Gopherus agassizii , Mojave desert tortoise, ectotherm, Draft · tortoises resume inactivity but...
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Temperature-independent, seasonal fluctuations in
immune-function in a reptile, the Mojave desert tortoise (Gopherus agassizii)
Journal: Canadian Journal of Zoology
Manuscript ID cjz-2016-0010.R1
Manuscript Type: Article
Date Submitted by the Author: 18-Apr-2016
Complete List of Authors: Sandmeier, Franziska; Lindenwood University Belleville Campus, Biology
Horn, Kelly; University of Nevada, Reno, Department of Biology Tracy, C. Richard; University of Nevada, Reno, Department of Biology
Keyword: <i>Gopherus agassizii</i>, Mojave desert tortoise, ectotherm, ecoimmunology, temperature-independent
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Canadian Journal of Zoology
Temperature-independent, seasonal fluctuations in immune-function in a reptile, the
Mojave desert tortoise (Gopherus agassizii)
F.C. Sandmeier1, K.R. Horn2, C.R. Tracy3
1. Department of Biology, Lindenwood University-Belleville, 2600 W Main St., Belleville,
Illinois, 62223, USA; [email protected]
2. Biology Department, University of Nevada, Reno, Mailstop 314 1664 N Virginia St, Nevada
89557, USA; [email protected]
3. Biology Department, University of Nevada, Reno, Mailstop 314 1664 N Virginia St, Nevada
89557, USA; [email protected]
Correspondence to: F.C. Sandmeier; Department of Biology, Lindenwood University-
Belleville, Belleville, Illinois, 62223, USA; phone: (775) 750-4092; fax (618) 277- 6001; email:
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Temperature-independent, seasonal fluctuations in immune-function in a reptile, the
Mojave desert tortoise (Gopherus agassizii)
F.C. Sandmeier, K.R. Horn, C.R. Tracy
Abstract
As long-lived reptiles, Mojave desert tortoises (Gopherus agassizii, Cooper 1861) are
expected to make substantial energetic investments in immune-defense. This species also
has many adaptations to living in an arid environment characterized by seasonal extremes
in temperature and resource-availability. By housing tortoises at a controlled, constant
ambient temperature, we quantified predominantly temperature-independent seasonal
fluctuations in innate immune function and circulating leukocytes in a reptile. We found a
decrease in bacteriocidal activity of the blood plasma in winter, with reduced function
lasting into the spring. Lymphocyte numbers were elevated in fall and winter, while
eosinophil numbers increased in summer. Thus, properties of the immune system were up
or down-regulated in different directions across the seasons. We found a much higher level
of variation of leukocyte profiles among individuals than has previously been reported for
other chelonians. Heterophil:lymphocyte ratios (indicative of chronic glucocorticoid levels)
were not associated with any measure of immune function, and thus glucocorticoid does
not seem to mediate the observed seasonal changes. We propose a new hypothesis to
explain seasonal changes in immune function, based on seasonal resource-limitation in the
Mojave Desert – including the availability of dietary protein, energy, and opportunities for
thermal regulation.
Key words: Gopherus agassizii; Mojave desert tortoise; ectotherm; ecoimmunology;
temperature-independent
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Introduction
Because immune defense often incurs some cost to an organism, seasonal trade-offs
with other physiological functions – such as reproduction, migration, etc. – appear to be
common in vertebrates (Lochmiller and Deerenber 2000; Martin et al. 2008; Weil and
Nelson 2012). Most studies in ecoimmunology have focused on endothermic vertebrates,
and the current theory of temperature-independent, seasonal changes in immune function
explains how small-bodied mammals up-regulate immune functions in the winter (Demas
and Nelson 2012; Weil and Nelson 2012). In contrast, research on seasonal immune
fluctuations in ectothermic vertebrates has focused on the effects of temperature on
immune-activity, especially in amphibians from temperate climates (Maniero and Carey
1997; Merchant et al. 2003; Raffell et al. 2006; Rollins-Smith and Woodhams 2012). One
important observation from this research is that seasonal acclimation to adjust immune
functions to cooler temperatures often occurs in fish and amphibians, sometimes with “lag
effects” when temperature changes rapidly (Raffel et al. 2006). While temperature drives
seasonality in some functions of the immune system, not all patterns of seasonal,
physiological change are explained by temperature in these ectotherms (Raffell et al. 2006;
Rollins-Smith and Woodhams 2012).
