were analysed for haematocrit, pH,
red blood cell concentration, haemoglobin content and plasma osmolality. Increased
plasma osmolality was observed in fish at low temperature (15C compared to 25C)
and at high salinity (≥30 ppt compared to ≤2 ppt). Blood pH was increased at 15C
compared to 25C, with the highest pH at 15C and low salinity. Haemoglobin, hae-
matocrit and red blood cell concentration were higher at 25C than 15C, with hae-
moglobin lowest at 15C and low salinity. For the low-temperature tolerance test, all
fish were acclimated to 15C for 2 weeks, then transferred to separate tanks where
temperature was gradually decreased at 0.9 ± 0.1C/h until fish lost equilibrium. Fish
at low salinity lost equilibrium more rapidly (1 ppt, 12.65 ± 0.46C) than fish at high
salinity (30 ppt, 11.26 ± 0.14C). The results indicate juvenile tarpon are susceptible
to low temperature, which is exacerbated by low salinity, findings useful in the
assessment of juvenile tarpon overwintering habitat.
KEYWORDS
acid-base regulation, Elopiformes, haematology, Megalopidae, osmolality, osmoregulation,
overwintering, plasma biochemistry, sábalo, survival
Received: 7 March 2023 Accepted: 12 May 2023
DOI: 10.1111/jfb.15451
FISH
J Fish Biol. 2023;1–8. wileyonlinelibrary.com/journal/jfb © 2023 Fisheries Society of the British Isles. 1

1|INTRODUCTION
Atlantic tarpon Megalops atlanticus (hereafter tarpon) are large
migratory, elopiform fish (Family Megalopidae) of ecological impor-
tance in tropical, subtropical and temperate waters of the Atlantic
Ocean where they support valuable recreational fisheries
(Crabtree et al., 1997;Crabtreeeet al., 1995;Wade,1962). Tarpon
demonstrate a dependence on upper estuarine habitats during
their early life history. These habitats include brackish lagoons,
tidal creeks, stagnant pools, backwaters, ephemeral coastal ponds,
hurricane and storm overwashes, swales, mangrove swamps,
marshes, man-made mosquito impoundments and artificial wet-
lands (Graham et al., 2017;Judet al., 2011; Mace III et al., 2018;
Rickards, 1968;Wade,1962;Wilsonet al., 2019)whichhavea
wide range of temperature, salinity, pH and dissolved oxygen con-
centrations (Geiger et al., 2000). Salinity is one of the most influen-
tial abiotic factors determining the composition of estuarine fish
communities (Barletta et al., 2005; Cyrus & Blaber, 1992). Estua-
rine ecosystems are subject to seasonal fluctuations in salinity, pri-
marily due to freshwater discharge (Conroy et al., 2020), and
vertical and horizontal salinity stratification is a common feature of
these systems (Schroeder et al., 1990). However, tarpon are facul-
tative air breathers and possess euryhaline osmoregulatory capa-
bilities that facilitate occupancy of these habitats (Adams
et al., 2013;Wade,1962). Sounds, bays and bayous are juvenile
tarpon habitats that provide access for later emigration as sub-
adults to deeper coastal waters (Ault, 2007). Juvenile and subadult
tarpon are thought to depend on these estuarine habitats for
approximately 7–10 years (Kurth et al., 2019), followed by an
ontogenetic habitat shift to coastal waters, which may range from
near- to offshore, as they approach maturity, where they presum-
ably join the adult stock.
The northern latitudinal range of Atlantic tarpon is primarily
limited by their thermophilic physiology (Howells & Garrett, 1992;
Overstreet, 1974). Estuaries in US waters that are utilized by tar-
pon as nursery areas extend into the northern Gulf of Mexico
(GOM) and along the South Carolina and North Carolina coasts
(Franks et al., 2013;Grahamet al., 2017;MaceIIIet al., 2020;
Wade, 1962), where juveniles can be exposed to lethally low water
temperatures during winter months (Franks et al., 2013;Graham
et al., 2017;MaceIIIet al., 2020;Rickards,1968). Mace III et al.
