Submitted 16 March 2018
Accepted 9 June 2018
Published 11 July 2018

Corresponding author
Wilfried Klein, wklein@usp.br

Academic editor
Donald Kramer

Additional Information and
Declarations can be found on
page 23

DOI 10.7717/peerj.5137

Copyright
2018 Trevizan-Baú et al.

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OPEN ACCESS

Effects of environmental hypoxia and
hypercarbia on ventilation and gas
exchange in Testudines
Pedro Trevizan-Baú1,2, Augusto S. Abe3 and Wilfried Klein1

1Departmento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São
Paulo, Ribeirão Preto, SP, Brazil

2Programa de Pós-graduação em Biologia Comparada, Universidade de São Paulo, Ribeirão Preto, SP, Brazil
3 Instituto de Biociências, Universidade Estadual Paulista, Rio Claro, SP, Brazil

ABSTRACT
Background. Ventilatory parameters have been investigated in several species of
Testudines, but few species have had their ventilatory pattern fully characterized by
presenting all variables necessary to understand changes in breathing pattern seen under
varying environmental conditions.
Methods. Wemeasured ventilation and gas exchange at 25 ◦C in the semi-aquatic turtle
Trachemys scripta and the terrestrial tortoise Chelonoidis carbonarius under normoxia,
hypoxia, and hypercarbia and furthermore compiled respiratory data of testudine
species from the literature to analyze the relative changes in each variable.
Results. During normoxia both species studied showed an episodic breathing pattern
with two to three breaths per episode, but the non-ventilatory periods (TNVP) were
three to four times longer in T. scripta than in C. carbonarius. Hypoxia and hypercarbia
significantly increased ventilation in both species and decreased TNVP and oxygen
consumption in T. scripta but not in C. carbonarius.
Discussion. Contrary to expectations, the breathing pattern in C. carbonarius did
show considerable non-ventilatory periods with more than one breath per breathing
episode, and the breathing pattern in T. scripta was found to diverge significantly from
predictions based on mechanical analyses of the respiratory system. A quantitative
analysis of the literature showed that relative changes in the ventilatory patterns of
chelonians in response to hypoxia and hyperbarbia were qualitatively similar among
species, although there were variations in the magnitude of change.

Subjects Animal Behavior, Zoology
Keywords Reptilia, Normoxia, Breathing pattern, Breathing mechanics, Oxygen consumption

INTRODUCTION
The order Testudines differs from the other groups of reptiles by the presence of a rigid shell,
impeding lung ventilation through movement of the ribs (Lyson et al., 2014). To overcome
thismorphological constraint, Testudines contract abdominalmuscles associated with their
legs, thereby compressing or expanding the body cavity and resulting in lung ventilation
(Gans & Hughes, 1967; Gaunt & Gans, 1969).

Testudines can be divided into two suborders. The 100 species of Pleurodira are
characterized by a retraction of the neck in the horizontal plane, whereas the 250 species

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of Cryptodira retract their neck in the vertical plane (Werneburg et al., 2015; Uetz, Freed &
Hošek, 2018). All Pleurodira occur in freshwater habitats, just as the majority of cryptodiran
species. However, some Cryptodira live in the marine environment and all representatives
of the family Testudinidae and some species of Emydidae are terrestrial.

Gas exchange and ventilation have been studied in several species of turtles, tortoises,
and terrapins, but few species were fully characterized regarding their breathing variables.
In particular, two species of the family Emydidae (Cryptodira), the semi-aquatic Trachemys
scripta and Chrysemys picta have been used in numerous respiratory studies (see Table S1).
Although oxygen consumption has been determined in many chelonian species (seeUltsch,
2013, for review), data on ventilatory parameters, such as overall breathing frequency,
tidal volume, and minute ventilation, are available only for a small number of species,
representing a limited range of the taxonomic diversity, especially when considering
responses to hypoxic and hypercarbic exposures (Table S1). While the number of studies
listed in Table S1 seems extensive, few have actually characterized the ventilatory pattern
by providing data such as inspiratory time, expiratory time, total duration of a ventilatory
cycle, duration of the non-ventilatory period, breathing frequency during breathing
episodes, frequency of breathing episodes, as well as breathing frequency, tidal volume, and
minute ventilation (Benchetrit & Dejours, 1980;Cordeiro, Abe & Klein, 2016). Furthermore,
most of these data have been obtained for Chrysemys picta (e.g., Milsom & Jones, 1980;
Milsom & Chan, 1986; Funk & Milsom, 1987;Wasser & Jackson, 1988). The totality of these
variables is needed to fully characterize the ventilatory behavior of a species under varying
environmental conditions, especially in ectothermic vertebrates where ventilation can show
highly episodic burst breathing or regular singlet breathing (for review see Shelton, Jones
& Milsom, 1986). Fong, Zimmer & Milsom (2009) suggested that an increasing respiratory
drive changes episodic into continuous breathing and Johnson, Krisp & Bartman (2015)
used duration of inspiration and expiration as measures for inspiratory and expiratory
drive, respectively. Furthermore, Milsom &Wang (2017) argued that the regulation of the
ventilatory responses is complex and cannot be totally understood with few variables
measured, especially because Testudines possess an undivided heart that allows for
intracardiac shunting of blood between the pulmonary and systemic circulations.

Among the Testudines, the terrestrial species belonging to the family Testudinidae
are also very poorly characterized regarding their ventilatory response to hypoxia or
hypercarbia, and only data on breathing frequency, tidal volume, minute ventilation, and
oxygen consumption are available (Altland & Parker, 1955; Benchetrit, Armand & Dejours,
1977; Benchetrit & Dejours, 1980; Burggren, Glass & Johansen, 1977; Glass, Burggren &
Johansen, 1978; Ultsch & Anderson, 1988). Burggren, Glass & Johansen (1977) and Glass,
Burggren & Johansen (1978) showed that under normoxic conditions, the terrestrial
Testudo pardalis employs regular single breaths separated by short breath-holds. A
regular singlet breathing behavior has also been shown by Burggren (1975) for the tortoise
Testudo graeca and by Benchetrit, Armand & Dejours (1977) for Testudo horsfieldi. The
semi-aquatic Pelomedusa subrufa, on the other hand, uses breathing episodes containing
several ventilations interspaced by longer breath-holds (Burggren, Glass & Johansen, 1977;
Glass, Burggren & Johansen, 1978). Such a pattern has been interpreted as adaptation to the

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aquatic life-style observed in P. subrufa and other aquatic or semi-aquatic species, where
the episodic breathing reduces the amount of time spent at the water surface, reducing
the risk of predation, as well as reducing the cost of ascending to the surface (Randall
et al., 1981).

Depending on the gas concentration, hypoxia, as well as hypercarbia, stimulates
breathing in turtles, with moderate concentrations of hypercarbia generally increasing
ventilation more than very low oxygen concentrations (Shelton, Jones & Milsom, 1986).
Interestingly,Altland & Parker (1955) found amore episodic breathing pattern inTerrapene
carolina carolina under normoxia that changed to a more regular singlet breathing pattern
under hypoxic conditions. The normal response to either hypoxia or hypercarbia results in
reduced non-ventilatory periods, but may or may not increase breathing frequency or tidal
volume (Shelton, Jones & Milsom, 1986). In a recent study, Cordeiro, Abe & Klein (2016)
demonstrated that two closely related pleurodirans exhibit different ventilatory responses to
hypoxia and hypercarbia.While both species reduce significantly the non-ventilatory period
and increase breathing frequency during hypoxic and hypercarbic exposures, Podocnemis
unifilis significantly increases the breathing frequency during breathing episodes during
hypercarbia but significantly decreases the breathing frequency during breathing episodes
during hypoxia, whereas Phrynops geoffroanus significantly decreases breathing frequency
during breathing episodes under hypoxia, but does not change this variable during
hypercarbia.

Given these variations in the breathing pattern during normoxia, hypoxia, and
hypercarbia among Testudines, and considering the very few ventilatory data available
for terrestrial species, the aim of the present study was to analyze the ventilatory
response to different gas mixtures in two cryptodirans, the red-eared slider Trachemys
scripta (Emydidae) and the South American red-footed tortoise Chelonoidis carbonarius
(Testudinidae). Trachemys scripta, the model species for cardiorespiratory studies, was
investigated because no previous study reported all ventilatory variables obtained from the
same animals and experimental protocols, both under hypoxic and hypercarbic conditions,
whereas C. carbonarius was chosen because it is a widespread South American tortoise that
has not had its respiratory physiology investigated previously. Furthermore, the present data
were compiled together with available data from the literature to characterize the general
response of Testudines to hypoxia and hypercarbia and to verify if terrestrial species show
a significantly different ventilatory pattern compared to semi-aquatic species.

