gopherus agassizii , mojave desert tortoise, ectotherm, draft · tortoises resume inactivity but...

Draft

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

https://mc06.manuscriptcentral.com/cjz-pubs

Canadian Journal of Zoology

Draft


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:

[email protected]

Page 1 of 35



Draft

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

Page 2 of 35



Draft

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

Page 3 of 35



Draft

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;

Page 4 of 35



Draft

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

Page 5 of 35



Draft

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

Page 6 of 35



Draft

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

Page 7 of 35



Draft

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

Page 8 of 35



Draft

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

Page 9 of 35



Draft

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

Page 10 of 35



Draft

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).

Page 11 of 35



Draft

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

Page 12 of 35



Draft

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;

Page 13 of 35



Draft

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).

Page 14 of 35



Draft

(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

Page 15 of 35



Draft

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

Page 16 of 35



Draft

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

Page 17 of 35



Draft

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

Page 18 of 35



Draft

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.

Page 19 of 35



Draft

References

Agha, M., Augustine, B., Lovich, J.E., Delaney, D., Sinervo, B., Murphy, M.O., Ennen, J.R.,

Briggs, J.R., Cooper, R, and Price, S.J. 2015. Using motion-sensor camera technology to infer

seasonal activity and thermal niche of the desert tortoise (Gopherus agassizii). J. Therm.

Biol. 49: 119-126. doi:10.1016/j.therbio.2015.02.009.

Alleman, A.R., Jacobson, ER, and Raskin R.E. 1992. Morphologic and cytochemical

characteristics of blood cells from the desert tortoise (Gopherus agassizii). Am. J. Vet. Res.

53(9): 1645-1651.

Altizer, S., Dobson, A., Hosseini, P., Hudson, P., Pascual, M., and Pejman, R. 2006. Seasonality

and the dynamics of infectious diseases. Ecol. Lett. 9(4): 467-484. doi.10.1111/j.1461-

0248.2005.00879.

Buehler, D.M., Piersma, T., Matson, K., and Tieleman, B.I. 2008. Seasonal redistribution of

immune function in a migrant shorebird: annual-cycle effects override adjustments to

thermal regime. Am. Nat. 172(6): 783-796. http://www.jstor.org/stable/10.1086/592865.

Christopher, M.M., Berry, K.H., Wallis, I.R., Nagy, K.A., Henen, B.T., and Peterson, C.C. 1999.

Reference intervals and physiologic alterations in hematologic and biochemical values of

free-ranging desert tortoises in the Mojave Desert. J. Wildl. Dis. 35(2): 212-238.

doi:10.7589/0090-3558-35.2.212.

Page 20 of 35



Draft

Cooper, J.G. 1861. New California animals. Proc. Calif. Acad. Sci. ser. 1 2: 118-123.

Davis, A.K., Maney, D.L., and Maerz, J.C. 2008. The use of leukocyte profiles to measure

stress in vertebrates: a review for ecologists. Funct. Ecol. 22(5): 760-772.

doi:10.1111/j.1365-2435.2008.01467.x.

Demas, G.E., and Nelson, R.J. (Editors). 2012. Ecoimmunology. Oxford University Press, New

York, NY.

Dickinson, V.M., Jarchow, J.L., and Trueblood, M.H. 2002. Hematology and plasma

biochemistry reference range values for free-ranging desert tortoises in Arizona. J. Wildl.

Dis. 38(1): 143-153. doi: 10.7589/0090-3558-38.1.143.

Drake, K.K., Nussear, K.E., Esque, T.C., Barber, A.M., Vittum, K.M., Medica, P.A., Tracy, C.R.,

and Hunter, K.W. 2012. Does translocation influence physiological stress in the desert

tortoise? Anim. Conserv. 15(6): 560-570. doi:10.1111/j.1469-1795.2012.00549.x.

French, S.S., DeNardo, D.F., Greives, T.J., Strand, C.R., and Demas, G.E. 2010. Human

disturbance alters endocrine and immune responses in the Galapagos marine iguana

(Amblyrhynchus cristatus). Horm. Behav. 58(5): 792-799.

doi:10.1016/j.yhbeh.2010.08.001.

Page 21 of 35



Draft

Green, N., and Cohen, N. 1977. Effect of temperature on serum complement levels in the

leopard frog, Rana pipiens. Dev. Comp. Immunol. 1: 59-64.

Hagerty, B.E., and Tracy, C.R. 2010. Defining population structure of the Mojave desert

tortoise. Conserv. Genet. 11(5): 1795-1807. doi:10.1007/s10592-010-0073-0.

Hayman, J.R., Bly, J.E., Levine, R.P., and Lobb, C.J. 1992. Complement deficiencies in channel

catifish (Ictalurus punctatus) associated with temperature and seasonal mortality. Fish

Shellfish Immunol. 2(3): 1830192. doi:10.1016/S1050-4648(05)80058-0.