(2017) investigated the thermal threshold requirements for juve-
nile tarpon in South Carolina estuaries and reported that their find-
ings, combined with all published data on the cold tolerance of
juvenile tarpon, resulted in an overall mean ± S.D. minimum lethal
temperature of 12.0 ± 2.8C. However, there is limited information
on the synergistic effects that salinity and temperature can have
on tarpon haematology, osmoregulation, and survival. Blood chem-
istry can be used to measure physiological well-being under differ-
ent environmental conditions (Dinken et al., 2020;Fajt&
Grizzle, 1998;Kirk,1974), and physiological adaptive capacity
plays a central role in responses to environmental change (Young
et al., 2006). The objective of this study was to examine the
combined effects of water temperature and salinity on the blood
physiochemical variables, osmoregulation and survival of juvenile
tarpon. Experimental temperatures and salinities selected for the
study (15 and 25C, ≤2and≥30 ppt) were based on environmental
metrics associated with reported juvenile tarpon collections in US
GOM and South Atlantic coastal habitats (Graham et al., 2017;
Mace III et al., 2020;SteinIIIet al., 2016).
2|MATERIALS AND METHODS
2.1 |Ethics statement
The capture and research use of wild tarpon for this project was per-
mitted by the Mississippi Department of Marine Resources (permit
no. SRP-010-22). In addition, research methodology and procedures
were conducted in compliance with the Mississippi State University
Institutional Animal Care and Use Committee.
2.2 |Fish source and acclimation
Wild juvenile tarpon (n=50) were collected from Mississippi
Sound tidal sloughs in September and October 2020 using mono-
filament cast nets (0.9–3.0 m radius, 0.9–1.6 cm mesh). Salinity in
these habitats is variable and known to range from <1 to >30 ppt
(Graham et al., 2021). The specimens were immediately placed in a
45 l holding container with aerated water from the collection sites,
transferred to an aquaculture facility located at the University of
Southern Mississippi, Gulf Coast Research Laboratory, then trans-
ported to Mississippi State University's South Farm Aquaculture
Facility for experimentation. Fish were reared over a period of
10 months in large (3600 l) recirculating aquaculture systems (RAS)
where they were fed to satiation initially daily and after several
months every other day using small pieces of fresh catfish, Icta-
lurus spp. During this period, temperature was maintained at 23C
and salinity at 19 ppt. Following this period and 2 months prior to
experiments, conditions were held at 28Cand16pptfora
1 month period, then conditions were gradually adjusted to 25C
at a rate of 1C/day and to either ≥30 or ≤2 ppt (n=2 tanks/treat-
ment) at a rate of 1 ppt/day.
All fish were implanted with a passive integrated transponder
(PIT) tag (8 mm long 1.4 mm in diameter, 0.03 g, 134.2 kHz, ISO
FDXB; Biomark Inc., Boise, ID, USA) in the left dorsal musculature
anterior to the dorsal fin using a MK165 implanter (Biomark Inc.) with
a 16 gauge, 50 mm stainless-steel injector needle (N165; Biomark
Inc.). Before implantation, food was withheld for 48 h and fish were
anaesthetized in a buffered anaesthetic bath (150 mg l
1
tricaine
methanesulfonate (MS-222), 9 g l
1
NaCl and 400 mg l
1
NaHCO
3
),
then measured for total length (nearest mm) and weighed (wet, near-
est 0.01 g).
Fish were held in four 3600 l RAS and acclimated to 25C, with
two tanks maintained at ≤2 ppt and two tanks maintained at ≥30 ppt
2COFFILL-RIVERA ET AL.
FISH

for >2 weeks. Following acclimation, blood was collected from fish in
the treatment tanks (n=7 fish/tank). Afterwards, temperatures were
decreased at a rate of 1C/day in all tanks until 15C was reached,
and blood was collected again (n=7 fish/tank) after fish were accli-
mated for >2 weeks. This methodology provided duplicate tanks for
each treatment combination. Water quality parameters (temperature,
dissolved oxygen and salinity) were monitored approximately every
other day (Pro2030; YSI Inc., Yellow Springs, OH, USA).