MATERIALS AND METHODS
Animals
Adults of both sexes of T. scripta (body mass: MB = 1.08± 0.10 kg; N = 8) and
C. carbonarius (MB = 3.77± 0.61 kg; N = 6) living under natural conditions were
obtained from the Jacarezário, Univeridade Estadual Paulista ‘‘Júlio de Mesquita Filho’’,
Rio Claro, SP, Brazil, transported to the laboratory at the University of São Paulo in
Ribeirão Preto, SP, and maintained for at least three months before experimentation to
acclimate to laboratory conditions. Experiments were performed between November 2014

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and February 2015 following approval by the Instituto Chico Mendes de Conservação
da Biodiversidade (SISBIO; license number 35221-1) and Comissão de Ética no Uso
de Animais (CEUA USP/Campus de Ribeirão Preto; protocol number 12.1.1541.53.0).
Animals were maintained under a 12 h light/dark photoperiod cycle, in a temperature-
controlled room at 25 ± 2 ◦C and received a mixed diet supplemented with amino acids,
vitamins and minerals (Aminomix Pet, Vetnil R©, Louveira, Brazil) three times a week.
T. scripta were housed in a box with a water reservoir for diving whereas C. carbonarius
were housed in boxes whose bottom was covered with wooden chips.

Setup
Animals were submitted to open respirometry following Glass, Wood & Johansen (1978),
Wang &Warburton (1995) and Silva et al. (2011) to measure ventilation and gas exchange.
Individuals of T. scripta were placed in an aquarium with a single access to an inverted
funnel, and each individual only needed to extend its neck and protrude its nostrils into the
chamber for air breathing. C. carbonarius were placed in a plastic box and a mask was fitted
to the head of each animal for respirometry and a collar was fixed to the neck to prevent
head retraction. The dead space of the funnel or the mask was never larger than 40 ml. The
exit of the funnel and the frontal tip of the mask were equipped with a pneumotach (Fleisch
tube), which was connected to a spirometer (FE141, ADInstruments, Sydney, Australia).
The gas inside the funnel or mask was sampled at 180 ml min−1, dried, and pulled to a gas
analyzer (ML206, ADInstruments, Sydney, Australia). Data were recorded and analyzed
using PowerLab 8/35 and LabChart 7.0 (ADInstruments, Sydney, Australia).

Both the funnel and the mask were calibrated by injections of known volumes, using an
Inspira ventilator (Harvard Apparatus, Cambridge, MA, USA), and concentrations of gas,
supplied by a Pegas 4000MF gas mixer (Columbus Instruments Columbus, OH, USA). Air
was used for the spirometer calibration with volumes ranging from 1 to 60 ml and different
volumes and concentrations of O2 were used to calibrate the gas exchange measurements.
In all cases, calibrations resulted in linear regressions with R2 >0.95.

Experimental protocols
Experimental temperature and photoperiod were the same as during maintenance, and all
animals were fasted for three to seven days before experimentation to avoid the confounding
effects of digestion onmetabolism. The animals were weighed one day before the beginning
of each experimental treatment. Before any measurements, animals were placed into the
experimental setup or equipped with a mask at least 12 h before initiation of experiments.
Experimentation started around 08:00, and ventilation and gas exchange were measured
first under normoxic conditions, followed by progressively decreasing hypoxic (9, 7, 5, 3%
O2) or progressively increasing hypercarbic (1.5, 3.0, 4.5, 6.0% CO2) exposures, and after
that animals were exposed again to normoxia. The exposure time of each gas mixture, as
well as normoxic conditions, was 2 h. Animals were first exposed to one randomly chosen
progressive gas exposure and the following day to the other one.

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Figure 1 Example of a single ventilatory cycle and episodic ventilation in Chelonoidis carbonarius
during normoxia showing respiratory variables measured. Traces of a single ventilatory cycle (A, B)
and episodic ventilation (C, D) in Chelonoidis carbonarius during normoxia showing respiratory variables
measured A, C: ventilation; B, D: oxygen consumption. For abbreviations see ‘Material and Methods’. The
red parts on parts A and B represent the areas integrated to determine tidal volume and oxygen consump-
tion, respectively.

Full-size DOI: 10.7717/peerj.5137/fig-1

Data analysis
The last hour of each exposure was used to extract the following data: breathing frequency
during breathing episodes (fRepi, breaths episode−1), frequency of breathing episodes (fE,
episodes.h−1), duration of non-ventilatory period (TNVP, s; defined as the time between
the end of an inspiration and the beginning of the following expiration), duration of
inspiration (TINSP, s), duration of expiration (TEXP, s), total duration of one ventilatory
cycle (TTOT=TEXP+TINSP, s), tidal volume (VT, ml kg−1), breathing frequency (fR, breaths
min−1), and oxygen consumption (VO2, mlO2 kg−1) (Fig. 1). During episodic breathing,
due to the slow response time of the oxygen analyzer, oxygen consumption was determined
by integrating the area above the oxygen trace for the entire episode, and then divided by the
number of expirations to obtain mean oxygen consumption per breath. From the extracted
data, the instantaneous breathing frequency (f′, breaths min−1), the relative duration of
expiration (TEXP/TTOT), the relation between inspiration and expiration (TINSP/TEXP), the
expiratory flow rate (VT/TEXP, ml s−1), minute ventilation (V̇E , ml kg−1 min−1), oxygen
consumption (V̇O2, mlO2 kg−1 min−1), and air convection requirement (V̇E / V̇O2, ml
mlO−12 ) were calculated.

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Data were analyzed using GraphPad Prism 6.0 and applying RepeatedMeasures ANOVA
followed by a Tukey’s multiple comparison test. Values of P < 0.05 were considered
significant.

To compare the results of the present study with previously published data, we searched
for relevant publications using Pubmed, Web of Science, and Google Scholar databases
using keywords such as ‘turtle’, ‘Testudines’, ‘hypoxia’, ‘hypercarbia’, ‘hypercapnia’,
‘ventilation’, ‘gas exchange’, etc. Values of respiratory variables of Testudines obtained
under exposure to environmental hypoxia or hypercarbia (but not anoxia or hypoxic-
hypercarbia) measured at temperatures between 20 and 30 ◦C were included (Table 1).
Data from animals that had their trachea cannulated, or that had their respiratory system
surgically manipulated, were not included. Values were directly obtained from the text
or tables given, or by extracting values from published figures using the free software
PlotDigitizer (version 2.6.2). To enable comparison among species, data were expressed as
changes relative to normoxic values. Due to the very low number of chelonian species with
a complete set of respiratory variables available and the varying experimental protocols
applied at different temperatures, levels of hypoxia or hypercarbia, phylogenetically
informed multivariate analysis was not possible.

RESULTS
Ventilation and oxygen consumption in T. scripta and C. carbonarius
During normoxia, both species showed an episodic breathing pattern with two to three
ventilatory cycles interspersed by non-ventilatory periods (Fig. 2). The TNVP was, on
average, three to four times longer in T. scripta than in C. carbonarius. In T. scripta,
hypoxia significantly increased fE, VT, V̇E and V̇E/V̇O2, whereas fRepi, TNVP and V̇O2 were
significantly reduced (Figs. 3–6). Under hypoxic exposure, C. carbonarius significantly
increased fE, TINSP, VT, fR, V̇E , and V̇E/V̇O2 (Figs. 3–6). Once the hypoxic exposure ended,
all variables returned to pre-hypoxic values within 1 hour, with the exception of fR in
C. carbonarius, which was significantly greater when compared to the pre-hypoxic value.
Exposure to CO2 increased V̇E and V̇E/V̇O2 significantly and decreased V̇O2 significantly
in T. scripta, whereas in C. carbonarius TINSP, TTOT, VT, fR, V̇E , and V̇E/V̇O2 significantly
increased but TNVP and f′ significantly decreased (Figs. 3–6). One hour after the withdrawal
of CO2, all variables had returned to pre-hypercarbic values. The relationships between
TEXP, TINSP and TTOT (i.e., TEXP/TTOT, and TINSP/TEXP respectively) were not significantly
affected by either hypoxia nor hypercarbia, just as expiratory flow rate (VT/TEXP), but the
latter did show a tendency to increase in both species with increasing levels of hypoxia and
hypercarbia (Fig. 5).