Hereford, R., Webb, R.H., and Longpré, C.I. 2006. Precipitation history and ecosystem

response to multidecadal precipitation variability in the Mojave Desert region, 1893-2001.

J, Arid Environ. 67(2006): 13-34. doi:10.1016/j.jaridenv.2006.09.019.

Hernandez-Divers, S.M., Hernandez-Divers, S.J., and Wyneken, J. 2002. Angiographic,

anatomic and clinical technique descriptions of a subcarapacial venipuncture site for

chelonians. J. Herpetol. Med. Surg. 12(2): 32–37.

Inman, R.D., Nussear, K.E., and Tracy, C.R. 2009. Detecting trends in desert tortoise

population growth: elusive behavior inflates variance in estimates of population density.

Endang. Species Res. 10: 295-304. doi:10.3354/esr00214.

Page 22 of 35



Draft

Jacobson, E.R. 2007. Infectious Diseases and Pathology of Reptiles. Taylor and Francis

Group, Boca Raton, FL.

Keppel, G. 1991. Design and Analysis, a Researcher’s Handbook, 3rd edition. Prentice Hall,

Upper Saddle River, NJ.

Lance, V.A., Grumbles, J.S., and Rostal, D.C. 2001. Sex differences in plasma corticosterone in

desert tortoises, Gopherus agassizii, during the reproductive cycle. J. Exp. Zool. 289(5): 285-

289. doi: 10.1002/1097-10X(20010415/30)289:5<285::AID-JEZ2>3.0CO;2-B.

Leceta, J., and Zapata, A. 1986. Seasonal-variations in the immune response of the turtle

Mauremys caspica. Immunology, 57(3): 483-487.

Lochmiller, R.L., and Deerenberg ,C. 2000. Trade-offs in evolutionary immunology: just

what is the cost of immunity? Oikos, 88(1): 87-98. doi:1034/j.1600-0706.2000.880110.x.

Lovich, J.E., and Ennen, J.R. 2011. Wildlife conservation and solar energy development in

the desert southwest, United States. BioScience 61(12): 982-992.

doi:10.1525/bio.2011.61.12.8.

Maniero, G.D., and Carey, C. 1997. Changes in selected aspects of immune function in the

leopard frog, Rana pipiens, associated with exposure to cold. J. Comp. Physiol. B 167(4):

256-263. doi:10.1007/s003600050072.

Page 23 of 35



Draft

Martin, L.B., Wei,l Z.M., and Nelson, R.J. 2008. Seasonal changes in vertebrate immune

activity: mediation by physiological trade-offs. Philos. Trans. R. Soc. Lond. B Biol. Sci. No.

363(1490): 321-339. doi:10.1098/rstb.2007.2142.

McEwen, B.S., Biron, C.A., Brunson, K.W., Bulloch, K., Chambers, W.H., Dhabhar, F.S., and

Weis, J.M. 1997. The role of adrenocorticoids as modulators of immune function in health

and disease: neural, endocrine, and immune interactions. Brain Res. Rev. 23(1): 79-133.

doi:10.1016/So165-0173(96)00012-4.

Merchant, M.E., Roche, C., Elsey, R.M., and Prudhomme, J. 2003. Antibacterial properties of

serum from the American alligator (Alligator mississippiensis). Comp. Biochem. Physiol. B

136(3): 505-513. doi:10.1016/S1096-4959(03)00256-2.

Muñoz, F.J., and De la Fuente, N. 2001. The effect of seasonal cycle on the splenic leukocyte

functon in the turtle Mauremys caspica. J. Comp. Physiol. B 171(5): 27-42.

doi:10.1086/323033.

Muñoz, F.J., Galvan, A., Lerma, M., and De la Fuente, N. 2000. Seasonal changes in peripheral

blood leukocyte functions of the turtle Mauremys caspica and their relationship with

corticosterone, 17-beta-estradiol and testosterone serum levels. Vet. Immunol.

Immunopathol. 77:27-42.

Page 24 of 35



Draft

Murphy, K, (Editor). 2012. Janeway’s Immunobiology, 8th ed. Garland Science, Taylor and

Francis Group, New York, NY.

Murphy, R.W., Berry, K.H., Edwards, T., Leviton, A.E., Lathrop, A., and Riedle, J.D. 2011. The

dazed and confused identity of Agassiz’s land tortoise, Gopherus agassizii (Testudines,

Testudinidae) with the description of a new species, and its consequences for conservation.

ZooKeys, 113(24): 39-71. doi:10.3897/zookeys.113.1353.

Naegle, S.R. 1976. Physiological responses of the desert tortoise Gopherus agassizii. M.Sc.

thesis, Las Vegas University, Las Vegas, NV.