2.3 |Blood collection
Fish were anaesthetized in a buffered bath (same as above), mea-
sured for total length (nearest mm) and weighed (wet, nearest
0.01 g). Fish were placed into the supine position onto a wet sur-
face and blood samples (0.5 mL) were collected from the caudal
vein using a heparinized syringe or vacutainer and a 22–25 gauge
hypodermic needle. Blood was then transferred to a 1.5 ml micro-
centrifuge tube and placed onto ice. Immediately following collec-
tion, pH was measured in whole blood using a microelectrode
(Accumet AB15; Fisher Scientific, Hampton, NH, USA) in a water
bath set at the treatment temperature. Haematocrit (Hct), haemo-
globin (Hb) and red blood cell count (RBC) were also measured
from whole blood. Hct was measured by filling blood into a 75 mm
heparinized capillary tube (Drummond Scientific Company, Broo-
mall, PA, USA), centrifuging at 6000 gfor 5 min and read using a
microcapillary tube reader as the ratio of red blood cells to the
total volume of blood. Hb was analysed with Drabkin's reagent
(Sigma-Aldrich; St. Louis, MO, USA) and RBC concentration was
determined at a 1:200 dilution with saline using a haemocytometer
and an automated cell counter (Cytosmart; Corning Cell Counter,
Glendale, AZ). From the whole-blood samples, 50–200 μlwas
transferred into a 0.6 ml microcentrifuge tube and centrifuged for
3 min at 5000 g. Plasma was collected, stored at 80Candlater
used to measure osmolality via a vapour pressure osmometer
(Vapro 5520; Wescor Inc., Logan, UT, USA).
2.4 |Loss of equilibrium
Following blood collection at 15C, temperatures were maintained
at 15C for an additional 3 weeks before loss of equilibrium (LOE)
experimentation. After 2 weeks, or approximately 1 week before
experimentation, most fish stopped feeding, presumably due to
exposure to the prolonged cool temperature. During this period,
mortality occurred in both treatments, with approximately 20%
mortality at ≥30 ppt and approximately 70% mortality at ≤2 ppt.
Mortality events during this time affected sample size availability
for LOE experiments.
For LOE experiments, four or five fish were transferred into
two 950 l, 1.5 m diameter tanks per trial. Fish were placed in the
tanks at the same salinity and temperature as the acclimation tanks
the night prior to experiments (17:00–21:00). During the night,
water was recirculated to the two tanks, which drained into a 340 l
sump tank where a 1700 W heater maintained water temperature
at 15C. A magnetic-drive pump moved water from the sump tank
to each of the two test tanks. Tanks were covered by a net and
blue 1.3 cm foam board insulation, which reduced light and exter-
nal stimuli, and helped to maintain water temperature. Each tank
had a 0.5 horsepower, 115 V chiller with a programmable thermo-
stat (Cyclone Drop-In; Aqualogic Inc., San Diego, CA, USA), and air
stones supplied with forced air. The water temperature in the
tanks was decreased at 0.9 ± 0.1Ch
1
starting at 08:00–09:00 on
the following day.
For both high salinity (n=20 fish) and low salinity (n=8 fish)
treatments, temperature and dissolved oxygen were measured every
30 min. Fish were monitored initially every 30 min and at approxi-
mately 10 min intervals after the first fish lost equilibrium. LOE was
TABLE 1 Mean (±s.e.) water quality parameters during the acclimation period of Atlantic tarpon Megalops atlanticus
Acclimation period Trt-Temp Trt-Sal DO (mg/L) DO (%) Temp (C) Salinity (ppt)
Week 1 High High 6.36 ± 0.06 88.8 ± 1.0 23.6 ± 0.6 30.6 ± 0.9
Week 2 6.08 ± 0.09 89.3 ± 1.5 25.0 ± 0.1 30.7 ± 0.9
Sample day 6.04 ± 0.15 89.9 ± 0.4 25.3 ± 0.2 30.0 ± 0.3
Week 1 High Low 7.18 ± 0.04 85.8 ± 0.8 23.8 ± 0.1 2.0 ± 0.2
Week 2 7.04 ± 0.02 87.5 ± 0.1 25.3 ± 0.3 1.0 ± 0.0
Sample day 7.08 ± 0.19 89.7 ± 1.8 25.7 ± 0.3 1.0 ± 0.0
Week 1 Low High 7.63 ± 0.14 94.5 ± 2.2 16.2 ± 0.4 31.2 ± 0.3
Week 2 8.25 ± 0.11 99.0 ± 1.2 14.8 ± 0.0 31.3 ± 0.2
Sample day 8.93 ± 0.33 107.3 ± 4.3 14.5 ± 0.0 31.4 ± 0.0
Week 1 Low Low 8.96 ± 0.00 92.7 ± 0.4 16.5 ± 0.1 1.1 ± 0.0
Week 2 9.93 ± 0.05 99.2 ± 0.2 14.9 ± 0.1 1.2 ± 0.0
Sample day 10.57 ± 0.03 105.1 ± 0.0 14.3 ± 0.2 1.1 ± 0.0
Note: Water samples were collected from two rearing tanks for each treatment seven to 12 times during acclimation. DO, dissolved oxygen; Sal, salinity;
Trt, treatment; Temp, temperature.