Relative changes in respiratory variables
Both hypoxia and hypercarbia increased ventilation. This increase was achieved by
increasing the number of breathing episodes, caused by decreasing the non-ventilatory
period (Fig. 7). TNVP at 3%O2, for example, consistently represented about 20%of the TNVP

seen during normoxia in all species investigated, whereas 6% CO2 roughly reduced TNVP

by 50%. Interestingly, hypercarbia about doubled fE, with the exception of P. geoffroanus,

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Table 1 Respiratory variables extracted from the literature.

Species Respiratory variables Reference

fRepi fE TNVP TINSP TEXP TTOT f ′ VT fR V̇E V̇O2 V̇E/V̇O2

Hypoxia
Chelonoidis carbonarius x x x x x x x x x x x x This study
Chelydra serpentina x Boyer (1963)
Chelydra serpentina x Boyer (1966)
Chelydra serpentina x x x Frische, Fago & Altimiras (2000)
Chelydra serpentina x x West, Smits & Burggren (1989)
Chrysemys picta x x x x x x Glass, Boutilier & Heisler (1983)
Chrysemys picta x x x x x x Milsom & Chan (1986)
Gopherus polyphemus x Ultsch & Anderson (1988)
Pelomedusa subrufa x x x Burggren, Glass & Johansen (1977)
Pelomedusa subrufa x x x x Glass, Burggren & Johansen (1978)
Phrynops geoffroanus x x x x x x x x x x x x Cordeiro, Abe & Klein (2016)
Podocnemis unifilis x x x x x x x x x x x x Cordeiro, Abe & Klein (2016)
Terrapene carolina x x x x Altland & Parker (1955)
Terrapene carolina x Ultsch & Anderson (1988)
Testudo horsfieldi x Benchetrit, Armand & Dejours (1977)
Testudo pardalis x x x Burggren, Glass & Johansen (1977)
Testudo pardalis x x x x Glass, Burggren & Johansen (1978)
Trachemys scripta x x Frankel et al. (1969)
Trachemys scripta x Hicks & Wang (1999)
Trachemys scripta x Jackson & Schmidt-Nielsen (1966)
Trachemys scripta x x x x x x x Lee & Milsom (2016)
Trachemys scripta x x x x x x x x x x x x This study
Trachemys scripta x x x x x Vitalis & Milsom (1986b)

Hypercarbia
Chelonia mydas x x x Jackson, Kraus & Prange (1979)
Chelonoidis carbonarius x x x x x x x x x x x x This study
Chelydra serpentina x x West, Smits & Burggren (1989)
Chrysemys picta x x x x x x x x Funk & Milsom (1987)
Chrysemys picta x x x x x x Milsom & Chan (1986)
Chrysemys picta x x x x x x x x x Milsom & Jones (1980)
Chrysemys picta x x x x x x x Silver & Jackson (1985)
Pelomedusa subrufa x x x Burggren, Glass & Johansen (1977)
Pelomedusa subrufa x x x x Glass, Burggren & Johansen (1978)
Phrynops geoffroanus x x x x x x x x x x x x Cordeiro, Abe & Klein (2016)
Podocnemis unifilis x x x x x x x x x x x x Cordeiro, Abe & Klein (2016)
Testudo pardalis x x x x Glass, Burggren & Johansen (1978)
Testudo horsfieldi x x x x x x x Benchetrit & Dejours (1980)
Testudo pardalis x x x Burggren, Glass & Johansen (1977)

(continued on next page)

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Table 1 (continued)

Species Respiratory variables Reference

fRepi fE TNVP TINSP TEXP TTOT f ′ VT fR V̇E V̇O2 V̇E/V̇O2

Trachemys scripta x x Frankel et al. (1969)
Trachemys scripta x Hicks & Wang (1999)
Trachemys scripta x x x Hitzig & Nattie (1982)
Trachemys scripta x x x x x Jackson, Palmer & Meadow (1974)
Trachemys scripta x x x x Johnson & Creighton (2005)
Trachemys scripta x x x x x x x x x x x x This study
Trachemys scripta x x x x x Vitalis & Milsom (1986b)

Notes.
Abbreviations: fRepi, breathing frequency during breathing episodes; fE, number of breathing episodes; TNVP, duration of non-ventilatory period; TINSP, duration of inspiration;
TEXP, duration of expiration; TTOT, total duration of one ventilatory cycle; f′, instantaneous breathing frequency; VT, tidal volume; fR, breathing frequency; V̇E , minute ventila-
tion; V̇O2, oxygen consumption; V̇E/V̇O2, air convection requirement.

and slightly increased fRepi (exceptions P. geoffroanus and C. carbonarius), whereas hypoxia
caused a greater increase in fE, but slightly decreased fRepi (exception C. carbonarius).
Neither hypoxia nor hypercarbia drastically altered TINSP, TEXP, TTOT, and f′ (Fig. 8), as
well as TEXT/TTOT and TINSP/TEXP (Fig. 9).

VT/TEXP increased two to five-fold under hypoxic and hypercarbic conditions (Fig. 9) in
all species studied, which was mainly caused by an about two to three-fold increase in VT

at severe levels of hypoxia and hypercarbia (Fig. 10). C. carbonarius, showing a 12-fold, and
P. geoffroanus, showing a six-fold increase in VT, were the only species showingmuch larger
increases in VT. Several species increased fR during hypercarbia about six to seven-fold, but
many species only doubled or tripled fR (Fig. 10). The only species that increased fR more
than three-fold during hypoxia were C. picta at 30 ◦C (Glass, Boutilier & Heisler, 1983) and
P. geoffroanus at 25 ◦C (Cordeiro, Abe & Klein, 2016). The product of VT and fR, minute
ventilation, showed the greatest relative increases, with P. geoffroanus increasing V̇E 42
times and C. carbonarius about 30 times, both at 6% CO2. The relative increase at 6% CO2

ranged from four to 12 times, whereas at 3% O2 the increase in V̇E ranged between three
and six or between 12 and 17 for C. picta, C. carbonarius and P. geoffroanus.

With the exception of P. geoffroanus, both under hypoxia and hypercarbia, and of
P. unifilis under hypercarbia, V̇O2 decreased or remained unaltered during both exposures
(Fig. 11). The resulting air convection requirement, however, increased about 10 to 30-fold
in T. scripta (Jackson (1973) and Lee & Milsom (2016) versus this study, respectively), in
C. picta (3% O2; Glass, Boutilier & Heisler, 1983), and in C. carbonarius (4.5 and 6% CO2;
this study) (Fig. 11). In the remaining species, V̇E/V̇O2 increased about three to 12 times
under both hypoxic and hypercarbic conditions.

DISCUSSION
Ventilation and oxygen consumption in T. scripta and C. carbonarius
Breathing pattern of both species followed the general reptilian behavior of intermittent
lung ventilation. Burggren (1975) and Glass, Burggren & Johansen (1978) observed
intermittent ventilation in Testudo graeca and T. pardalis, respectively, but in both
species breathing pattern consisted of just one ventilatory cycle interspersed by short and

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Figure 2 Example traces of ventilation in Trachemys scripta and Chelonoidis carbonarius. Traces of
ventilation in Trachemys scripta (A–C) and Chelonoidis carbonarius (D–F) during normoxia (A, D), 6%
CO2 (B, E), and 3% O2 (C, F).