In reptiles, seasonal variation in adaptive immunity (induced, specific immune-
functions; sensu Martin et al. 2008) has long been recognized as being influenced by
temperature, but also by photoperiod and hormonal fluctuations (e.g., glucocorticoids and
sex hormones) - with different components of the immune system changing in different
directions throughout the year (Zapata et al. 1992; Muñoz and De la Fuente 2001; Origgi et
al. 2007). Most studies have not been able to differentiate between the effects of
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temperature and season. Such a differentiation may become increasingly important in wild
populations that are experiencing the effects of climate change and increased extremes in
temperature (Rohr and Raffel 2010). Additionally, evidence suggests that different
vertebrate species modulate seasonality in response to different environmental cues, but
the most reliable and predominant signals appear to be both temperature and photoperiod
(Rollins-Smith and Woodhams 2012; Weil and Nelson 2012). One of the only studies of
seasonal changes in innate immunity (constitutive, mainly non-specific immune functions;
sensu Martin et al. 2008) in a reptile described both seasonal up and down-regulation of
different immune-functions and showed that temperature alone cannot account for these
changes (Zimmerman et al. 2010b).
Like other chelonians (turtles and tortoises), the Mojave desert tortoise (Gopherus
agassizii, Cooper 1861) is a long-lived ectotherm, and therefore expected to invest
substantial resources in immune function – particularly in innate immunity (Zimmerman et
al. 2010a; Sandmeier and Tracy 2014). This species is a good candidate for the study of
seasonality in vertebrates, due to tortoises’ dramatic changes in physiology throughout the
year – mostly due to the Mojave Desert’s extreme seasonality in terms of temperature and
food availability (Nagy and Medica 1986, Peterson 1996a; Nagy et al. 1998). Activity and
field metabolic rates of tortoises are closely associated with each other and tied to
favorable environmental conditions, including availability of suitable thermal
environments, forage, and drinking water supplied by rare rainfall-events (Zimmerman et
al. 1994; Nagy and Medica 1986; Peterson 1996a; Agha et al. 2015). While drought reduces
all tortoise-activity, on average, tortoises emerge from hibernation at the beginning of
spring (late March/early April) as surface temperatures increase (Nagy and Medica 1986;
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Zimmerman et al. 1994; Peterson 1996a). Typically this is also the only time of the year
with availability of green forage, tortoises’ main source of dietary protein (Nagy and
Medica 1986; Peterson 1996a). In some regions, green forage may also become available
with summer monsoons, but this source of protein is less predictably available (Nagy and
Medica 1986). As surface temperatures increase at the beginning of summer (June),
tortoises resume inactivity but are able to remain within the range of preferred body
temperatures inside their burrows (Zimmerman et al. 1994; Snyder 2014). Surface
temperatures decrease at the beginning of fall (September), and tortoises may emerge
more frequently than in the summer to consume dry forage (Nagy and Medica 1986;
Peterson 1996a). As temperatures cool, most tortoises retreat to deep burrows to
commence winter brumation, or winter dormancy, at temperatures ranging from 6.5 –
16.3° C (Nussear et al. 2007).
Likely due to the extreme seasonal changes in the Mojave Desert, tortoises have the
ability to tolerate short-term seasonal “anhomeostasis” – or relatively extreme fluctuations
in body chemistry and blood osmolality (Woodbury and Hardy 1948; Nagy and Medica
1986; Peterson 1996b). For example, desert tortoises mainly accumulate protein from
springtime forage while experiencing a net loss in energy and increase in blood osmolality,
which is only reversed by obtaining water and dry forage later in the year (Peterson
1996b). Thus homeostatic balance is achieved in asynchrony, throughout the year.
Both blood corticosterone levels and field metabolic rates – often associated with
seasonal changes in vertebrates – have been quantified in desert tortoises, yet fluctuations
in immune function have not been previously studied (Martin et al. 2008; Sandmeier and
Tracy 2014). Importantly, G. agassizii is listed as threatened under the Endangered Species
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Act, and a respiratory disease has been implicated as a possible factor in population
declines (USFWS 1994). Knowledge of seasonal changes in immune-function is necessary
to more fully understand susceptibility to disease in this species. Here, we use a controlled
experiment to elucidate changes in immune function not driven by temperature, which in
the future can be compared to changes observed in natural populations that are subject to
different thermal regimes, but similar photoperiod regimes/seasonal cycles.