Nagy, K.A., and Medica, P.A. 1986. Physiological ecology of desert tortoises in southern

Nevada. Herpetologica 42(1): 73-92. http://www.jstor.org/stable/3892239.

Nagy, K.A., Henen, B.T., and Vyas, D.B. 1998. Nutritional quality of native and introduced

food plants of wild desert tortoises. J. Herpetol. 42(1): 260-278.

http://www.jstor.org/stable/3892239.

Nussear, K.E., Esque, T.C., Haines, D.F., and Tracy, C.R. 2007. Desert tortoise hibernation:

temperatures, timing, and environment. Copeia, 2007(2): 378-386. doi:1643/0045-

8511(2007)7[378:DTHTTA]2.0.CO;2).

Page 25 of 35



Draft

O’Connor, M.P., Grumbles, J.S., George, R.H., Zimmerman, L.C., and Spotila, J.R. 1994.

Potential hematological and biochemical indicators of stress in free-ranging desert

tortoises and captive tortoises exposed to a hydric stress gradient. Herpetol. Monogr.

8(1994): 5-26. doi:10.2307/1467067.

Origgi, F.C. 2007. Reptile immunology. In Infectious Diseases and Pathology of Reptiles.

Edited by E.R. Jacobson. Taylor & Francis Group, Boca Raton, FL. pp. 131-218.

Ott, J.A., Mendonça, M.T., Guyer, C., and Michener, W.K. 2000. Seasonal changes in sex and

adrenal steroid hormones of gopher tortoises (Gopherus polyphemus). Gen. Comp.

Endocrinol. 117(2): 299-312. doi:10.1006/gcen.1999.7419.

Pasmans, F., Herdt, P.D., Nerom, A.V., and Haesebrouck, F. 2001. Induction of respiratory

burst in turtle peritoneal macrophages of the turtle Trachemys scripta scripta. Dev. Comp.

Immunol. 26(2): 156-168. doi:10.1016/S0145-305X(00)00051-3.

Peterson, C.C. 1996a. Ecological energetics of the desert tortoise (Gopherus agassizii):

effects of rainfall and drought. Ecology, 77(6): 1831-1844.

http://www.jstor.org/stable/2265787.

Peterson, C.C. 1996b. Anhomeostasis: Seasonal water and solute relations in two

populations of the desert tortoise (Gopherus agassizii) during chronic drought. Physiol.

Zool. 69(6): 1324-1358. http://www.jstor.org/stable/30164263.

Page 26 of 35



Draft

Plytycz, B., and Jozkowicz, A. 1994. Differential effects of temperarture on macrophages of

ectothermic vertebrates. J. Leukoc. Biol. 56(6): 729-731.

http://www.jleukbio.org/content/56/6/729.full.pdf+html [accessed January 14, 2016].

Raffell, T.R., Rohr, J.R., Kiesecker, J.M., and Hudson, P.J. 2006. Negative effects of changing

temperatures on amphibian immunity under field conditions. Funct. Ecol. 20(5): 819-828.

doi.10.1111/j.1365-2435.2006.01159.x.

Rohr, J.R., and Raffell, T.R. 2010. Linking global climate and temperature variability to

widespread amphibian declines putatively caused by disease. Proc. Natl. Acad. Sci. U.S.A.

107(18): 8269-8274. doi:10.1073/pnas.0912883107.

Rollins-Smith, L.A., and Woodhams, D.C. 2012. Amphibian immunity: staying in tune with

the environment. In Ecoimmunology. Edited by R.J. Nelson and G.E. Demas. Oxford

University Press, Inc., New York, NY. pp. 92-143.

Sandmeier, F.C., Tracy, C.R., Dupré, S., and Hunter, K. 2009. Upper respiratory tract disease

(URTD) as a threat to desert tortoise populations: a reevaluation. Biol. Conserv. 142(7):

1255-1268. doi:10.1016/j.biocon.2009.02.001.

Page 27 of 35



Draft

Sandmeier, F.C., and Tracy, C.R. 2014. The metabolic pace-of-life model: incorporating

ectothermic organisms into the theory of vertebrate ecoimmunology. Integr. Comp. Biol.

2014: icu21. doi:1093/icb/icu021.

Sapolsky, R.M., Romero, L.M., and Munck, A.U. 2000. How do glucocorticoids influence

stress responses? Integrating permissive, suppressive, stimulatory, and preparative

actions. Endocr. Rev. 21(1): 55-89. doi:10.1210/edrv.21.1.0389.

Shaffer, B.H., Minx, P., Warren, D., Shedlock, A.M., Thomson, R.C., Valenzuela, N., Abraymyan

et al. 2013. The western painted turtle genome, a model for the evolution of extreme

physiological adaptations in a slowly evolving lineage. Genome. Biol. 14: R28.

doi:10.1186/gb-2013-14-3-r28.