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regular non-ventilatory periods. In the present study, both, T. scripta and, unexpectedly,
C. carbonarius, showed more than one ventilatory cycle per breathing episode, but the
mean duration of the non-ventilatory periods was lower in C. carbonarius when compared
to T. scripta. Vitalis & Milsom (1986a) consider episodic breathing an adaptive mechanism
that decreases the energetic cost of ventilation in ectotherms, and Randall et al. (1981)
consider such a breathing behavior advantageous for aquatic species, since it reduces the
energetic cost to surface and also reduces the exposure time at the surface, possibly lessening
risks of predation. Since episodic breathing with long non-ventilatory periods leads to a
significant change in arterial blood gases, decreasing PaO2 and pH and increasing PaCO2

(Glass, Burggren & Johansen, 1978), as well as decreasing the efficiency of pulmonary CO2

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Figure 3 Breathing frequency during breathing episodes, number of breathing episodes, and dura-
tion of the non-ventilatory period in Trachemys scripta and Chelonoidis carbonarius. Breathing fre-
quency during breathing episodes (A, D), number of breathing episodes (B, E), and duration of the non-
ventilatory period (C, F) in Trachemys scripta (triangle) and Chelonoidis carbonarius (circle) during nor-
moxia, hypoxia (A–C) and hypercarbia (D–F) (open symbols) and 1 hour after exposure to the different
gas mixtures (closed symbols). * (T. scripta) and + (C. carbonarius) indicate values significantly different
from initial normoxic values

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excretion (Malte, Malte & Wang, 2013), it should be more advantageous for a terrestrial
species to ventilate regularly and thereby maintain homeostasis of arterial blood gases. It is
therefore interesting to ask why the terrestrial C. carbonarius employs episodic breathing
under normoxic conditions, thereby possibly increasing variation in arterial blood gases
instead of maintaining a regular breathing pattern, such as seen in this species only
under severe levels of hypoxia or hypercarbia (Fig. 2). C. carbonarius does frequently

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Figure 4 Duration of inspiration, duration of expiration, total duration of one ventilatory cycle, and
instantaneous breathing frequency in Trachemys scripta and Chelonoidis carbonarius. Duration of in-
spiration (A, E), duration of expiration (B, F), total duration of one ventilatory cycle (C, G), and instan-
taneous breathing frequency (D, H) in Trachemys scripta (triangle) and Chelonoidis carbonarius (circle)
during normoxia, hypoxia (A–D) and hypercarbia (E–H) (open symbols) and 1 hour after exposure to the
different gas mixtures (closed symbols). * (T. scripta) and+ (C. carbonarius) indicate values significantly
different from initial normoxic values.

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Figure 5 Relation between expiration and total duration of one ventilatory cycle, the relation between
inspiration and expiration, and the expiratory flow rate in Trachemys scripta and Chelonoidis car-
bonarius. The relation between expiration and total duration of one ventilatory cycle (A, D), the relation
between inspiration and expiration (B, E), and the expiratory flow rate (C, F) in Trachemys scripta (trian-
gle) and Chelonoidis carbonarius (circle) during normoxia, hypoxia (A–C) and hypercarbia (D–F) (open
symbols) and 1 hour after exposure to the different gas mixtures (closed symbols).

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seek shelter in shallow burrows or other small spaces and remains non-ventilatory for
long periods (AS Abe, pers. obs., 2000), possibly explaining the episodic breathing seen
in this terrestrial species, but currently a physiological explication for this behavior is
lacking. Interestingly, other ectothermic terrestrial species such as varanid (Thompson &
Withers, 1997) and agamid lizards (Frappell & Daniels, 1991) also breathe intermittently.
However concomitant blood gas analyses have not been performed in these species to
verify accompanying variations in blood gases or pH.

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Figure 6 Tidal volume, breathing frequency, minute ventilation, oxygen consumption, and air con-
vection requirement in Trachemys scripta and Chelonoidis carbonarius Tidal volume (A, F), breathing
frequency (B, G), minute ventilation (C, H), oxygen consumption (D, I), and air convection requirement
(E, J) in Trachemys scripta (triangle) and Chelonoidis carbonarius (circle) during normoxia, hypoxia (A–
E) and hypercarbia (F–J) (open symbols) and 1 hour after exposure to the different gas mixtures (closed
symbols). * (T. scripta) and+ (C. carbonarius) indicate values significantly different from initial normoxic
values. # denotes a post-hypoxia normoxic value significantly different from the initial normoxia.

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Figure 7 Relative changes in breathing frequency during breathing episodes, number of breathing
episodes, and duration of the non-ventilatory period in Testudines under hypoxic and hypercarbic ex-
posures. Relative changes in breathing frequency during breathing episodes (A, D), number of breath-
ing episodes (B, E), and duration of the non-ventilatory period (C, F) in Testudines under hypoxic (A–
C) and hypercarbic (D–F) exposures. Chelonia mydas: 25 ◦C, Jackson, Kraus & Prange, 1979; Chelonoidis
carbonarius: 25 ◦C, this study; Chelydra serpentina: 25 ◦C, Boyer, 1966; 22–24 ◦C,West, Smits &
Burggren, 1989; 20 ◦C, Frische, Fago & Altimiras, 2000); Chrysemys picta: 20–23 ◦C,Milsom & Jones,
1980; 20 ◦C, Glass, Boutilier & Heisler, 1983; 30 ◦C, Glass, Boutilier & Heisler, 1983; 20 ◦C, Silver
& Jackson, 1985; 22–23 ◦C,Milsom & Chan, 1986; 20 ◦C, Funk & Milsom, 1987; 30 ◦C, Funk &
Milsom, 1987; Gopherus polyphemus: 22 ◦C, Ultsch & Anderson, 1988; Pelomedusa subrufa: 25 ◦C,
Burggren, Glass & Johansen, 1977; 25 ◦C, Glass, Burggren & Johansen, 1978); Phrynops geoffroanus:
25 ◦C, Cordeiro, Abe & Klein, 2016; Podocnemis unifilis: 25 ◦C, Cordeiro, Abe & Klein, 2016; Terrapene
carolina : 20–23 ◦C, Altland & Parker, 1955; 22 ◦C, Ultsch & Anderson, 1988; Testudo horsfieldi:
25 ◦C, Benchetrit, Armand & Dejours, 1977; 30 ◦C, Benchetrit, Armand & Dejours, 1977; 23–25 ◦C,
Benchetrit & Dejours, 1980; Testudo pardalis: 25 ◦C, (continued on next page. . . )

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Figure 7 (. . .continued)
Burggren, Glass & Johansen, 1977; 25 ◦C, Glass, Burggren & Johansen, 1978; Trachemys scripta:
24 ◦C, Jackson & Schmidt-Nielsen, 1966; 28 ◦C, Frankel et al., 1969; 20 ◦C, Jackson, 1973; 30 ◦C,
Jackson, 1973; 20 ◦C, Jackson, Palmer & Meadow, 1974; 30 ◦C, Jackson, Palmer & Meadow, 1974;
20 ◦C, Hitzig & Nattie, 1982; 25 ◦C, Hicks & Wang, 1999; 27–28 ◦C, Johnson & Creighton, 2005;
20–23.5 ◦C, Lee & Milsom, 2016; 25 ◦C, this study.

Comparing our data with previous studies on the effect of hypoxia or hypercarbia on
ventilation and gas exchange in Trachemys scripta, Frankel et al. (1969) found values for
TTOT about three times larger during normoxia, hypoxia and hypercarbia when compared
to our study. However animals in their study had their tracheas cannulated which may
have influenced the length of the ventilatory cycle, since TTOT values reported by Vitalis &
Milsom (1986b) (calculated from their f′: 1.7 s during normoxia and 4%O2 and 1.8 s during
3–5% CO2) are similar to ours. Reyes & Milsom (2009) report similar values for fE as in the
present study (from 8.4 ± 1.6 in normoxia during winter up to 37.1 ± 2.3 episodes.h−1 in
summer), but found considerable variation in fRepi through different seasons, ranging from
3.6± 0.4 breaths.episode−1 in normoxia during winter up to 26.1± 5.4 breaths.episode−1

in hypoxic-hypercarbia during autumn, thereby demonstrating considerable seasonal
variation in breathing pattern in T. scripta. Lee & Milsom (2016) report nearly identical
values as in the present study for fRepi and fE during normoxia and hypoxia, and Frankel
et al. (1969) report a comparable fRepi during normoxia. Johnson & Creighton (2005), on
the other hand, report greater values of fRepi during both normoxia and hypercarbia, and
Frankel et al. (1969) found fRepi at 10–12% CO2 to be 5.6 ± 1.0 at 28 ◦C.