We used a colony of adult, captive tortoises to establish minimum, temperature-
independent seasonal fluctuations in multiple aspects of immune function. We manipulated
air temperature to be within the range of preferred body temperature (25 – 35° C), except
during 1.5 months at the start of winter when temperature was gradually brought down to
12-13° C to maintain hibernation (sensu Nussear et al. 2007; Snyder 2014). We mimicked
the natural photoperiod with UV and visible light lamps, and thus were able to test the
hypothesis that aspects of the immune system of the G. agassizii experience inherent,
predictable changes across the year. Animals were not actively breeding, thus also
eliminating the effects of reproductive investment competing with investments in immune
function. To test multiple aspects of the immune system, we measured innate bacteriocidal
activity of the blood plasma (“bacteriocidal activity”) and conducted differential white
blood cell (leukocyte) counts (Origgi 2007; Zimmerman et al. 2010b). Briefly, bacteriocidal
assays of blood plasma quantify the functional effect of proteins in the peripheral blood,
primarily natural antibodies and complement proteins, to kill or inhibit the growth of a
standard strain of Escherichia coli (French et al. 2010; Zimmerman et al. 2010a). Natural
antibodies and complement proteins are constitutively produced, can directly destroy a
wide variety of pathogens, and activate other branches of the immune system (Murphy
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2012). The predominance of different types of leukocytes indicates the strength of certain
types of immunological capabilities, such as antibody production (some lymphocytes:
plasma cells), phagocytosis (heterophils, monocytes, and some lymphocytes), and oxidative
responses (basophils and eosoinophils) (Strik et al. 2007; Zimmerman et al. 2010b; Murphy
2012). We also used heterophil:lymphocyte (H:L) ratios to be indicative of chronic levels of
corticosterone, which has previously been shown to be most closely associated with
activity levels and not chronic stress in desert tortoises (Davis et al. 2008, Inman et al.
2009; Drake et al. 2012).
Specifically, we tested five hypotheses. (1) There will be a general decrease in both
H:L ratios and bacteriocidal activity in the winter, consistent with an involution of immune
organs during winter in other species of turtles (Leceta and Zapata 1986; Muñoz et al.
2000). (2) In addition to larger changes during brumation/hibernation, there will be an
additional, significant variation in bacteriocidal activity across the active seasons (spring,
summer, and fall)– likely reaching a maximum in summer (Maniero and Carey 1997; Raffell
et al. 2006; Zimmerman et al. 2010a). If there is evidence of acclimation to cold winter
temperatures, there will not be a decrease in bacteriocidal activity in the spring. Conversely
if this type of innate immunity does not acclimate to functioning well in colder
temperatures, there may be a “lag effect” in immune-function in the spring (sensu Raffell et
al. 2006). (3) H:L ratios (indicative of corticosterone levels associated with activity/field
metabolic rate; sensu Drake et al. 2012) will be positively associated with bacteriocidal
activity, if favorable conditions are coupled to increased immune function. (4) White blood
cell profiles will change with season, with a general reduction in lymphocytes during
winter. (5) Desert tortoises will exhibit an inherently high individual variation in leukocyte
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profiles. Similar high individual variability has been measured for other physiological and
hematological values (Peterson 1996b).
Materials and Methods
Manipulation of photoperiod/temperature and sample-collection
Thirty captive, adult desert tortoises (14 females and 16 males) were used in this
experiment. All tortoises had been reared in captivity for 20-22 years, were never exposed
to wild tortoises, and had no known infections. Tortoises were offspring from parents
originating from wild populations in Nevada, and were hatched as part of two earlier
studies (Spotila et al. 1994; Lewis-Winokur and Winokur 1995). They represent one of
three genetic sub-groups of G. agassizii (“Las Vegas” genotype) (Hagerty and Tracy 2010).
The tortoises were fed a diet of alfalfa and green vegetables several times weekly, and once
weekly were offered water for hydration in a tub of shallow water. Animals were kept in an
indoor facility with a controlled environment, in cattle tanks that allowed for daily
movement and access to heat lamps (during daylight hours) and a cooler, covered burrow.
Each tank housed one to three tortoises, usually of the same gender. We sorted individuals
among tanks to minimize aggressive and mating behavior.