COFFILL-RIVERA ET AL.3
FISH

determined by an inability to maintain an upright position in the water
column. When a fish lost equilibrium, the water temperature in the
tank was measured and the fish was quickly removed, scanned for PIT
tag number and released into the acclimation tank. Dissolved oxygen
concentration during the LOE experiments was maintained above
7mgl
1
and above 80% saturation for both salinity treatments.
2.5 |Statistical analyses
Statistical analyses were performed using Rstudio 4.0.3 (R Core
Team, 2022) at a significance level of α=0.05. Normality and homo-
geneity of variance were tested on residuals with Shapiro–Wilk and
Levene's tests, respectively. Data were log
10
transformed to meet
parametric assumptions, as needed. A two-way analysis of variance
(ANOVA), with factors of temperature (15 and 25C) and salinity (≤2
and ≥30 ppt), was used to analyse total length, weight and blood phy-
siochemical variables. Following a significant ANOVA, group means
were compared using Tukey's honestly significant difference test.
A Student's t-test was used to compare temperature at LOE between
different salinities (≤2 and ≥30 ppt). Data are reported as mean
± standard error (S.E.).
3|RESULTS
Water-quality variables in rearing tanks during the acclimation period
are shown in Table 1. There were no differences in total length
(F
1,52
=0.132, P=0.7179) or wet weight (F
1,52
=0.438, P=0.5110)
among temperature and salinity treatments at the beginning of experi-
mentation (Table 2).
For blood physiochemical variables, the interaction between
temperature and salinity was significant for pH (F
1,52
=24.433,
P< 0.001) (Figure 1a). The pH varied between salinity concentra-
tions at cold temperature (15C) but not at high temperature
(25C). The interaction between temperature and salinity was not
significant for Hb (F
1,42
=0.441, P=0.5100), Hct (F
1,52
=0.176,
P=0.6770), RBC concentration (F
1,24
=0.081, P=0.7785), mean
corpuscular volume (F
1,24
=2.213, P=0.1499), mean corpuscular
haemoglobin (F
1,24
=1.4982, P=0.2328), mean corpuscular hae-
moglobin concentration (F
1,42
=0.2931, P=0.5911) or plasma
osmolality (F
1,52
=1.451, P=0.2339). However, a main effect of
TABLE 2 Mean (±s.e.) blood characteristics of Atlantic tarpon Megalops atlanticus acclimated to 15 and 25C temperature and ≤2 ppt and
≥30 ppt salinity
Trt
temp (C)
Trt
salinity (ppt)
Total
length (mm)
Wet
weight (g) Hb (g/dL) Hct (%)
RBC (10
9
cells/L)
MCH
(pg/cell) MCV (fL)
MCHC
(g/dL)
15 ≤2 462 ± 12 888 ± 82 11.17 ± 1.77
a
29 ± 1
a
2.52 ± 0.15
a
45.3 ± 9.5 106 ± 11 39.5 ± 6.5
15 ≥30 425 ± 17 694 ± 91 12.52 ± 0.73
b
28 ± 2
a
2.32 ± 0.10
a
59.6 ± 1.1 125 ± 2 45.5 ± 2.4
25 ≤2 446 ± 14 743 ± 80 12.80 ± 0.54
c
36 ± 1
b
2.91 ± 0.12
b
45.4 ± 2.9 130 ± 4 35.4 ± 1.1
25 ≥30 420 ± 21 663 ± 94 12.96 ± 0.55
c
34 ± 1
b
2.79 ± 0.13
b
47.8 ± 3.4 124 ± 9 38.2 ± 1.2
Note: Due to partial clotting of blood prior to quantification of RBC concentration in some samples, the number of samples for that particular assay, and
calculations directly related (MCH and MCV) were reduced to n=4–10/treatment. Different letters (a, b, c) indicate statistical differences among
temperature and salinity treatments (ANOVA; P< 0.05, n=14/treatment). Hb, haemoglobin; Hct, haematocrit; MCH, mean corpuscular haemoglobin;
MCHC, mean corpuscular haemoglobin concentration; MCV, mean corpuscular volume; RBC, red blood cell count; Temp, temperature; Trt, treatment.