More data are available regarding VT, fR, V̇E , V̇O2, and V̇E/V̇O2 during both, hypoxic
and hypercarbic exposures. In general, data obtained in the present study for normoxia
are similar to the ones obtained by other authors, such as V̇O2, which at 25 ◦C varies
from 0.82 (Hicks & Wang, 1999; this study) to 1.1 mlO2 kg−1 min−1 (24 ◦C; Jackson &
Schmidt-Nielsen, 1966), whereas the values given by Vitalis & Milsom (1986b) for VT and
V̇E are the lowest ones reported forT. scripta exposed to hypoxia or hypercarbia. The overall
changes observed in the ventilatory responses of T. scripta to hypoxia and hypercarbia are
also comparable between the present study and data from the literature. Only V̇E/V̇O2 in
the present study, both during hypoxia and hypercarbia, was greater when compared to
data from the literature. This difference was caused by a much lower V̇O2 during hypoxic
and hypercarbic exposures when compared to data from other authors, since V̇E was very
similar to data obtained by others at similar temperatures (Jackson, Palmer & Meadow,
1974; Lee & Milsom, 2016). The oxygen consumption measured by us during hypercarbia
was similar to the one obtained by Jackson, Palmer & Meadow (1974) at 10 ◦C, a 15 ◦C
difference, that may represent a variation in chemosensivity seen in this species during
different seasons (Reyes & Milsom, 2009), as we found similarly low V̇O2 values during
hypoxic exposures. Interestingly, our normoxic V̇O2 values werewell within the range forT.
scripta at 25 ◦C reported in the literature (Hicks & Wang, 1999; Jackson & Schmidt-Nielsen,
1966). A significant drop in oxygen consumption during hypoxia has also been described
before (Jackson & Schmidt-Nielsen, 1966; Jackson, 1973; Lee & Milsom, 2016), whereas other
studies did not find a pronounced fall in metabolism during hypercarbia (Hicks & Wang,

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Figure 8 Relative changes in duration of inspiration and expiration, total duration of one ventilatory
cycle, and instantaneous breathing frequency in Testudines under hypoxic and hypercarbic exposures.
Relative changes in duration of inspiration (A, E), duration of expiration (B, F), total duration of one ven-
tilatory cycle (C, G), and instantaneous breathing frequency (D, H) in Testudines under hypoxic (A–D)
and hypercarbic (E–H) exposures. For symbols see Fig. 7.

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Figure 9 Relative changes in the relation between expiration and total duration of one ventilatory cy-
cle, the relation between inspiration and expiration, and expiratory flow rate. Relative changes in the re-
lation between expiration and total duration of one ventilatory cycle (A, D), the relation between inspira-
tion and expiration (B, E), and the expiratory flow rate (C, F) in Testudines under hypoxic (A–C) and hy-
percarbic (D–F) exposures. For symbols see Fig. 7.

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1999; Jackson, Palmer & Meadow, 1974). One motive for the observed variations could
lie in the significant seasonal variations in metabolism, gas exchange, and, consequently,
ventilation found in T. scripta (Reyes & Milsom, 2009), variations that possibly were not
eliminated by maintaining the animals at a constant temperature of 25 ◦C. Furthermore,
exposing animals for 2 hours to each gas mixture may not have been sufficient to reach
a physiological steady-state, as suggested by Malte, Malte & Wang (2016). Another reason
for this discrepancy could be the species physiological phenotypic plasticity, since animals
used in the previous studies were native to the North American continent and thereby

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Figure 10 Relative changes in tidal volume, breathing frequency, andminute ventilation in Testudines
under hypoxic and hypercarbic exposures. Relative changes in tidal volume (A, D), breathing frequency
(B, E), and minute ventilation (C, F) in Testudines under hypoxic (A–C) and hypercarbic (B–F) expo-
sures. For symbols see Fig. 7.

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subject to a more temperate climate than the animals used in the present study, that have
been bred under the subtropical climate of southeastern Brazil.

The values for minute ventilation in T. scripta at 8% CO2 found by Hitzig & Nattie
(1982) seem somewhat low, when compared to the values found by Johnson & Creighton
(2005) at the same CO2 concentration at a different temperature (20 versus 27–28 ◦C,
respectively), but are somewhat similar to the values found by Jackson, Palmer & Meadow
(1974) at 20 ◦C and 6% CO2 (135.0 versus 215 ml kg−1 min−1, respectively). The general
response of T. scripta to reducing oxygen concentrations can be described by a moderate,
when compared to the response during hypercarbia, increase in minute ventilation, mainly

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Figure 11 Relative changes in oxygen consumption and air convection requirement in Testudines un-
der hypoxic and hypercarbic exposures. Relative changes in oxygen consumption (A, C), and air convec-
tion requirement (B, D) in Testudines under hypoxic (A–B) and hypercarbic (C–D) exposures. For sym-
bols see Fig. 7.

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caused by increasing VT, and a reduction in oxygen consumption, thereby increasing the
air convection requirement. These changes are generally more pronounced below 5% O2.
The response to hypercarbia also includes an increase in ventilation due to an increase
in VT and fR. In T. scripta neither hypoxia nor hypercarbia caused significant changes in
TINSP, TEXP, TTOT, f′, and fRepi, whereas fE and TNVP, increased and decreased significantly,
respectively.

In respect to C. carbonarius during hypoxic or hypercarbic exposures, only data on VT,
fR, V̇E , and V̇O2 are available for other terrestrial Testudines belonging to the Emydidae
and Testudinidae. Despite comparing different species, the ventilatory variables are similar
among the terrestrial species studied, with the exception of the normoxic V̇O2 value
given by Altland & Parker (1955) for Terrapene carolina carolina, possibly indicating that
animals in their study may not have been resting quietly during normoxia. However,
their V̇O2 value reported for 3–5% O2 is identical to the values from other studies at
similar oxygen concentrations. VT in C. carbonarius is on the lower end of data available
for terrestrial Testudines, which may have been influenced by the relative large amount
of bone tissue present in adult individuals of this species (AS Abe, pers. obs., 2005).
Breathing frequency and V̇E , on the other hand, were very similar to the data obtained on
other terrestrial Emydidae and Testudinidae (Altland & Parker, 1955, Benchetrit, Armand

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& Dejours, 1977, Burggren, Glass & Johansen, 1977, Glass, Burggren & Johansen, 1978 and
Benchetrit & Dejours, 1980).

Ultsch & Anderson (1988), studying Gopherus polyphemus and Terrapene carolina, found
values of oxygen consumption very similar to those ofC. carbonarius during both normoxia
and hypoxia. Interestingly, G. polyphemus spends a significant amount of time in burrows
that may show hypoxia as well as hypercarbia, and whose critical oxygen level (percentage
of O2 where V̇O2 starts decreasing) can be found at approximately 1.5% O2, whereas the
exclusively terrestrial T. carolina shows a somewhat larger critical oxygen tension of 3.5%
O2 (Ultsch & Anderson, 1988). Since C. carbonarius did not show any significant changes
in V̇O2during hypoxia down to 3% O2, the critical oxygen level of this species seems to be
similar to the one seen in the former two species, but V̇O2 was consistently lower at any
oxygen concentration when compared to G. polyphemus and T. carolina and e.g., at 3%
O2 (0.08 mlO2 kg−1 min−1) was similar to the lowest V̇O2 given for G. polyphemus (0.05
mlO2 kg−1 min−1) and T. carolina (0.08 mlO2 kg−1 min−1) at less than 1% O2 (Ultsch &
Anderson, 1988). C. carbonarius is not known to use burrows and therefore may not show
a critical oxygen level as low as G. polyphemus, but Chelonoidis chilensis has been reported
to use shallow burrows for retreat during cold days (Pritchard, 1979) and therefore other
species of the Testudinidae may possess a similarly low oxygen level as the testudinidid G.
polyphemus.

