Temperature-Dependent Sex Determination in the Diamond-backed Terrapin

ABSTRACT.Ã¢Â€Â”Most Emydidae, including Diamond-backed Terrapins (Malaclemys terrapin), have temperature-dependent sex determination. However, the full relationship between incubation temperature and offspring sex in the Diamond-backed Terrapin has not been reported, and the pivotal temperature, range of transitional temperatures, and the temperature-sensitive period have not been investigated fully. Here we report on these parameters, comparing our data with data from other emydid turtles, and also comparing our laboratory data to field data collected previously for this population. Copyright 2014 Society for the Study of Amphibians and Reptiles
About Jack Berlin

RUSSELL L. BURKE AND ARTHUR M. CALICHIO - Journal of Herpetology, Vol. 48, No. 4, 466Ã¢Â€Â“470, 2014
temperature sex incubation terrapins data diamond-backed model determination
About Jack Berlin

RUSSELL L. BURKE AND ARTHUR M. CALICHIO - Journal of Herpetology, Vol. 48, No. 4, 466Ã¢Â€Â“470, 2014
temperature sex incubation terrapins data diamond-backed model determination
Journal of Herpetology, Vol. 48, No. 4, 466â€“470, 2014
Copyright 2014 Society for the Study of Amphibians and Reptiles
Temperature-Dependent Sex Determination in the Diamond-backed Terrapin
(Malaclemys terrapin)
RUSSELL L. BURKE1 AND ARTHUR M. CALICHIO
Department of Biology, Hofstra University, Hempstead, New York 11549 USA
ABSTRACT.â€”Most Emydidae, including Diamond-backed Terrapins (Malaclemys terrapin), have temperature-dependent sex determination.
However, the full relationship between incubation temperature and offspring sex in the Diamond-backed Terrapin has not been reported, and
the pivotal temperature, range of transitional temperatures, and the temperature-sensitive period have not been investigated fully. Here we
report on these parameters, comparing our data with data from other emydid turtles, and also comparing our laboratory data to field data
collected previously for this population.
RESUMEN.â€”La mayorÄ±Â´a de Emydidae, incluyendo a las Diamond-backed Terrapins (Malaclemys terrapin), tienen determinacioÂ´n de sexo por
temperatura. Sin embargo, la relacioÂ´n entre temperatura de incubacion y sexo de crÄ±Â´as en el Diamond-backed Terrapin no ha sido reportada, ni
el umbral de temperatura ni el perÄ±Â´odo sensible a la temperatura han sido investigados. AquÄ±Â´ reportamos ambos fenoÂ´menos y los compararmos
con otras Emydidae, asi como con datos de campo de eÂ´sta poblacioÂ´n previamente colectados.
In their landmark paper, Ewert and Nelson (1991) reported
sex determination data for a wide range of turtle species, laying
the groundwork for multispecies analyses of sex determination
patterns in reptiles. Of particular interest was the Emydidae,
wherein both temperature-dependent sex determination (TSD)
and genotypic sex determination (GSD). Among TSD turtles,
there are two patterns known, TSD Ia and TSD II, but their
evolutionary and ecological significance remains enigmatic
(Ewert et al., 2004).
Although Ewert and Nelson (1991) reported detailed infor-
mation for many emydids, their data for Diamond-backed
Terrapins (Malaclemys terrapin) were limited. Their article
showed only that Diamond-backed Terrapins have TSD, with
all female hatchlings from eggs (Accomac County, Virginia;
Hotaling, pers. comm.) incubated at 308C and all male
hatchlings from eggs (from Bergen County, New Jersey, E.
Hotaling, pers. comm.) incubated at 248C. Later, Jeyasuria et al.
(1994) incubated Diamond-backed Terrapin eggs from St.
Maryâ€™s County, Maryland, and found that high temperatures
(31 and 328C) resulted in 100% female hatchlings and low
incubation temperatures (26 and 278C) resulted in 100% male
hatchlings and that eggs incubated at 29.58C resulted in 13%
male hatchlings. Thus, Diamond-backed Terrapins seem to be a
typical TSD Ia species (Ewert and Nelson, 1991), with a single
transitional temperature range; incubation temperatures below
this range result in males, whereas incubation temperatures
above this range produce females. The TSD I pattern is evident
in most but not all emydid turtles. However, without sex ratio
data from a wide range of temperatures, it is possible that a
second transitional range exists, as is seen in TSD II species
(Ewert and Nelson, 1991).
