ArticlePDF Available

Abstract and Figures

Uricotelic species, such as squamate reptiles, birds and insects, effectively eliminate nitrogen as uric acid in a solid form commonly called urates. Observations made over a decade suggested that the voided urates produced by colubroids (modern snake species) exhibit remarkable differences from those of boids and pythons (ancient snake species). Here, we compare the urates generated by eight captive snake species fed the same diet. Although all fresh urates were wet at the time of excretion, those produced by modern snakes dried to a powdery solid, whereas those of ancient species dried to a rock-hard mass that was tightly adherent to surfaces. Powder X-ray diffraction and infrared spectroscopy analyses performed on voided urates produced by five modern and three ancient snakes confirmed their underlying chemical and structural differences. Urates excreted by ancient snakes were amorphous uric acid, whereas urates from modern snakes consisted primarily of ammonium acid urate, with some uric acid dihydrate. These compositional differences indicate that snakes have more than one mechanism to manage nitrogenous waste. Why different species use different nitrogen-handling pathways is not yet known, but the answer might be related to key differences in metabolism, physiology or, in the case of ancient snakes, the potential use of urates in social communication. ADDITIONAL KEYWORDS: ammonium acid urate-behaviour-chemistry-ecology-powder X-ray diffraction-renal-Reptilia-snakes-uric acid.
Content may be subject to copyright.
1
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, XX, 1–10
Biological Journal of the Linnean Society, 2021, XX, 1–10. With 4 figures.
Urates of colubroid snakes are different from those of
boids and pythonids
ALYSSA M. THORNTON1,, GORDON W. SCHUETT2,3,* and JENNIFER A. SWIFT1,*
1Department of Chemistry, Georgetown University, Washington, DC 20057, USA
2Chiricahua Desert Museum, Rodeo, NM 88056, USA
3Department of Biology and Neuroscience Institute, Georgia State University, Atlanta, GA 30303, USA
Received 21 January 2021; revised 7 March 2021; accepted for publication 10 March 2021
Uricotelic species, such as squamate reptiles, birds and insects, effectively eliminate nitrogen as uric acid
in a solid form commonly called urates. Observations made over a decade suggested that the voided urates
produced by colubroids (modern snake species) exhibit remarkable differences from those of boids and pythons
(ancient snake species). Here, we compare the urates generated by eight captive snake species fed the same diet.
Although all fresh urates were wet at the time of excretion, those produced by modern snakes dried to a powdery
solid, whereas those of ancient species dried to a rock-hard mass that was tightly adherent to surfaces. Powder
X-ray diffraction and infrared spectroscopy analyses performed on voided urates produced by five modern and
three ancient snakes confirmed their underlying chemical and structural differences. Urates excreted by ancient
snakes were amorphous uric acid, whereas urates from modern snakes consisted primarily of ammonium acid
urate, with some uric acid dihydrate. These compositional differences indicate that snakes have more than one
mechanism to manage nitrogenous waste. Why different species use different nitrogen-handling pathways is not
yet known, but the answer might be related to key differences in metabolism, physiology or, in the case of ancient
snakes, the potential use of urates in social communication.
ADDITIONAL KEYWORDS: ammonium acid urate – behaviour – chemistry – ecology – powder X-ray diffraction
– renal – Reptilia – snakes – uric acid.
INTRODUCTION
Every living organism needs a mechanism to manage
excess nitrogen. Among vertebrates, nitrogen that
cannot be utilized is excreted in the form of ammonia
(NH3), urea [CO(NH2)2] or uric acid (C5H4N4O3) (Walsh &
Wright, 1995). Different animal species can access these
interrelated nitrogenous waste products to various
extents, such that species are typically classified as
ammonotelic, ureotelic or uricotelic based on the dominant
metabolic pathway. The production of uric acid as a
final nitrogenous waste material is more energetically
demanding compared with urea and ammonia, but its
excretion as a solid requires significantly less water.
The prevalence of uricotelism across reptiles, birds and
insect species is largely thought to be an evolutionary
adaptation to meet water conservation needs. In fact,
some amphibian species, such as foam-nest tree frogs
(Chiromantis xerampelina), have been shown to alter
the activity of their waste nitrogen metabolism enzymes
in response to the availability of water (Balinsky, 1972;
Balinsky et al., 1976).
The early study of metabolic wastes in squamate
reptiles (lizards, amphisbaenians and snakes),
particularly excess nitrogen, was focused primarily on
understanding the structural and chemical properties
of ureteral–cloacal urine and voided urine (Khalil,
1948a, b; Minnich, 1972; Minnich & Piehl, 1972).