Relative changes in respiratory variables
Analyzing the respiratory variables available in the literature for chelonians exposed to
hypoxia and hypercarbia (Figs. 7–11), one notices the discrepancy in data availability
between commonly studied parameters such as VT, fR, V̇E , and V̇O2, and less frequently
reported ones such as TEXP, TTOT, or fE, for example. Furthermore, only very few terrestrial
species have been studied, when compared to the wealth of data available for T. scripta
and C. picta. Based on the data analyzed, it seems clear that the breathing pattern of
terrestrial chelonians does not significantly differ from aquatic or semi-aquatic species
when considering the responses to hypoxia and hypercarbia. With few exceptions, both
hypoxia and hypercarbia elicit similar respiratory responses, showing variation mainly in
the magnitude of the species’ responses. The different patterns seen in fE and fRepi during
hypoxia and hypercarbia may suggest varying degrees of chemosensivity between species
and towards different gas exposures. Previous experimental manipulations transforming
episodic breathing into continuous single ventilations in T. scripta were vagotomy (Vitalis
& Milsom, 1986b) and dissection of the spinal cord (Johnson & Creighton, 2005). Recently,
Johnson, Krisp & Bartman (2015) changed episodic breathing in T. scripta from episodic
to singlet breathing through pharmacological manipulation of serotonin 5-HT3 receptors.
Studying the participation of serotonin in central chemoreception under hypoxia and
hypercarbia in phylogenetically distant species, as well as species occupying different
habitats, might help to elucidate the varying responses to hypoxia and hypercarbia seen
in chelonian breathing pattern, since the switch from episodic to singlet breathing under
hypoxia has been suggested to be caused by an increased respiratory drive (Fong, Zimmer
& Milsom, 2009). However, under hypercarbia nearly all species increase the number of

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breaths per episode, and do not decrease fRepi as under hypoxia, suggesting that CO2

exposure increases respiratory drive by different regulatory pathways than under hypoxia.
Interestingly, Herman & Smatresk (1999) demonstrated that in T. scripta hypoxia and
hypercarbia cause different changes in pulmonary ventilation and perfusion. During
hypoxia, lung ventilation and perfusion increased, whereas under hypercarbia only lung
ventilation increased, but not pulmonary perfusion, resulting in a ventilation/perfusion
mismatch during exposure to CO2. Burggren, Glass & Johansen (1977) found a similar
cardiovascular response in Pelomedusa subrufa and in Testudo pardalis, suggesting a
common testudine response, but its importance or relation with the differences observed
in fRepi remains unclear.

P. geoffroanus and C. carbonarius seem to be more sensitive regarding TINSP, TEXP,
TTOT, f′, TEXT/TTOT, and TINSP/TEXP, with the former species increasing these variables
mainly during hypercarbia, but the latter one increasing all variables with increasing levels
of hypoxia and hypercarbia. Such increases in TINSP and TEXP have been interpreted by
Johnson, Krisp & Bartman (2015) as a stronger respiratory drive from central respiratory
neurons, whose intensity, however, seems to vary among species. The absolute and relative
decrease in instantaneous breathing frequency seen in C. carbonarius implies that breathing
mechanics may be more variable than previously anticipated for Testudines, since Vitalis
& Milsom (1986b) found f′ to be unaffected by either hypoxia or hypercarbia in T. scripta
and suggested (Vitalis & Milsom, 1986a; Vitalis & Milsom, 1986b) that T. scripta breathes
at combinations of volume and frequency to keep the mechanical work of breathing at
a minimum. In the present study, f′ in T. scripta, as well as in C. carbonarius, did show
larger variations than reported for T. scripta in earlier studies (Frankel et al., 1969; Vitalis
& Milsom, 1986b). Vitalis & Milsom (1986a) found, based on mechanical analyses of the
respiratory system of T. scripta, that the mechanical work of breathing is minimal at
ventilation frequencies of 35 to 45 cycles min−1 for different levels of minute pump
ventilation (100, 200, 300 ml min−1), meaning that for a minute pump ventilation of 200
ml min−1. Animals should therefore ventilate at a frequency of 40 breaths min−1 and a
tidal volume of 5 ml to ventilate the respiratory system with the lowest mechanical work,
but such a breathing pattern would result in severe alkalosis due to increased CO2 excretion
(Vitalis & Milsom, 1986b). In the present study, however, T. scripta reached the greatest
level of minute ventilation (215.9 ml min−1 kg−1) at 6% CO2, using a tidal volume of
57.2 ml kg−1 and an instantaneous breathing frequency of 18.3 breaths min−1 (fR= 3.0
breaths min−1), values much different from mechanical predictions. The significance of
this variation in breathing pattern versus the mechanical predictions of work of breathing
needs to be investigated to better understand the mechanical work of breathing of the
Testudines respiratory system, since mechanical work of breathing increases markedly with
increasing tidal volume, e.g., from 57 to 272 ml cmH2Omin−1 kg−1 at 6.2 ml kg−1 and 3.0
breaths min−1 in undisturbed T. scripta versus 34.2 and 0.8 breaths min−1 in vagotomized
T. scripta, each at 4% O2 (Vitalis & Milsom, 1986b).

The relatively large increases seen in VT/TEXP of C. carbonarius and P. geoffroanus can be
explained by very low values of VT under normoxic conditions. P. geoffroanus (3.1 ml kg−1;
Cordeiro, Abe & Klein, 2016) and C. carbonarius (3.98 ml kg−1; this study) show much

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smaller tidal volumes during normoxia than other chelonians (mostly between 10 and 20
ml kg−1), resulting in relatively larger increases in VT during hypoxia and hypercarbia than
the other species. Both species also showed relatively larger increases in V̇E , which are again
attributable to the low values seen in fR and VT under normoxic conditions.

Whereas V̇E increases largely in all species, V̇O2 remains unaltered or even decreases
under both hypoxia and hypercarbia in nearly all species investigated. The relative increase
seen in P. geoffroanus under both hypoxia and hypercarbia can be explained by the very
low oxygen consumption under normoxic conditions, which could be a consequence of
significant extra-pulmonary gas exchange or hypometabolism in this species (Cordeiro,
Abe & Klein, 2016). Increases in V̇E/V̇O2 have been linked both under hypoxia (e.g., Glass,
Boutilier & Heisler, 1983) and hypercarbia (e.g., Funk & Milsom, 1987) to regulation of
arterial PO2, PCO2, and pH, as all turtles investigated maintain control of these variables
under varying environmental conditions.

The chelonian respiratory system shows significant variations in lung structure, as well
as in associated structures such as the post-pulmonary septum (PPS; Perry, 1998). The
PPS is a membrane that partially or completely separates the lungs from the other viscera
(Lambertz, Böhme & Perry, 2010). As a testudinid, C. carbonarius possesses a complete
post-pulmonary septum (W Klein, pers. obs., 2015), when compared to the incomplete
PPS of the emydid T. scripta (Lambertz, Böhme & Perry, 2010). The presence or absence
of a PPS may significantly influence the mechanics of the respiratory system, as has been
shown for the post-hepatic septum of the lizard Salvator (Tupinambis) merianae, whose
static breathing mechanics was significantly affected by the removal of their post-hepatic
septum (Klein, Abe & Perry, 2003). Similarly, a complete PPS in Testudinidae could alter
the mechanics of the respiratory system by reducing the impact of the viscera onto the
lungs, when compared to species with an incomplete PPS such as T. scripta.

CONCLUSION
This is the first study to present all the different variables necessary to fully characterize the
breathing pattern in the terrestrial C. carbonarius and the semi-aquatic T. scripta during
hypoxic and hypercarbic conditions. Contrary to most previous reports on breathing
pattern in terrestrial Testudines, C. carbonarius did show considerable non-ventilatory
periods with more than one breath per episode. While our data confirm previous data
on the general response of T. scripta to hypoxia and hypercarbia, breathing pattern has
been found to diverge significantly from predictions based on mechanical analyses of the
respiratory system.

Our meta-analysis demonstrates general trends regarding ventilatory parameters of
Testudines when exposed to hypoxia or hypercarbia, but a multivariate analysis of the
taxons respiratory physiology will need a complete set of ventilatory parameters from
a much larger number of species. To date it is not possible to associate the variations
in the magnitude of different respiratory variables with phylogeny, habitat, behavior,
and/or lung structure, which could provide important information regarding the evolution
of cardiorespiratory physiology in chelonians. Cardiovascular data regarding intracardiac

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shunt, pulmonary and systemic perfusion, and blood gases during hypoxia and hypercarbia
are particularly needed from more species to fully understand blood gas homeostasis in
such an important group of intermittent breathers.

ADDITIONAL INFORMATION AND DECLARATIONS

Funding
Financial support was provided by the Fundação de Amparo à Pesquisa do Estado de São
Paulo (FAPESP) to Wilfried Klein (2012/18652-1) and Augusto Shinya Abe and Wilfried
Klein (2008/57712-4), by the Conselho Nacional de Desenvolvimento Tecnológico e
Científico (CNPq) to Augusto Shinya Abe and Wilfried Klein (573921/2008-3). Pedro
Trevizan-Baú received a scholarship from the Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior (CAPES). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.