Pivotal (=threshold; Bull et al., 1982) temperature is the
constant incubation temperature that results in 1:1 offspring sex
ratios in TSD species (Mrosovsky and Pieau, 1991). This value
has become an important component of models that relate
incubation temperatures to offspring sex ratios (e.g., Georges et
al., 1994; Doody et al., 2006) and is a common repeatable metric
for intraspecific (e.g., Bull et al., 1982; Ewert et al., 2005) and
interspecific (Sarre et al., 2004) comparisons. Less frequently
calculated, yet perhaps as important for understanding TSD
(e.g., Hulin et al., 2009), is the transitional range of temperature
(TRT). The TRT is the range of constant incubation temperatures
that produce both sexes and may be associated with the
likelihood of evolutionary response to selective pressures
because of changing thermal environments (Hulin et al., 2009).
Girondot (1999) pointed out that calculation of TRT depends on
statistical technique, number of incubation temperatures, and
samples sizes used.
There is significant genetic structuring across the Diamond-
backed Terrapin range (Hart, 2005; Hauswaldt and Glen, 2005)
that spans 2,000 km northâ€“south and could include substantial
differences in incubation conditions. Thus, different populations
may have evolved different pivotal temperatures (Bull et al.,
1982; Ewert et al., 1994, 2005) and different TRTs. Unfortunately,
Jeyasuria et al. (1994) did not report obtaining males and
females at more than a single incubation temperature, so their
data cannot be used to estimate pivotal temperature or TRT
(Girondot, 1999).
The temperature-sensitive period (TSP) (Bull and Vogt, 1981)
is that portion of the incubation period during which incubation
temperature can affect the eventual sex of hatchlings. Identify-
ing the TSD for a species is critical for models that are used to
explore the relationship between temperatures in natural nests
and the sex of resulting offspring (e.g., Georges et al., 2004). The
TSP has not been reported for Diamond-backed Terrapins,
although Jeyasuria et al. (1994) and Jeyasuria and Place (1997)
estimated the TSP for Diamond-backed Terrapins to start at
embryonic stage 12â€“14, both without explanation. The TSP has
been reported for four other emydid turtles: European Pond
Turtle (Emys orbicularis) (stages 16â€“21; Pieau and Dorizzi, 1981),
Red-eared Slider (Trachemys scripta scripta (stages 16â€“19 or 20;
Wibbels et al., 1991), Ouachita Map Turtle (Graptemys ouachi-
tensis) (stages 16â€“22; Bull and Vogt, 1981); and Painted Turtle
(Chrysemys picta) (stages 16â€“22; Bull and Vogt, 1981). All five of
these studies used Yntema (1968) to identify embryonic stages,
so their results are comparable.
Our goals were to 1) determine whether the Diamond-backed
Terrapin is a TSD type Ia species as is common in emydid
turtles, 2) calculate the pivotal temperature of sex determination
and TRT for the northern Diamond-backed Terrapin, 3) explore
geographic variation in pivotal temperature of sex determina-
tion and TRT in Diamond-backed Terrapins, and 4) test the
hypothesis of Jeyasuria et al. (1994) regarding the TSP and
1Corresponding Author. E-mail: biorlb@hofstra.edu
DOI: 10.1670/13-188
developmental stage for Diamond-backed Terrapins. We ex-
plored these phenomena in Diamond-backed Terrapins because
of our investigations into the relationship between temperatures
in wild nests and offspring sex ratios (e.g., Scholz, 2007) in this
species. We used constant temperature incubation temperatures
to address goals 1â€“3 and shift-temperature experiments to
address goal 4.
MATERIALS AND METHODS
We collected Diamond-backed Terrapinsâ€™ freshly laid eggs
from nests in 2009 and 2010 on Rulers Bar Hassock, a 463-ha
island (408570N, 738500W) in the mainly estuarine Jamaica Bay
Wildlife Refuge. This site is located in Kings and Queens
counties, New York, and near the center of the range of the
Northern Diamond-backed Terrapin subspecies Malaclemys
terrapin terrapin. In both years, we transported eggs to the
laboratory; there they were marked and mass was determined.