During spring, summer, and fall tortoises were held under a natural photoperiod
using lamps emitting both visible and UV light. Photoperiod was adjusted every three
months, with the onset of the seasons. From fall through summer, light:dark hours were
12:12, 10:14, 12:12, 14:10. The air temperature was kept between 25.5 and 28° C, which is
within the range of preferred body temperatures (25 – 35° C) of both captive and free-
living desert tortoises (Naegle 1976; Zimmerman et al. 1994; Snyder 2014). Hibernation
(at 12-13 °C) was gradually induced in December, and animals were allowed to hibernate
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for one and a half months in total darkness. Length of hibernation varies for both individual
tortoises and populations across their geographic range, and one and half months
represents a short period of hibernation (Nussear et al. 2007).
Blood samples (0.5 ml) were drawn from the subcarapacial sinus at the beginning of
each season, within one week of each equinox or solstice in September (2011), December
(2011), March (2012), and June (2012) (Hernandez-Divers et al. 2002). These time periods
were chosen because they represent noticeable changes in behavior and activity of free-
ranging tortoises (Nagy and Medica 1986; Zimmerman et al. 1994). One blood smear was
made per blood sample and stained with Wright-Giemsa (Jacobson et al. 2007). Whole
blood was collected in heparinized tubes, kept on ice, and centrifuged within two hours of
collection, and plasma was frozen at −30°C. All work was conducted in accordance with
permits issued by the Institutional Animal Care and Use Committee (A06/07-49), the
Nevada Division of Wildlife (S33080), and the U.S. Fish and Wildlife Service (TE076710-8).
Bacteriocidal assay
Standard protocol for bacteriocidal assays was followed (French et al. 2010;
Zimmerman et al. 2010a). We determined that a pooled sample of plasma from five desert
tortoises used in this study, killed roughly 50% of bacteria (unpubl. data). Briefly, 10 µl of
tortoise plasma was added to 10 µl of E. coli (ATCC #8739), equivalent to 500-600 colony-
forming units, and 180 µl of LB broth under sterile conditions. One negative control (10 µl
of LB broth) and one standard sample were run with each set of 15 samples of
experimental tortoise plasma. Samples were then incubated for 30 minutes at 37° C. After
incubation, two 75 µl portions were spread onto each of two separate agar plates. The agar
plates were then incubated overnight at 37° C. The total number of colonies was counted
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for each of the plates. The resulting number of E. coli colonies present on the sample plates
was subtracted from the mean number of colonies present on control plates and divided by
the control plate number to determine the proportion of bacteria killed by the plasma
(Zimmerman et al. 2010).
Differential white blood cell counts
We counted the first 100 white cells encountered for each sample and classified
cells as lymphocytes, heterophils, eosinophils, basophils, monocytes, or azurophils based
on their known morphology (Alleman et al. 1992; Jacobson et al. 2007). To differentiate
between lymphocytes and thrombocytes, a subset of blood samples were used to make a
second smear, which was stained with Periodic Acid-Shiff stain – which allowed for the
clear differentiation of lymphocyte and thrombocyte morphologies (Alleman et al. 1992;
Strik et al. 2007).
Statistical analyses
To verify that sex was indeed not affecting the immune system, we first tested
whether there was not a difference between females and males in H:L ratios, levels of all
types of white blood cells, and bacteriocidal activity, using repeated measure ANOVAs.
Since all analyses were insignificant (see Results), we excluded sex from any further
statistical analyses. A correlation matrix, and subsequent regression analyzes were used to
assess relationships among the different types of leukocytes. Because all leukocytes were
strongly correlated to lymphocyte numbers (Table 1), we were not able to perform a
MANOVA. Instead, repeated- measure ANOVAs were used to analyze differences in H:L
ratios and the quantity of each type of leukocyte among the four seasons. We used a
modified Bonferonni correction, according to the following formulas, to account for
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multiple comparisons among leukocytes and H:L ratios (Keppel 1991).
αFW = (degrees of freedom)(0.05)
α individual comparison = αFW /(number of comparisons)
Thus we used an αFW (familywise α) of 0.15 and an α of 0.025 for individual comparisons.
Tukey’s HSD tests were used for pair-wise comparisons when ANOVAs were significant.