Grant Disclosures
The following grant information was disclosed by the authors:
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP): 2012/18652-1,
2008/57712-4.
Conselho Nacional de Desenvolvimento Tecnológico e Científico (CNPq): 573921/2008-3.
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Competing Interests
The authors declare there are no competing interests.

Author Contributions
• Pedro Trevizan-Baú conceived and designed the experiments, performed the
experiments, analyzed the data, prepared figures and/or tables, authored or reviewed
drafts of the paper, approved the final draft.
• Augusto S. Abe conceived and designed the experiments, contributed reagents/materi-
als/analysis tools, authored or reviewed drafts of the paper, approved the final draft.
• Wilfried Klein conceived and designed the experiments, performed the experiments,
analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or
tables, authored or reviewed drafts of the paper, approved the final draft.

Animal Ethics
The following information was supplied relating to ethical approvals (i.e., approving body
and any reference numbers):

Experiments were performed between November 2014 and February 2015 following
approval by the Instituto Chico Mendes de Conservação da Biodiversidade (SISBIO;
license number 35221-1) and Comissão de Ética no Uso de Animais (CEUA USP/Campus
de Ribeirão Preto; protocol number 12.1.1541.53.0).

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Data Availability
The following information was supplied regarding data availability:

Klein, Wilfried (2018): C. carbonarius and T. scripta ventilation. figshare. Fileset.
Available at https://doi.org/10.6084/m9.figshare.5936296.v1.

Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.5137#supplemental-information.

REFERENCES
Altland PD, Parker M. 1955. Effects of hypoxia upon the box Turtle. American Journal of

Physiology 180:421–427.
Benchetrit G, Armand J, Dejours P. 1977. Ventilatory chemoreflex drive in the tortoise,

Testudo horsfieldi. Respiration Physiology 31:183–191
DOI 10.1016/0034-5687(77)90101-3.

Benchetrit G, Dejours P. 1980. Ventilatory CO2 drive in the tortoise Testudo horsfieldi.
Journal of Experimental Biology 87:229–236.

Boyer DR. 1963.Hypoxia: Effects on heart rate and respiration in the snapping turtle.
Science 140:813–814 DOI 10.1126/science.140.3568.813.

Boyer DR. 1966. Comparative effects of hypoxia on respiratory and cardiac function in
reptiles. Physiological Zoology 39:307–316 DOI 10.1086/physzool.39.4.30152354.

BurggrenWW. 1975. A quantitative analysis of ventilation tachycardia and its control in
two chelonians, Pseudemys scripta and Testudo graeca. Journal of Experimental Biology
63:367–380.

BurggrenWW, Glass ML, Johansen K. 1977. Pulmonary ventilation: perfusion rela-
tionships in terrestrial and aquatic chelonian reptiles. Canadian Journal of Zoology
55:2024–2034 DOI 10.1139/z77-263.

Cordeiro TEF, Abe AS, KleinW. 2016. Ventilation and gas exchange in two turtles:
Podocnemis unifilis and Phrynops geoffroanus (Testudines: Pleurodira). Respiration
Physiology & Neurobiology 224:125–131 DOI 10.1016/j.resp.2014.12.010.

Fong AY, ZimmerMB, MilsomWK. 2009. The conditional nature of the ‘‘Central
Rhythm Generator’’ and the production of episodic breathing. Respiration Physiology
& Neurobiology 168:179–187 DOI 10.1016/j.resp.2009.05.012.

Frankel HM, Spitzer A, Blaine J, Schoener EP. 1969. Respiratory response of turtles
(Pseudemys scripta) to changes in arterial blood gas composition. Comparative
Biochemistry and Physiology 31:535–546 DOI 10.1016/0010-406X(69)90055-3.

Frappell PB, Daniels CB. 1991. Ventilation and oxygen consumption in agamid lizards.
Physiological Zoology 64:985–1001 DOI 10.1086/physzool.64.4.30157953.

Frische S, Fago A, Altimiras J. 2000. Respiratory responses to short term hypoxia in the
snapping turtle, Chelydra serpentina. Comparative Biochemistry and Physiology Part A
126:223–231 DOI 10.1016/S1095-6433(00)00201-4.

Trevizan-Baú et al. (2018), PeerJ, DOI 10.7717/peerj.5137 24/27

https://peerj.com
https://doi.org/10.6084/m9.figshare.5936296.v1
http://dx.doi.org/10.7717/peerj.5137#supplemental-information
http://dx.doi.org/10.7717/peerj.5137#supplemental-information
http://dx.doi.org/10.1016/0034-5687(77)90101-3
http://dx.doi.org/10.1126/science.140.3568.813
http://dx.doi.org/10.1086/physzool.39.4.30152354
http://dx.doi.org/10.1139/z77-263
http://dx.doi.org/10.1016/j.resp.2014.12.010
http://dx.doi.org/10.1016/j.resp.2009.05.012
http://dx.doi.org/10.1016/0010-406X(69)90055-3
http://dx.doi.org/10.1086/physzool.64.4.30157953
http://dx.doi.org/10.1016/S1095-6433(00)00201-4
http://dx.doi.org/10.7717/peerj.5137


Funk GD,MilsomWK. 1987. Changes in ventilation and breathing pattern produced by
changing body temperature and inspired CO2 concentration in turtles. Respiration
Physiology 67:37–51 DOI 10.1016/0034-5687(87)90005-3.

Gans C, Hughes GM. 1967. The mechanism of lung ventilation in the tortoise Testudo
graeca Linné. Journal of Experimental Biology 47:1–20.

Gaunt AS, Gans C. 1969.Mechanics of respiration in the snapping turtle, Chelydra ser-
pentina (Linné). Journal of Morphology 128:195–227 DOI 10.1002/jmor.1051280205.

Glass ML, Boutilier RG, Heisler N. 1983. Ventilatory control of arterial PO2 in the turtle
Chrysemys picta belli: effects of temperature and hypoxia. Journal of Comparative
Physiology 151:145–153 DOI 10.1007/BF00689912.

Glass ML, BurggrenWW, Johansen K. 1978. Ventilation in an aquatic and a terrestrial
chelonian reptile. Journal of Experimental Biology 72:165–179.

Glass ML,Wood SC, Johansen K. 1978. The application of pneumotachography on small
unrestrained animals. Comparative Biochemistry and Physiology Part A 59:425–427
DOI 10.1016/0300-9629(78)90191-3.

Herman JK, Smatresk NJ. 1999. Cardiorespiratory response to progressive hypoxia
and hypercapnia in the turtle Trachemys scripta. Journal of Experimental Biology
202:3205–3213.

Hicks JW,Wang T. 1999.Hypoxic hypometabolism in the anesthetizes turtle, Trachemys
scripta. American Physiological Society 277:R18–R23.

Hitzig BM, Nattie EE. 1982. Acid-base stress and central chemical control of ventilation
in turtles. Journal of Applied Physiology 53:1365–1370
DOI 10.1152/jappl.1982.53.6.1365.

Jackson DC. 1973. Ventilatory response to hypoxia in turtles at various temperatures.
Respiration Physiology 18:178–187 DOI 10.1016/0034-5687(73)90048-0.

Jackson DC, Kraus DR, Prange HD. 1979. Ventilatory response to inspired CO2 in the
sea turtle: effects of body size and temperature. Respiration Physiology 38:71–81
DOI 10.1016/0034-5687(79)90007-0.

Jackson DC, Palmer SE, MeadowWL. 1974. The effects of temperature and carbon diox-
ide breathing on ventilation and acid-base status of turtles. Respiration Physiology
20:131–146 DOI 10.1016/0034-5687(74)90102-9.

Jackson DC, Schmidt-Nielsen K. 1966.Heat production during diving in the fresh water
turtle, Pseudemys scripta. J. Cellular Physiol. 67:225–231 DOI 10.1002/jcp.1040670204.

Johnson SM, Creighton RJ. 2005. Spinal cord injury-induced changes in breathing are
not due to supraspinal plasticity in turtles (Pseudemys scripta). American Journal of
Physiology. Regulatory, Integrative and Comparative Physiology 289:R1550–R1561
DOI 10.1152/ajpregu.00397.2005.

Johnson SM, Krisp AR, BartmanME. 2015.Hypoxia switches episodic breath-
ing to singlet breathing in red-eared slider turtles (Trachemys scripta) via a
tropisetron-sensitive mechanism. Respiration Physiology & Neurobiology 207:48–57
DOI 10.1016/j.resp.2014.12.015.