Clutches were divided so as to maximize representation of
clutches among treatments. They were buried in vermiculite
that had been mixed 2 parts distilled water:1 part vermiculite by
weight and placed in constant temperature incubators within 48
h of oviposition. The mass of eggs and containers was
determined weekly during incubation, and evaporated water
was replaced.
In 2009, we incubated eggs at constant temperatures (24, 25,
26, 27, 28, 29, 30, 31, and 328C) until hatching. We added data
for eggs incubated at constant 348C from Giambanco (2003)
where applicable; Giambanco collected eggs in 2000 from the
same population and used the same methodology described
here for constant-temperature experiments. After pipping, eggs
were removed from the incubators and hatchlings were allowed
to emerge fully from the eggs. Hatchlings were then euthanized
via chemical injection 3â€“4 d postemergence. Sex was identified
by gross inspection of gonads using the presence or absence of
MuÂ¨llerian ducts (indicating female and male, respectively), and
both microscopic and macroscopic examinations of the external
appearance of the gonads.
In 2010, we collected eggs and incubated them as described
above, except we conducted shift experiments (Valenzuela,
2001) to identify the TSP. We used baseline temperatures of 25
and 318C. Eggs were held at one of the single baseline
temperatures through most of incubation, but they were shifted
to a different temperature for a specific length of time (Figs. 1
and 2). The baseline temperatures for the shift experiments (25
and 318C) were chosen as likely to be male producing and
female producing, respectively, based on results from 2009
experiments. We predicted incubation duration for the shift
experiments based on results from 2009 experiments, and we
divided the predicted incubation duration for the 318C baseline
into 5-d intervals and the predicted incubation duration for the
258C baseline into 7-d intervals (Figs. 1 and 2). At each time
interval when eggs were moved from their assigned baseline
incubation temperature to the shift temperature, five eggs were
chosen randomly for preservation. With these eggs, we used a
bright light to locate an area within the shells without blood
vessels; there, we used forceps to peel back the eggshells and
expose the embryos. The embryos were removed and placed in
Bouinâ€™s solution for preservation. Preserved embryos were
staged according to Yntema (1968) and Greenbaum (2002).
We analyzed the constant temperature and sex of offspring
data (including that of Giambanco [2003]) to assess sex
determination mode and estimate both TSP and TRT with the
data from Diamond-back Terrapins from New York and
Maryland (Jeyasuria et al., 1994) using the program TSD 4.0.3
(Girondot, 1999, 2012; Godfrey et al., 2003). Sex ratio data were
fitted using several models (GSD, logistic, Hill, and Richards)
using maximum likelihood with binomial distributions. The
GSD model for genotypic sex determination is a model in which
sex ratio has a predicted value of 0.5 and sex ratio is
independent of incubation temperature. The logistic and Hill
models both use a sigmoidal equation with two parameters,
whereas the Richards model uses a sigmoidal equation with
three parameters. Goodness of fit testing was based on
likelihood ratios between the saturated model (df1 = 0) and
fitted model (df2 = [number of temperatures] â€“ [number of
estimated parameters]). Comparisons between different models
were conducted using Akaike Information Criterion (AIC), a
method to rank different models by order of merit by penalizing
for too many parameters. Akaike weight is a method that
converts AIC into probabilities assessing whether each model is
really the best choice among the tested models (Burnham and
Anderson, 2013).
RESULTS
The GSD model can be excluded from fitting the New York
data on the basis of a very high AIC ranking and very low
weight (goodness of fit < 0.001, Table 1). The logistic and Hill
FIG. 1. Shift experiments with 258C baselines, indicating shift periods
and resulting sex ratios (male:female).
TSD IN DIAMOND-BACKED TERRAPINS 467
models both performed better than the Richards model, based
on AIC ranking and Akaike weight. However, although the
logistic and Hill models cannot be distinguished, we used the
logistic based on parsimony because the Hill model is a special
case of the logistic model with the x-axis being log transformed.
Goodness of fit testing for New York data rejected the model
as fitting the data correctly (p = 10-5). Observations at four
incubation temperatures were particularly unlikely, such as the
male observed at 348C. However, even if this observation is
removed from the data set, the model is still rejected (p =
2.10-3), because there are significant variations in sex ratio
among incubation temperatures that are not well represented by
this model (Fig. 3). This could be due to fluctuation or
nonstability of temperature within incubators or maternal
effects.