FIGURE 1 Mean (+S.E.) blood pH (a) and plasma osmolality (b) of
Atlantic tarpon Megalops atlanticus acclimated to different
temperatures and salinities. In (a) different letters indicate statistical
differences among treatment groups. In (b) different letters indicate
statistical differences between salinities, while the asterisk denotes
statistical differences between temperatures (both main effects)
(ANOVA, P< 0.05, n=14/treatment). ,≤2; , ≥30.
4COFFILL-RIVERA ET AL.
FISH

temperature was observed for Hct (F
1,52
=28.622, P< 0.001) and
RBC concentration (F
1,24
=8.349, P=0.0081), with lower Hct and
RBC concentration at 15Cthanat25
C(Table2). For plasma
osmolality, main effects of both temperature (F
1,52
=11.661,
P< 0.001) and salinity (F
1,52
=49.211, P< 0.001) were observed,
with higher plasma osmolality at 15Cthanat25
C, and higher
plasma osmolality at ≥30 ppt than at ≤2ppt(Figure1b).
For the LOE test, there was a significant difference between
salinity treatments (t
8.45
=2.90, P< 0.05). LOE occurred at a lower
temperature for fish acclimated to 30 ppt salinity and at a higher tem-
perature for fish acclimated to 1 ppt salinity (Figure 2).
4|DISCUSSION
Temperature and salinity are essential physical components forming
abiotic environmental conditions for aquatic fauna. Each fish species
has a range of optimal conditions (e.g., fundamental niche) typically
defining the habitats they occupy (Bacheler et al., 2009; Craig &
Crowder, 2002; Stevens et al., 2022), although habitats with condi-
tions outside of optimal ranges may be occupied due to the energetic
benefits of increased forage opportunities or refugia (e.g., realized
niche) (Brownscombe et al., 2022; Freitas et al., 2016). Temperatures
and salinities above and below the optimum range can affect meta-
bolic and immune mechanism performance, hormonal control, blood
composition, osmoregulatory energy expenditure, gill Na
+
/K
+
-ATPase
activity, feed consumption rate and efficiency, digestion times and
survival (Cuesta et al., 2005; Galkanda-Arachchige et al., 2021; Ims-
land et al., 2001; Kolok & Sharkey, 1997; Resley et al., 2006;
Sampaio & Bianchini, 2002; Saoud et al., 2007; Soegianto et al., 2017;
Stuenkel & Hillyard, 1981).
For euryhaline fishes, the capacity to regulate internal solutes in
the face of daily and seasonally changing environments is vital for
function and survival, particularly for those species inhabiting estua-
rine environments. In this context, blood plasma osmolality is an
excellent measure of overall regulatory ability and well-being (Kolok &
Sharkey, 1997; Sampaio & Bianchini, 2002). In this study, the plasma
osmolality of juvenile tarpon was elevated at low temperature (15C
compared to 25C) and at high salinity (≥30 ppt compared to ≤2 ppt).