Trevizan-Baú et al. (2018), PeerJ, DOI 10.7717/peerj.5137 25/27

https://peerj.com
http://dx.doi.org/10.1016/0034-5687(87)90005-3
http://dx.doi.org/10.1002/jmor.1051280205
http://dx.doi.org/10.1007/BF00689912
http://dx.doi.org/10.1016/0300-9629(78)90191-3
http://dx.doi.org/10.1152/jappl.1982.53.6.1365
http://dx.doi.org/10.1016/0034-5687(73)90048-0
http://dx.doi.org/10.1016/0034-5687(79)90007-0
http://dx.doi.org/10.1016/0034-5687(74)90102-9
http://dx.doi.org/10.1002/jcp.1040670204
http://dx.doi.org/10.1152/ajpregu.00397.2005
http://dx.doi.org/10.1016/j.resp.2014.12.015
http://dx.doi.org/10.7717/peerj.5137


KleinW, Abe AS, Perry SF. 2003. Static lung compliance and body pressures in
Tupinambis merianae with and without post-hepatic septum. Respiration Physiology
& Neurobiology 135:73–86 DOI 10.1016/S1569-9048(03)00063-6.

Lambertz M, BöhmeW, Perry SF. 2010. The anatomy of the respiratory system in
Platysternon megacephalum Gray, 1831 (Testudines: Cryptodira) and related species,
and its phylogenetic implications. Comparative Biochemistry and Physiology. A:
Comparative Physiology 156:330–336 DOI 10.1016/j.cbpa.2009.12.016.

Lee SY, MilsomWK. 2016. The metabolic cost of breathing in red-eared sliders: an
attempt to resolve an old controversy. Respiration Physiology & Neurobiology
224:114–124 DOI 10.1016/j.resp.2015.10.011.

Lyson TR, Schachner ER, Botha-Brink J, Scheyer TM, Lambertz M, Bever GS, Rubidge
BS, De Queiroz K. 2014. Origin of the unique ventilatory apparatus of turtles.
Nature Communications 5:5211 DOI 10.1038/ncomms6211.

Malte CL, Malte H,Wang T. 2013. Episodic ventilation lowers the efficiency of
pulmonary CO2 excretion. Journal of Applied Physiology 115:1506–1518
DOI 10.1152/japplphysiol.00808.2013.

Malte CL, Malte H,Wang T. 2016. The long road to steady state in gas exchange:
metabolic and ventilatory responses to hypercapnia and hypoxia in Cuvier’s dwarf
caiman. Journal of Experimental Biology 219:3810–3821 DOI 10.1242/jeb.143537.

MilsomWK, Chan P. 1986. The relantioship between lung volume, respiratory drive
and breathing pattern in the turtle, Chrysemys picta. Journal of Experimental Biology
120:233–247.

MilsomWK, Jones DR. 1980. The role of vagal afferent information and hypercapnia
in control of the breathing pattern in Chelonia. Journal of Experimental Biology
87:53–63.

MilsomWK,Wang T. 2017. Is the hypoxic ventilatory response driven by blood oxygen
concentration? Journal of Experimental Biology 220:956–958 DOI 10.1242/jeb.151316.

Perry SF. 1998. Lungs: comparative anatomy, functional morphology, and evolution.
In: Gans C, ed. Biology of the reptilia. Vol. 19. Ithaca: Society for the Study of
Amphibians and Reptiles, 1–92.

Pritchard PCH. 1979. Encyclopedia of turtles. Neptune City: TFH Publications, 895.
Randall D, BurggrenWW, Farrell A, Haswell MS. 1981. The evolution of air breathing in

vertebrates. Cambridge: Cambridge University Press, 133.
Reyes C, MilsomWK. 2009. Daily and seasonal rhythms in the respiratory sensitivity

of red-eared sliders (Trachemys scripta elegans). Journal of Experimental Biology
212:3339–3348 DOI 10.1242/jeb.027698.

Shelton G, Jones DR, MilsomWK. 1986. Control of breathing in ectothermic verte-
brates. In: Fishman AP, Chermiak NS, Widdicombe JG, Geiger SR, eds. Handbook
of physiology, section 3. The respiratory system, volume II. Control of breathing, Part 2,
Am. Physiol. Soc. Bethesda, 857–909.

Silva GSF, Giusti H, Branco LGS, Glass ML. 2011. Combined ventilatory responses to
aerial hypoxic and temperature in the South American lungfish Lepidosiren paradoxa.
Journal of Themal Biology 36:521–526 DOI 10.1016/j.jtherbio.2011.09.004.

Trevizan-Baú et al. (2018), PeerJ, DOI 10.7717/peerj.5137 26/27

https://peerj.com
http://dx.doi.org/10.1016/S1569-9048(03)00063-6
http://dx.doi.org/10.1016/j.cbpa.2009.12.016
http://dx.doi.org/10.1016/j.resp.2015.10.011
http://dx.doi.org/10.1038/ncomms6211
http://dx.doi.org/10.1152/japplphysiol.00808.2013
http://dx.doi.org/10.1242/jeb.143537
http://dx.doi.org/10.1242/jeb.151316
http://dx.doi.org/10.1242/jeb.027698
http://dx.doi.org/10.1016/j.jtherbio.2011.09.004
http://dx.doi.org/10.7717/peerj.5137


Silver RB, Jackson DC. 1985. Ventilatory and acid-base response to long-term hyper-
capnia in the freshwater turtle, Chrysemys picta bellii. Journal of Experimental Biology
144:661–672.

Thompson GG,Withers PC. 1997. Patterns of gas exchange and extended non-
ventilatory periods in small goannas (Squamata: Varanidae). Comparative Biochem-
istry and Physiology 118A:1411–1417.

Uetz P, Freed P, Hošek J. 2018. The reptile database . Available at http://www.reptile-
database.org (accessed on 10 May 2018).

Ultsch GR. 2013.Metabolic scaling in turtles. Comparative Biochemistry and Physiology.
A: Comparative Physiology 164:590–597 DOI 10.1016/j.cbpa.2013.01.012.

Ultsch GR, Anderson JF. 1988. Gas exchange during hypoxia and hypercarbia of
terrestrial turtles: a comparison of a fossorial species (Gopherus polyphemus)
with a sympatric nonfossorial species (Terrapene carolina). Physiological Zoology
61:142–152 DOI 10.1086/physzool.61.2.30156145.

Vitalis TZ, MilsomWK. 1986a. Pulmonary mechanics and the work of breathing in the
semi aquatic turtle, Pseudemys scripta. Journal of Experimental Biology 125:137–155.

Vitalis TZ, MilsomWK. 1986b.Mechanical analysis of spontaneous breathing in the
semi-aquatic turtle, Pseudemys scripta. Journal of Experimental Biology 125:157–171.

Wang T,Warburton SJ. 1995. Breathing pattern and cost of ventilation in the American
alligator. Respiration Physiology 102:29–37 DOI 10.1016/0034-5687(95)00043-D.

Wasser JS, Jackson DC. 1988. Acid-base balance and the control of respiration during
anoxic-hypercapnic gas breathing in turtles. Respiration Physiology 71:213–226
DOI 10.1016/0034-5687(88)90017-5.

Werneburg I, Hinz JK, Gumpenberger M, Volpato V, Natchev N, JoyceWG. 2015.
Modeling neck mobility in fossil turtles. Journal of Experimental Zoology Part B
324:230–243 DOI 10.1002/jez.b.22557.

West NH, Smits AW, BurggrenWW. 1989. Factors terminating nonventilatory
periods in the turtle, /Chelydra serpentina/. Respiration Physiology 77:337–350
DOI 10.1016/0034-5687(89)90121-7.

Trevizan-Baú et al. (2018), PeerJ, DOI 10.7717/peerj.5137 27/27

https://peerj.com
http://www.reptile-database.org
http://www.reptile-database.org
http://dx.doi.org/10.1016/j.cbpa.2013.01.012
http://dx.doi.org/10.1086/physzool.61.2.30156145
http://dx.doi.org/10.1016/0034-5687(95)00043-D
http://dx.doi.org/10.1016/0034-5687(88)90017-5
http://dx.doi.org/10.1002/jez.b.22557
http://dx.doi.org/10.1016/0034-5687(89)90121-7
http://dx.doi.org/10.7717/peerj.5137