A regression analysis, with repeated measures, was used to test whether H:L ratios
were associated with bacteriocidal activity. Since bacteriocidal assays involved just blood
plasma, and not leukocytes, we used a separate, repeated-measure ANOVA to test for
significance among seasons, with a Tukey’s HSD to test for pair-wise comparisons if the
ANOVA was significant. All analyses were run in JMP 10.0.2 (SAS Institute, Inc.) with a
significance level of α = 0.05, with the exception of Bonferonni-corrected multiple
comparisons.
We compared individual-variation in differential leukocyte counts with comparable
data available from a natural population of red-eared slider turtles (Trachemys scripta
Schoepff 1792), also sampled in late June (Zimmerman et al. 2013).
Results
All distributions of variables met assumptions of normality, except for H:L ratios,
which were square-root transformed to allow for parametric statistical analyses.
There were no differences between the two sexes in H:L ratios (F1, 35 = 0.511 p =
0.479), spring H:L ratios (F1, 35 = 1.081, p = 0.306), heterophils (F1, 35 = 1.081, p = 0.306),
basophils (F1, 28 = 1.651, p = 0.209), lymphocytes (F1, 35 = 1.365, p = 0.250), monocytes (F1, 35
= 1.371, p = 0.250), eosinophils (F1, 35 = 1.779, p = 0.191), nor bacteriocidal activity (F1, 27 =
0.301, p > 0.5).
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H:L ratios were significantly different among the four seasons (F3, 21 = 3.332, p =
0.025) (Fig. 1a). Among seasons, spring differed from winter (p = 0.037), but other pair-
wise comparisons were not significantly different. Only lymphocytes (F3, 21 = 6.250, p <
0.001) and eosinophils (F3, 21 = 4.434, p = 0.004) differed among seasons (Fig. 1b, c).
Lymphocytes were significantly higher in fall than in spring and summer (p = 0.003; p =
0.046) and in winter than in spring (p =0.009) (Fig. 1b). Eosinophils were higher in
summer than in fall and winter (p = 0.009; p = 0.025) (Fig. 1c). Heterophils, basophils, and
monocytes did not vary with seasons (F3, 21 = 1.308, p = 0.280; F3, 21 = 1.489, p = 0.226; F3, 21
= 2.661, p = 0.056, respectively). Only lymphocytes were correlated with other cell types in
a correlation matrix (Table 1). In a repeated measures, multiple regression model,
heterophils, basophils, monocytes, and eosinophils all were significant predictors of
lymphocytes, and negatively correlated to lymphocyte numbers (adjusted R2 = 0.988, p =
0.001). In all repeated-measures ANOVAs, the identity of the animal was always significant
(p < 0.05). Similarly, we found high individual variation for numbers of specific leukocytes,
and June values are compared those reported for T. scripta in Table 2.
Bacteriocidal activity was significantly different among seasons (F3,16 = 4.591, p =
0.007), with winter levels lower than those in summer (p = 0.031) and fall (p = 0.006)
levels (Fig. 2). In a repeated-measures regression, H:L ratios were not associated with
bacteriocidal activity (p = 0.980).
While the pooled, tortoise plasma used to optimize the bacteriocidal assay killed
about 50% of E. coli cells, the unexpectedly high variation in killing ability among
individuals is evident in the much lower mean bacteriocidal activity of all animals included
in the study. In fact, some bacteria grew better in tortoise plasma and media than in the
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media alone (leading to negative values). We were limited by the amount of blood we were
able to draw from each animal, but in the future may adjust parameters of this assay to
reflect the generally low bacteriocidal activity seen in this population of tortoises.
However, results for each individual tortoise were highly reproducible, and were only
included in analyses if both the negative and positive controls were consistent with
expectations and showed no evidence of contamination.
Discussion
We quantified minimal, seasonal rhythms in immune-function in captive tortoises
provided with optimal levels of resources and exposed to a natural photoperiod. We were
able to sample most tortoises at all time-points, including during brumation and behavioral
inactivity in burrows. Studies on wild tortoises thus far have not been able to sample
tortoises during inactivity in deep burrows (such as during brumation), and this may
explain some differences between the results of our study and those focused on wild
animals (Christopher et al. 1999; Lance et al. 1999; Drake et al. 2012). We detected no
difference between the sexes, likely due to reduced reproduction/reproductive behavior.