Although moderately increased osmolality in hyperosmotic environ-
ments is typical in fishes (Edwards & Marshall, 2013; Holmes &
Donaldson, 1969; Marshall, 2012), high blood osmolality can indicate
an osmoregulatory imbalance, which can relate directly to limitations
in habitat occupancy (Kammerer et al., 2010; McCormick et al., 1987;
Tsui et al., 2012). Low temperature has been shown to be problematic
for osmoregulation in fishes (Allen et al., 2017; Anderson &
Scharf, 2014; Thomsen et al., 2007) due in part to effects on cellular
membrane fluidity (Hochachka & Somero, 2002) and gill Na
+
/K
+
-
ATPase activity (Hansen et al., 2022; Sardella et al., 2007). The effects
of cold stress in warm-water euryhaline species may be reduced by
acclimation to seawater, in part through reduced effects on gill Na
+
/
K
+
-ATPase activity (Kang et al., 2015). The relative level of low tem-
perature where effects are incurred is species-specific and may be
exacerbated by short-term exposure to rapid decreases in tempera-
ture (Anderson & Scharf, 2014; Beitinger et al., 2000; Donaldson
et al., 2008). For juvenile tarpon, survival has been shown to diminish
at low temperatures under laboratory conditions (Mace III et al., 2017)
and during rapidly dropping temperatures from cold fronts in the wild
(Franks et al., 2013; Graham et al., 2017; Overstreet, 1974; Storey &
Gudger, 1936).
Haematological and blood physiochemical variables are helpful for
understanding osmoregulatory ability and adaptive capacity. Perhaps
the most informative blood variable in this study was pH, which was
highest at low temperature and low salinity but also elevated at low
temperature and high salinity. Acid–base balance, which is indicated by
blood pH, is extremely important to physiological processes, and
extreme pH has been related to temperature effects on survival in other
species (Dinken et al., 2022;Smitet al., 1981; Stewart et al., 2019).
Blood pH is known to generally relate inversely to temperature (e.g.,
decrease with increasing temperature) in water-breathing and air-
breathing fishes (Cameron, 1978; Howell et al., 1970;Rahn&
Baumgardner, 1972). In air-breathing fishes, blood pH is also related to
use of the air-breathing organ with decreased pH resulting from greater
reliance on air-breathing (Graham, 1997;Rahnet al., 1971). This is due
to a reduction of blood flow to the gills, the primary site of CO
2
excre-
tion, therefore increasing retention of CO
2
and reducing blood pH
(Shartau & Brauner, 2014). Thus, the observed increase in blood pH at
low temperatures in tarpon could be due to the inverse relationship
between temperature and blood pH, as well as diminished use of the
air-breathing organ at cold temperatures as evidenced in other fishes
with bimodal respiration (Damsgaard et al., 2018;Rahnet al., 1971;
Smatresk & Cameron, 1982). Although not quantified, our observations
indicated that air-breathing frequency in tarpon was greatly diminished
at 15C compared to 25C, which has also been reported by Geiger
et al.(
2000). Additionally, the highest blood pH at low temperature and
low salinity may be due partly to impaired osmoregulation at low tem-
perature (Smit et al., 1981), possibly manifested in part by reduced
internal HCO
3

exchange for external Cl

(Cameron, 1978). In air-
breathing fishes, although several studies have found increased blood
FIGURE 2 Mean (+S.E.) loss of equilibrium (LOE) of Atlantic
tarpon Megalops atlanticus acclimated to different salinities. Asterisk
indicates statistical differences between salinity treatments (t-test,
P< 0.05, n=8 for 1 ppt and n=20 for 30 ppt)
COFFILL-RIVERA ET AL.5
FISH

pH with decreasing temperature (Damsgaard et al., 2018;Gam
et al., 2020; Smatresk & Cameron, 1982; Vinh Thinh et al., 2018), the
effects of extreme low temperatures and concomitant salinity change
on blood pH are not well known.
Several other blood variables also had diminished values at
low temperature, with decreases in RBC concentration, Hct and
Hb, with the lowest value of Hb in the low-temperature and low-
salinity treatment. These measures all relate to blood oxygen
capacity, although lower temperatures are typically associated
with higher oxygen availability and a decreased metabolic oxygen
demand in ectothermic species. Changes in blood composition due
to temperature, salinity or a combination of both have been
reported for various fishes (Adeyemo et al., 2003;Soegianto
et al., 2017;Toneys&Coble,1980; Ziegeweid & Black, 2010).