Subsequent work emphasized renal morphology and
physiological (osmotic and ionic) regulation (King &
Goldstein, 1985; Ditrich, 1996; Dantzler & Bradshaw,
2008; Urity et al., 2012) and attempts to understand
ecological and evolutionary trends (Schmidt-Nielsen,
1997). More recent work has concentrated on social
aspects, such as territorial marking via scats (urates
and faeces) in lizards (Duvall et al., 1987; Bull et al.,
1999a, b; Shah et al., 2006; Baeckens et al., 2019).
applyparastyle “g//caption/p[1]” parastyle “FigCapt”
*Corresponding authors. E-mail: jas2@georgetown.edu;
gwschuett@yahoo.com
Downloaded from https://academic.oup.com/biolinnean/advance-article/doi/10.1093/biolinnean/blab052/6261016 by Georgetown University user on 30 April 2021
2 A. M. THORNTON ET AL.
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, XX, 1–10
This study was prompted by a series of general
observations made on a large number of snakes over
the course of the past decade. It was noted that urate
wastes (urates or urate pellets) produced and excreted
by modern taxa (e.g. colubrids, elapids and viperids)
and ancient snakes (e.g. boids and pythonids) differ
qualitatively in several key aspects, including the
timing of excretion, their physical appearance and their
mechanical strength. Notably, modern snake species
always excrete wet urates and faeces simultaneously,
and the urates dry quickly (< 1 day) to a powdery, sand-
like solid. In contrast, ancient species typically expel
urates in two intervals: the first void alone and the
second in tandem with faeces. Especially from large-
bodied taxa, the freshly voided ancient snake urates
initially have a thick, wet, toothpaste-like consistency
but solidify to a very hard material over several days
to weeks depending on mass (e.g. several grams in
small-sized individuals to > 400 g in giant species).
Furthermore, these solidified urates adhere tightly to
hard surfaces (e.g. plastics, wood and stone) on which
they are deposited, with the removal of such deposits
typically requiring sharp tools.
Prompted by these general observations, we sought
to establish whether the qualitative differences in
urates produced by these different snake species
were associated with differences in their chemical
composition and/or structure. Several species of similar
length from modern (Colubridae and Viperidae) and
ancient (Boidae and Pythonidae) snake lineages
were fed the same controlled diet and maintained
in common conditions (e.g. temperature and water
availability). Using X-ray diffraction and infrared
spectroscopic methods, we show, for the first time,
that the physicochemical properties of voided urates
from modern and ancient snake taxa are chemically
distinct. These results add to the growing literature
illustrating that snakes (Greene, 1997; Burbrink
et al., 2020) are not a monolithic group insofar as
behavioural, morphological and physiological traits
are concerned (Secor & Diamond, 1998; Castoe et al.,
2013; Lillywhite, 2014; Booth & Schuett, 2016; Gamble
et al., 2017; Perry et al., 2019) and that they might
have an important role as unconventional models in
addressing research questions related to efficient
nitrogen management.
MATERIAL AND METHODS
Study SpecieS
Postprandial voided uric acid waste samples (urates)
of adult snakes were obtained from eight different
species (N = 15 animals) of ancient (lineages Boidae
and Pythonidae) and modern (Colubroidea: lineages
Colubridae and Viperidae) taxa (see Greene, 1997;
Burbrink et al., 2020) housed at the Chiricahua Desert
Museum (Rodeo, NM, USA). The three species of
ancient snakes studied included two species of African
pythonids (Angolan python, Python anchietae, and
ball python, Python regius) and one species of boid
from Madagascar (Madagascan tree boa, Sanzinia
madagascariensis). The five modern snakes, all New
World taxa from North America, included three species
of colubrids (Trans-Pecos rat snake, Bogertophis
subocularis, desert kingsnake, Lampropeltis splendida,
and the Mexican hog-nosed snake, Heterodon kennerlyi)
and two species of viperids (western diamond-backed
rattlesnake, Crotalus atrox, and Mojave rattlesnake,
Crotalus scutulatus). All snakes were of similar size,
ranging from ~80 to 100 cm in length.
production and collection of SampleS
All snakes at the Chiricahua Desert Museum were
offered one to four frozen (thawed) adult laboratory
mice (a strain from a common local facility). Water
(from a common source) was available from glass
bowls ad libitum. Room temperature was 72–74 °F
(~22–23 °C), and cage temperature permitted
thermoregulation via a commercial heat strip at one
end maintained at 90 °F (32 °C). The semi-arboreal boid
(Madagascan tree boa) had an incandescent basking
light (90–95 °F) and was housed in a commercial snake
enclosure (61 cm × 61 cm × 61 cm). All other snakes
studied were housed and maintained in identical
enclosure conditions with the same dimensions
(59 cm × 41 cm × 15 cm). Lighting and supplementary
heat were maintained on a 12 h light–12 h dark cycle.