Test of our hypotheses
(1) Decrease of both H:L ratios and bacteriocidal activity in the winter - We found a general
decrease in H:L ratios and bacteriocidal activity in the winter (Figs. 1a, 2). As expected, H:L
ratios, indicators of circulating levels of corticosterone, decreased in winter with lower
body temperature, lower metabolic rates, and no behavioral activity (Fig. 1a; Peterson
1996a; Davis et al. 2008; Drake et al. 2012). The production of complement proteins is
known to be sensitive to low temperature in other species, consistent with our observation
of low bacteriocidal activity in winter (Green and Cohen 1977; Hyaman et al. 1992;
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Maniero and Carey 1997).
(2) Variation in bacteriocidal activity across the active season: evidence of acclimation or a
“lag effect” in spring - This hypothesis was only partially supported. Although there was a
trend in increasing bacteriocidal activity throughout the active season with relatively low
levels in the spring, levels in summer and fall were only significantly different from those in
winter (Fig. 2). Additionally, unlike in T. scripta, levels did not drop in fall (Zimmerman et
al. 2010a). Low bacteriocidal activity in the winter suggests that acclimation does not
occur, and somewhat suppressed bacteriocidal activity in the spring indicates that there is
a weak “lag-effect” in the spring (Maniero and Carey 1997; Raffel et al. 2006; Rohr and
Raffell 2010). Possibly, both the temperature-dependent depression of complement
production in the winter and lower lymphocyte levels in the spring (potentially reducing
antibody-production) are responsible for continued, low bactericidal activity in spring.
(3) Association between H:L ratios and immune function - H:L ratios did not correlate with
bacteriocidal activity. This indicates that higher metabolic rates do not appear to be
positively associated with this immune function in tortoises. Neither did we detect
evidence of seasonal stress nor a negative association beween H:L ratios and immune
function. Unlike in many other seasonally-active animals, corticosterone levels in desert
tortoise were shown to fluctuate very little throughout the active season in a three-year
study (Drake et al. 2012). The closely-related gopher tortoise (Gopherus polyphemus,
Daudin 1802) also shows no significant variation in corticosterone across the active season
(Ott et al. 2000). Thus, corticosterone may not modulate seasonality in tortoises, including
seasonality in immune function, in the same ways as has been described for mammals
(McEwen et al. 1997; Martin et al. 2008; Drake et al. 2012).
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(4) Variation in white blood cell profiles among all seasons, with a general reduction in
lymphocytes during winter - This hypothesis was partially supported, and we only found
significant variation in relative numbers of lymphocytes and eosinophils (Figs. 1b, c).
Unexpectedly, lymphocytes were significantly elevated in both fall and winter, in contrast
to what has been reported for free-ranging desert tortoises (Christopher et al. 1999) and
other species of chelonians (Leceta and Zapata 1985; Muñoz et al. 2000; Wilkinson 2004;
Strik et al. 2007; Zimmerman et al. 2013). In the closely related Sonoran desert tortoise
(Gopherus morafkai, Murphy et al. 2011), which experiences higher food and water
availability in its geographic range, lymphocyte levels also increase in fall (Christopher et
al. 1999; Dickinson et al. 2002). Possibly, the ad libitum availability of food and water in
our experiment may have influenced maximum levels of lymphocytes. Indeed, lymphocyte
levels in wild G. agassizii also decrease in years with low rainfall, and thus may be
especially sensitive to food-availability (Christopher et al. 1999). Eosinophil numbers were
significantly greater in summer than in fall and winter, similar to patterns reported for
amphibians, but not reptiles (Christopher et al. 1999; Wilkinson 2004; Raffel et al. 2006;
Strik et al. 2007) (Fig. 1c).
Therefore, we found weaker seasonal patterns than expected from studies done on
wild desert tortoises (Christopher et al. 1999), but a stronger pattern of different immune
cells and innate immune functions changing in different directions with each other across
the year. This trend has also been found in other species of turtles (Muñoz and De la Fuente
2001; Origgi 2007; Zimmerman et al. 2010). We also found much higher levels of
eosinophils than reported in Alleman et al (1992) – a study based on a group of wild
tortoises from the driest region of the Mojave Desert (the season of sampling was not
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reported). Variation among values reported from wild populations highlights the
importance of using captive animals, housed under controlled environmental conditions, to
interpret possible effects of site and season on these baseline values (Alleman et al. 1992;
Christopher et al. 1999; Wilkinson 2004).