Spotila, J.R., Zimmerman, L.C., Binckly, C.A., Grumbles, J.S., Rostal, D.C., List, Jr.A., Beyer, E.C.,

Philips, K.M., and Kemp, S.J. 1994. Effects of incubation conditions on sex determination,

hatching success, and growth of hatchling desert tortoises, Gopherus agassizii. Herpetol.

Monogr. 1994: 103-116. doi:10.2307/1467074.

Strik, N.I., Alleman, A.R., and Harr, K.E. 2007. Circulating inflammatory cells. In Infectious

Diseases and Pathology of Reptiles. Edited by E.R. Jacobson. Taylor & Francis Group, Boca

Raton, FL. pp. 167-218.

Page 28 of 35



Draft

Snyder, S. 2014. Effects of fire on desert tortoise (Gopherus agassizii) thermal ecology. Ph.D.

dissertation, Program in Ecology, Evolution, and Conservation Biology, The University of

Nevada, Reno, Reno, NV.

Tracy, C.R., Nussear, K.E., Esque, T.C., Dean-Bradley, K., Tracy, C.R., DeFalco, L.A., Castle, K.T.,

Zimmerman, L.C., Espinoza, E., and Barber, A.M. 2006. The importance of physiological

ecology in conservation biology. Integr. Comp. Biol. 46(6): 1191-1205.

doi:10.1093/icb/icl054.

U.S. Fish and Wildlife Service [USFWS]. 1994. Desert Tortoise (Mojave Population)

Recovery Plan. US Fish and Wildlife Service, Portland, OR.

Weil, Z.M., and Nelson, R.J. 2012. Neuroendocrine mechanisms of seasonal patterns in

immune function. In Ecoimmunology. Edited by R.J. Nelson and G.E. Demas. Oxford

University Press, Inc., New York, NY. pp. 297-325.

Wilkinson, R. 2004. Clinical pathology. In Medicine and Surgery of Turtles and Tortoises.

Edited by S. McArthur, R. Wilkinson, and J. Meyer. John Wile and Sons, Ltd., Sussex, UK. pp.

141-186.

Winokur-Lewis, V., and Winokur, R.M.1995. Incubation temperature effects sexual

differentiation, incubation time, and posthatching survival in desert tortoises (Gopherus

agassizii). Can. J. Zool. 73(11): 2091-2097. doi:10.1139/z95-246.

Page 29 of 35



Draft

Woodbury, A.M., and Hardy, R. 1948. Studies of the desert tortoise, Gopherus agassizii. Ecol.

Mongr. 18(2):145-200. http://www.jstor.org/stable/1948638.

Zapata, A.G., Varas, A., and Torroba, M. 1992. Seasonal variations in immune system of

lower vertebrates. Immunol. Today, 13(4): 142-147. doi:10.1016/0167-5699(92)90112-K.

Zimmerman, L.C., O’Connor, M.P., Bulova, S.J., Spotila, J.R., Kemp, S.J., and Salice, C.J. 1994.

Thermal ecology of desert tortoises in the eastern Mojave Desert: seasonal patterns of

operative and body temperatures, and microhabitat utilization. Herpetol. Monogr. 8(1994):

45-59. doi:10.2307/1467069.

Zimmerman, L.M., Paitz, R.T., Vogel, L.A., and Bowden, R.M. 2010a. Variation in seasonal

patterns of innate and adaptive immunity in the red-eared slider (Trachemys scripta). J.

Exp. Biol. 213(9): 1477-1483. doi:10.1242/jeb.037770.

Zimmerman, L.M., Vogel, L.A., and Bowden, R.M. 2010b. Understanding the vertebrate

immune system: insights from the reptilian perspective. J. Exp. Biol. 213(5): 661-671.

doi:10.1242/jeb.038315.

Zimmerman, L.M., Vogel, L.A., Edwards, K.A., and Bowden, R.M. 2010c. Phagocytic B cells in

a reptile. Biol. Lett. 2010(6): 270-273. doi:10.1098/rsbl.2009.0692.

Page 30 of 35



Draft

Zimmerman, L.M., Clairardin, S.G., Paitz, R.T., Hicke, J.W., LaMagdeleine, K.A., Vogel, L.A., and

Bowden, R.M. 2013. Humoral immune responses are maintained with age in a long-lived

ectotherm, the red-eared slider. J. Exp. Biol. 216(4): 633-640. doi:10.1242/jeb.078832.

Page 31 of 35



Draft

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)

Page 32 of 35



Draft

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.

Page 33 of 35



Draft

(a)

(c)

(b)

Page 34 of 35



Draft

Page 35 of 35



gopherus agassizii , mojave desert tortoise, ectotherm, draft · tortoises resume inactivity but...

Documents