The pivotal temperature for the New York data was 28.168C
and the TRT was 25.88â€“30.458C (Fig. 3). Incubation duration at
constant 258C averaged 69.4 d (SD = 2.0); incubation duration at
constant 318C averaged 43.2 d (SD = 1.3).
Only two incubation temperatures are available for the New
Jersey and Virginia populations, and none of these temperatures
produced mixed sex ratios. With such a situation, it was
impossible to fit these data to any model, and it did not make
sense to add them to our other data sets.
The GSD model can be excluded from fitting the Maryland
data on the basis of a very high AIC ranking and very low
Akaike weight (goodness of fit < 0.001; Table 1). The logistic
model (goodness of fit = 1.00) performed better than the Hill or
Richards models, based on AIC ranking and Akaike weight.
The pivotal temperature for the Maryland data was 29.258C and
the TRT was 28.85â€“29.648C.
The combined data set (New York data and Maryland data)
had a high AIC value and low Akaike weight (Table 1),
especially in comparison with the two data sets analyzed
separately (Table 1). This indicates significant differences
between the data sets (goodness of fit < 0.001).
Our shift experiment with the 258C baseline indicated that the
TSP occurred between incubation days 28 and 35, 40.3â€“50.4% of
incubation (Fig. 1). All five embryos preserved from 258C
baseline after 28 days of incubation were at developmental
stage 16 as described in Yntema (1968) and Greenbaum (2002).
FIG. 2. Shift experiments with 318C baselines, indicating shift periods
and resulting sex ratios (male:female).
TABLE 1. Comparison of model fits among the populations of M. terrapin using AIC and Akaike weight.
Population Model AIC Akaike weight
New York GSD 300.01 2.785 Â· 10-55
New York Logistic 50.52 4.165 Â· 10-1
New York Hill 50.46 4.303 Â· 10-1
New York Richards 2.07 1.532 Â· 10-1
Maryland GSD 176.141 8.44 Â· 10-38
Maryland Logistic 7.129 4.237 Â· 10-1
Maryland Hill 7.14 4.20 Â· 10-1
Maryland Richards 9.13 1.56 Â· 10-1
New York and Maryland combined Logistic 69.18 3.13 Â· 10-3
New York and Maryland separate Logistic 57.65 9.97 Â· 10-1
FIG. 3. Relationship between constant incubation temperature and
offspring sex (percentage of males) in the Diamond-backed Terrapin.
Plotted points indicate actual data; curves indicate the Hill equation
(dashed line) fit to data bracketed by confidence interval of 2 SDs (solid
lines).
468 R. L. BURKE AND A. M. CALICHIO
The shift experiment with the 318C baseline indicated that the
TSP occurred between incubation days 30 and 35, 69.4â€“81.0% of
incubation (Fig. 2). Four of the five embryos preserved from
318C baseline after 30 d of incubation were malformed, and we
could not stage them reliably. The remaining embryo was at
developmental stage 23. The embryos from the previous shift
(removed at day 25) were at developmental stage 22; the
embryos from the subsequent shift (removed at day 35) were at
developmental stage 24.
DISCUSSION
We found that Diamond-backed Terrapins have TSD Ia
pattern, as originally suggested, based on limited data, by
Ewert and Nelson (1991). The pivotal temperature for the New
York population is 1.098C cooler than Diamond-backed Terra-
pins from populations from Maryland. These results span the
two most northern of the six genetically based management
units delineated by Hart (2005) and a range of ~400 km, which
is only ~20% of the Diamond-backed Terrapin northâ€“south
distribution. Because of the large fraction of the Diamond-back
Terrapin range for which TSD data are unknown, there is still
considerable possibility for even more variation in pivotal
temperature and TRT in this species, as has been demonstrated
in other turtles (Bull et al., 1982; Ewert et al., 1994, 2004, 2005).
However, our results indicate that pivotal temperatures and
TRT for both populations of Diamond-backed Terrapins are
within the range of other emydid turtles (28â€“298C and 1â€“28C,
respectively, e.g., Pieau, 1976; Bull et al., 1982; Crews et al., 1991;
Ewert and Nelson, 1991; Ewert et al., 2004; Hulin et al., 2009).