(5) High individual variation in immune parameters measured - Desert tortoises showed
extremely high individual variation in levels of leukocytes, much higher than that reported
for T. scripta (Table 1) (Zimmerman et al. 2013). In fact, without our relatively large sample
size, many of the patterns that we quantified for populations of tortoises would not have
been detectable. Similar high individual variation has been documented for other
hematological values, and may be a common characteristic of desert tortoises (Peterson et
al. 1996a, b; O’Connor et al. 1994). Whether this variation is genetically determined or due
to phenotypic variation is unknown. Given the high annual variation of conditions in the
Mojave Desert, it is possible that the survivorship of young is dependent on their
physiology to survive extreme conditions (Hereford et al. 2006). If physiological trade-offs
exist among the abilities to survive such conditions as disease, predation, drought, or low
nutrient availability, annual environmental conditions may influence the recruitment of
different phenotypes of tortoises across time, leading to high phenotypic variation in the
relatively resilient adult population (e.g., Peterson 1996b).
New model for seasonal variation in tortoise immune function
Bacteriocidal activity, lymphocytes, and eosinophils all reached their highest levels
during different times of the year, and we hypothesize these seasons correspond to times
during which specific resources – protein, energy, temperature – are available to maximize
specific immunological functions. Specifically, protein availability in the diet occurs in
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spring, the availability of dry forage and water to increase energy stores occurs in summer
and fall, and the ability to reach optimal thermal preference occurs in late spring through
the early fall (Nagy and Medica 1986; Zimmerman et al. 1994; Peterson 1996a; Snyder
2014). Thus, various immune functions are up-regulated according to their reliance on
specific resources: antibody-production requires protein-availability, oxidizing reactions
and inflammation (which may involve eosinophils) have high energy demands,
complement activity functions best at warmer temperatures, and lymphocyte proliferation
requires energy and the availability of protein, lipids, and other structural “building blocks”
of cells (Maniero and Carey 1997; Murphy 2012). We know that desert tortoises temporally
separate the acquisition protein, the acquisition of chemical energy, and homeostatic
balance (Peterson 1996a, b). Our model – described in detail below - suggests that desert
tortoises are so constrained in the acquisition of seasonally-available resources, that they
also de-couple necessary immune activities by season.
In this model, tortoises forage aggressively for protein-rich foods in spring, in lieu of
up-regulating any aspect of the immune system (Fig. 1a; Peterson 1996a; Tracy et al. 2006;
Inman et al. 2009). Even though our captive animals were offered food throughout the
year, their H:L ratios (indicative of behavioral and physiological activity) were highest in
the springtime.
In summer, wild tortoises can optimize body temperatures for most of the day, and
they can accumulate additional energy resources in dry forage (Peterson et al. 1996a;
Snyder 2014). At the same time, they increase eosinophil numbers, which are thought to
reduce parasite-loads (Pasmans et al. 2001; Strik et al. 2007). (Fig. 1c). Eosinophils
function in respiratory bursts (oxidative reactions) and inflammation, which are expensive
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in terms of oxidative stress, damage to host tissue, and energy (Murphy 2012). In snapping
turtles, eosinophils also are phagocytic, and they may also be important phagocytes in
tortoises during this time of year (Strik et al. 2007). Tortoises up-regulate complement
proteins and/or antibody production, leading to elevated bacteriocidal activity (Fig. 2).
Lymphocyte levels may start to increase during this time as well.
During fall, animals have acquired all the nutrition available over the active season
and achieving preferred body temperature becomes difficult, and they enter cooler
burrows for longer proportions of the day (Snyder 2014). Eosinophil numbers decrease in
the fall, consistent with these cells’ inability to acclimate, or to function efficiently, at cool
temperatures (Plytycx and Jozkowicz 1994). Lymphocytes have longer lifespans than other
white blood cells (weeks-months), and their up-regulation in the fall may ensure high cell
populations in the winter as well (Murphy 2012). This may be especially important if some
functions of lymphocytes can acclimate to cooler temperatures (Zimmerman et al. 2010c).
For example, NK cells (a type of lymphocyte) reach high levels in hibernating pond turtles,
and some turtle B lymphocytes are known to be phagocytic (Muñoz and De la Fuente 2001;
Zimmerman et al. 2013). It is now important to know if the phagocytic activity involving
these B lymphocytes acclimates to winter temperatures (Zimmerman et al. 2012). In
addition, if antibody production continues in winter at a reduced level, it may provide
protection to the hibernating animal at a relatively low energetic cost (Buehler et al. 2008).