Interestingly, salinity had no effect on haematological variables at
the higher temperature treatment (25C), likely because Megalops
spp. are broadly adapted to tropical and subtropical temperatures
(>20C) (Chen et al., 2008;Luoet al., 2020; Luo & Ault, 2012).
Results from the LOE experiment indicate tarpon can tolerate lower
temperatures under a hyper-osmotic environment (≥30 ppt) than in a
hypo-osmotic environment (≤2 ppt), as evidenced in other fishes
(Anweiler et al., 2014;Atwoodet al., 2001;Zale&Gregory,1989). In pre-
dominantly marine species, this may be partially due to the increased diffi-
culty in osmoregulating in a hypo-osmotic environment, which leads to a
passive osmotic influx of water and diffusive loss of ions, which can lead
to mortality (e.g., red drum Sciaenops ocellatus,Crockeret al., 1981;cobia
Rachycentron canadum,Resleyet al., 2006; rabbitfish Siganus rivulatus,
Saoud et al., 2007; red snapper Lutjanus campechanus, Galkanda-
Arachchige et al., 2021). Our results are similar to prior lower thermal tol-
erance studies conducted on smaller (length range 83–239 mm standard
length) juvenile tarpon (mean ± S.D. 12.0 ± 2.8C; Mace III et al., 2017).
Euryhaline capabilities allow juvenile tarpon to inhabit many
estuarine habitats regardless of ambient salinity. Juveniles are able
to maximize prey availability and seek refuge from predators, cool
temperatures and natural events (e.g.,stormsandplanktonblooms)
by moving across salinity gradients. Osmoregulation in fishes can
have high energy demands (Bœuf & Payan, 2001), therefore the
ability to regulate plasma osmolality is an important physiological
mechanism that allows fish to perform routine activities under
varying environmental salinities.
This research concludes that low salinity has a synergistic effect
with low temperature on the survival of juvenile tarpon. Juveniles can
tolerate lower temperatures when inhabiting hyper-osmotic environ-
ments, suggesting they can increase overwintering survival by utilizing
these habitats during winter months when temperatures drop <20C
in the latitudinal range of their nursery distribution. Alternatively, mor-
tality may be high if juvenile tarpon are trapped in low-salinity habi-
tats during extreme cold weather events.
AUTHOR CONTRIBUTIONS
J.W.N., S.B.C. and P.J.A. contributed to funding and project con-
ception/design. M.E.C.-R., Y.D.P.M., L.L. and P.J.A. contributed to
data generation, graphic design and data analysis. P.M.G. and
J.S.F. collected the fish and contributed ecological insight. All
authors contributed to the data interpretation and manuscript
preparation.
ACKNOWLEDGEMENTS
We thank the NOAA Puerto Rico Sea Grant Program (Grant
no. 2020-2021-007) and Mississippi State University's Undergraduate
Research Scholars Program (URSP) program for funding. We also
thank the Mississippi Agricultural and Forestry Experimental Station
and the US Department of Agriculture (USDA) Agricultural Research
Service (Grant no. 58-6066-5-042) for facility funding, and USDA
National Institute of Food and Agriculture (Grant no. 1005154 to
P.J.A. and Project no. MISZ-081700 to S.B.C.). We also thank Jacob
A. Moreland, Abby J. Vaughn, Joshua J. Neary, Brandon J. Gerhart,
Amanda Daulong, Alicia Santiago, McKinley Owens and the South
Farm Aquaculture Facility staff for their assistance and support with
fish care and experiments. We acknowledge staff of the University of
Southern Mississippi Thad Cochran Marine Aquaculture Center for
the temporary holding and maintenance of the juvenile tarpon imme-
diately following their capture. Finally, we thank the two anonymous
reviewers and assistant editor for their suggestions, which improved
the manuscript.
ORCID
Manuel E. Coffill-Rivera https://orcid.org/0000-0003-0284-0030
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How to cite this article: Coffill-Rivera, M. E., Paez Mendez,
Y. D., Little, L., Graham, P. M., Franks, J. S., Correa, S. B., Neal,
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