Our finding that the TSP for Diamond-backed Terrapins
corresponds with embryonic stages 16â€“23 is not surprising
because it is consistent with other emydid turtles (Bull and Vogt,
1981; Pieau and Dorizzi, 1981; Wibbels et al., 1991). Mrosovsky
and Pieau (1991) suggested that the TSP in turtles occurred in
the middle third of incubation. Although our findings confirm
this for eggs incubated at the 258C baseline, the TSP occurred in
the last third of incubation for eggs incubated at 318C. The cause
of this striking variation is unclear, and we are unaware of
similar results in any other species. In all cases, the same guide
to staging embryos (Yntema, 1968) was used.
Our results have potentially valuable applications. For
example, Scholz (2007) recorded temperatures in 82 Diamond-
backed Terrapin nests throughout incubation at our study site in
2004. Average temperatures for these nests varied considerably
(18.9â€“27.08C, mean = 25.28C, SD = 1.6). Therefore, these nests
apparently spent relatively little time near or above the pivotal
temperature and were probably biased strongly toward male
hatchlings. Further analysis is needed to investigate the
relationships between constant temperature incubation results
reported here and variable temperature incubation patterns
observed in the field.
Acknowledgments.â€”We are grateful to B. Clendening, J.
Joseph, and C. Peterson for assistance with techniques and
data collection. E. Hotaling shared unpublished data, M.
Godfrey helped interpret TSD output, M. Girondot was
extraordinarily helpful with analytical assistance and use of
his model, and L. Rodriguez helped with the Spanish
translation. This work was permitted under New York State
License to Collect and Possess #383 and the Hofstra University
Institutional Animal Care and Use Committee.
LITERATURE CITED
BULL, J. J., AND R. C. VOGT. 1981. Temperature-sensitive periods of sex
determination in emydid turtles. Journal of Experimental Zoology.
218:435â€“440.
BULL, J. J., R. C. VOGT, AND C. J. MCCOY. 1982. Sex determining
temperatures in turtles: a geographic comparison. Evolution 36:
326â€“332.
BURNHAM, K. P., AND D. R. ANDERSON. 2013. Model Selection and
Multimodel Inference: A Practical Information-Theoretic Approach.
2nd ed. Springer, USA.
CREWS, D., J. J. BULL, AND T. WIBBELS. 1991. Estrogen and sex reversal in
turtles: a dose-dependent phenomenon. General and Comparative
Endocrinology 81:357â€“364.
DOODY, J. S., E. GUARINO, A. GEORGES, B. COREY, G. MURRAY, AND M. EWERT.
2006. Nest site choice compensates for climate effects on sex ratios in
a lizard with environmental sex determination. Evolutionary
Ecology 20:307â€“330.
EWERT, M., AND C. NELSON. 1991. Sex determination in turtles: diverse
patterns and some possible adaptive values. Copeia 1991:50â€“69.
EWERT, M. A, C. R. ETCHBERGER, AND C. E. NELSON. 2004. Turtle sex-
determining modes and TSD patterns, and some TSD pattern
correlates. In N. Valenzuela and V. Lance (eds.), Temperature-
dependent Sex Determination in Vertebrates, pp. 21â€“32. Smithsonian
Books, USA.
EWERT, M. A., D. R. JACKSON, AND C. E. NELSON. 1994. Patterns of
temperature-dependent sex determination in turtles. Journal of
Experimental Zoology 270:3â€“15.
EWERT, M. A., J. W. LANG, AND C. E. NELSON. 2005. Geographic variation in
the pattern of temperature-dependent sex determination in the
American snapping turtle (Chelydra serpentina). Journal of Zoology
265:81â€“95.
GEORGES, A., C. LIMPUS, AND R. STOUTJESDIJK. 1994. Hatchling sex in the
marine turtle Caretta caretta is determined by proportion of
development at a temperature, not daily duration of exposure.
Journal of Experimental Zoology 270:432â€“444.
GEORGES, A., S. DOODY, K. BEGGS, AND J. YOUNG. 2004. Thermal models of
TSD under laboratory and field conditions. In N. Valenzuela and V.
Lance (eds.), Temperature-dependent Sex Determination in Verte-
brates, pp. 79â€“89. Smithsonian Books, USA.