Our model emphasizes that the tortoise immune system is controlled by more
factors than temperature alone. However, we still know little about the controls and effects
of this seasonality in wild populations. One caveat of our hypothesis is that it excludes
possible seasonal changes in the abundance of different pathogens – which currently is not
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known for the desert tortoise (Altizer et al. 2006; Rollins-Smith and Douglas 2012).
Another necessary next step is to quantify the functional activity of different immune
parameters of wild desert tortoises (e.g., bacteriocial activity, phagocytosis, etc.)
experiencing natural temperature patterns and to test for temperature-by-season
interactions on immune function (Raffell et al. 2012; Zimmerman et al. 2010a). In addition,
the physiological control of seasonality in tortoises is not understood. We do not
understand the extent to which it depends on photoperiod and whether that dependence is
mediated by a hormone such as melatonin (Weil and Nelson 2011).
Our study emphasizes the importance of expanding research to a diversity of
organisms to understand broader physiological mechanisms. Reptiles in general are under-
represented in the immunological literature (Zimmerman et al. 2010b). Chelonians
comprise an interesting group of study organisms, as different species exhibit extreme
physiological adaptations to the environment, such as anoxia in T. scripta and
“anhomeostasis” in water and energy balance in the desert tortoise (Peterson et al. 1996b;
Shaffer et al. 2013). Understanding how these patterns and adaptations in physiology
relate specifically to immune function in natural environments is an unexplored field in
ecological immunology.
Acknowledgements
The authors thank John Gray for assistance with animal care and the collection of blood
samples and Lindsay Parton and Kristen Pietrzyk for assistance with laboratory work.
Funding was provided by the Bureau of Land Management (USA); United States federal
grant number L11C20384.
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Tables
Table 1. Correlation matrix of leukocyte numbers. Bold indicates significance at α = 0.05.
Leukocyte Heterophils Basophils Lymphocytes Monocytes Eosinophils
Heterophils 1.0000 -0.0620 -0.4358 0.0737 0.0634
Basophils -0.0620 1.0000 -0.5704 0.0340 0.0046
Lymphocytes -0.4358 -0.5704 1.0000 -0.5966 -0.4037
Monocytes 0.0737 0.0340 -0.5966 1.0000 -0.0011
Eosinophils 0.0634 0.0046 -0.4037 -0.0011 1.0000
Table 2. Blood cell counts (specific leukocyte/100 leukocytes counted) in summer
compared between G. agassizii (n = 22) and T. scripta, both sampled in late June
(replicated, with permission from Zimmerman et al. (2013).
G. agassizii T. scripta
Leukocyte Mean SEM Range Mean SEM Range
Lymphocyte 37.7 3.88 (6-75) 43.66 1.29 (40-56)
Basophil 23.6 2.51 (8-54) 5.25 0.23 (4-7)
Heterophil 14.6 1.33 (6-26) 38.4 0.81 (33-42)
Monocyte/azurophil 12.95 2.15 (3-44) 7.23 0.57 (5-11)
Eosoinophil 11 1.61 (0-32) 6.21 0.33 (4-8)
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Figure legends
Fig. 1. Three measures of white blood cell counts were significantly different among
seasons in repeated-measures ANOVAs (displayed as mean diamonds: the broadest line of
each diamond represents the mean and the height of the diamond represent 95%
confidence intervals) (n = 22). We considered analyses significant at p = 0.025, due to a
modified Bonferonni correction for multiple comparisons. Letters indicate similarity by
Tukey’s HSD pair-wise comparisons. (a) H:L ratios (F3,21 = 3.332, p = 0.025), with spring
ratios significantly elevated over winter ratios (p = 0.037). (b) Number of lymphocytes (per
100 leukocytes counted) (F3,21 = 6.250, p < 0.001). (c) Number of eosinophils (per 100
leukocytes counted)(F3,21 = 4.434, p = 0.004).
Fig. 2. Bacteriocidal activity was significantly different by season in a repeated measures
ANOVA (F3,16 = 4.591; p = 0.007), with letters indicating similarity by Tukey’s HSD pair-wise
comparisons. Bacteriodical activity was calculated as the percentage of bacteria killed were
calculated in reference to the negative control, and negative values indicate that bacteria
grew better in media with a plasma sample than in media alone.
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(a)
(c)
(b)
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