GIAMBANCO, M. R. 2003. Comparison of Viability Rates, Hatchling
Survivorship and Sex Ratios of Laboratory- and Field-incubated
Nests of the Estuarine Emydid Turtle Malaclemys terrapin. Unpubl.
M.S. thesis, Hofstra University, USA.
GIRONDOT, M. 1999. Statistical description of temperature-dependent sex
determination using maximum likelihood. Evolutionary Ecology
Research 1:479â€“486.
â€”â€”â€”. 2012. TSD (Version 3.0.4). CNRS et UniversiteÂ´ Paris-Sud, UMR-
8079-Ecologie, SysteÂ´matique et Evolution, Department dâ€™Ecologie
des Populations et des CommunauteÂ´s, Groupe de Conservation des
Populations et des communauteÂ´s, France.
GODFREY, M. H., V. DELMAS, AND M. GIRONDOT. 2003. Assessment of
patterns of temperature-dependent sex determination using maxi-
mum likelihood model selection. Ecoscience 10:265â€“272.
GREENBAUM, E. 2002. A standardized series of embryonic stages for the
emydid turtle Trachemys scripta. Canadian Journal of Zoology 80:
1350â€“1370.
HART, K. M. 2005. Population Biology of Diamond-backed Terrapins
(Malaclemys terrapin): Defining and Reducing Threats across Their
Range. Ph.D. diss., Duke University, USA.
HAUSWALDT, J. S., AND T. C. GLEN. 2005. Population genetics of the
diamond-backed terrapin (Malaclemys terrapin). Molecular Ecology
14:723â€“732.
HULIN, V., V. DELAMAS, M. GIRONDOT, M. H. GODFREY, AND J.-M. GUILLON.
2009. Temperature-dependent sex determination and global climate
change: are some species at greater risk? Oecologica 160:493â€“506.
JEYASURIA, P., AND A. R. PLACE. 1997. Temperature-dependent aromatase
expression in developing diamondback terrapin (Malaclemys terra-
pins) embryos. Journal of Steroid Biochemistry and Molecular
Biology 61:415â€“425.
JEYASURIA, P., W. M. ROOSENBURG, AND A. R. PLACE. 1994. Role of P-450
aromatase in sex determination of the diamondback terrapin,
Malaclemys terrapin. Journal of Experimental Zoology 270:85â€“111.
MROSOVSKY, N., AND C. PIEAU. 1991. Transitional range of temperature,
pivotal temperatures and thermosensitive stages for sex determina-
tion in reptiles. Amphibia-Reptilia 12:169â€“179.
PIEAU, C. 1976. DonneÂ´es reÂ´centes sur la diffeÂ´renciation sexuelle en
function de la temperature chez les embryons dâ€™Emys orbicularis L.
TSD IN DIAMOND-BACKED TERRAPINS 469
(CheÂ´ lonien). Bulletin of the Zoological Society of France
101(Suppl.)4:46â€“53.
PIEAU, C., AND M. DORIZZI. 1981. Determination of temperature sensitive
stages for sexual differentiation of the gonads in embryos of the
turtle (Emys orbicularis). Journal of Morphology 170:373â€“382.
SARRE, S. D., A. GEORGES, AND A. QUINN. 2004. The end of a continuum:
genetic and temperature-dependent sex determination in reptiles.
Bioessays 26:639â€“645.
SCHOLZ, A. L. 2007. Impacts of Nest Site Choice and Nest Characteristics
on Hatchling Success in the Diamondback Terrapins of Jamaica Bay,
New York. Unpubl. M.S. thesis, Hofstra University, USA.
VALENZUELA, N. 2001. Constant, shift, and natural temperature effects on
sex determination in Podocnemus expansa turtles. Ecology 82:3010â€“
3024.
WIBBELS, T., J. J. BULL, AND D. CREWS. 1991. Chronology and morphology
of temperature-dependent sex determination. Journal of Experimen-
tal Zoology 260:371â€“381.
YNTEMA, C. L. 1968. A series of stages in the embryonic development of
Chelydra serpentina. Journal of Morphology 125:219â€“252.
Accepted: 12 January 2014.
470 R. L. BURKE AND A. M. CALICHIO
Copyright of Journal of Herpetology is the property of Society for the Study of Amphibians &
Reptiles and its content may not be copied or emailed to multiple sites or posted to a listserv
without the copyright holder's express written permission. However, users may print,
download, or email articles for individual use.