After feeding, the snakes were inspected every 12 h
for potential wastes (urates and/or faeces). Owing to
sample size, we analysed data from 20 trials on the
timing of postprandial excretion in the ball pythons
(N = 4) and the two species of rattlesnakes (N = 4).
Urates naturally excreted by each individual were
deposited on clean, fresh, commercial paper towelling.
Only the urates (white material) that could be
separated from the faeces (if present) cleanly and
reliably were used in subsequent analyses. All urate
samples were maintained in ambient temperature and
humidity conditions and kept out of direct light until
the time of analysis (Supporting Information, Fig. S1).
X-ray diffraction analySiS
Powder X-ray diffraction (PXRD) data were collected
on urate samples from all eight species of snakes
~2 months after excretion using a Bruker Apex
DUO X-ray diffractometer (Cu Kα radiation, 50 kV,
30 mA current). Samples ground with a mortar and
pestle were mounted in Kapton capillaries, with data
Downloaded from https://academic.oup.com/biolinnean/advance-article/doi/10.1093/biolinnean/blab052/6261016 by Georgetown University user on 30 April 2021
URATE DIFFERENCES IN SNAKES 3
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, XX, 1–10
collection from 2θ = 5 to 50°. The PXRD patterns were
compared against all known crystal forms of uric
acid and its urate salts available in the Cambridge
Structure Database (Groom et al., 2016), including
anhydrous uric acid [refcode: URICAC (Ringertz,
1966)], uric acid monohydrate [GEJQAO (Schubert
et al., 2005)], uric acid dihydrate [ZZZPPI02 (Parkin
& Hope, 1998)], sodium urate monohydrate [NAURAT
(Mandel & Mandel, 1976)], calcium urate hexahydrate
[YODJAE (Presores et al., 2013)], magnesium
urate [BADTEX10 (Dubler et al., 1986)], potassium
quadriurate [PABRIW (Bazin et al., 2016)] and
ammonium acid urate [HOZSUL (Friedel et al., 2006)].
Two single crystals large enough for single-
crystal X-ray diffraction were isolated from the
Mojave rattlesnake sample. Single-crystal data were
collected on a Bruker D8 Quest diffractometer (Mo
Kα radiation = 0.71073) equipped with a Photon
100 CMOS detector (Bruker AXS) at 100 K. APEX 3
software and SHELX were used for structure solution
and refinement. Both single crystals had a unit cell
consistent with uric acid dihydrate (Parkin & Hope,
1998). Refinement of the higher-quality data set
yielded a complete structure with a final R-factor of
4.67%. Whole-molecule disorder and twinning were
observed. The CCDC deposition number is 2041488.
infrared SpectroScopy
Fourier-transformed infrared (FT-IR) spectra of ground
(mortar and pestle) snake urate samples, ~20 months
after excretion, were recorded on a Perkin Elmer
Spectrum-Two FT-IR spectrophotometer equipped
with a UATR-TWO diamond ATR attachment. Scans
were collected on each sample over a 600–4000 cm−1
range, with each spectrum representing an average of
ten scans.
data availability
Optical micrographs and powder X-ray data are
provided in the Supporting Information, Fig. S1. Any
other data may be obtained from the corresponding
authors on reasonable request.
RESULTS
timing of poStprandial urate eXcretion
After feeding, without exception, the adult ball pythons
(N = 4 subjects) excreted urates at two different time
points. In all 20 trials (20/20), the first excretion (urate-
1) consisted only of urates (no faeces) and occurred
within 3–7 days of feeding (N = 20 trials, z-test, z =
4.248; null = 0.5, two-tailed, P < 0.01). The second
excretion of urates (urate-2) also involved a bowel
movement (faeces) and occurred 7–15 days after urate-
1. The second urate excretions were typically smaller in
size (mass). Regardless of the time at which they were
excreted (i.e. urate-1 or urate-2), all ball python urates
exhibited the same initial toothpaste-like consistency
and dried to a hard mass within several days (Fig.
1A). The pattern of urate excretion was similar in the
Madagascan tree boa and Angolan python (i.e. urate-1
and urate-2) we examined.
In the conditions of this study, the two species of
adult rattlesnakes (N = 4 subjects) always excreted
urate and faecal wastes in tandem within 6–10 days
after feeding (N = 20 trials, z-test, z = 4.248; null = 0.5,
two-tailed, P < 0.01). Occasionally, but not reliably, a
second void of urates and faeces occurred 5–8 days
after the first one, especially when meals were large
(e.g. two or three mice). Urates dried quickly over a
period of ~1 day, yielding a sand-like consistency
(Fig. 1B). Postprandial urate excretion in the other
Figure 1. Optical micrographs of snake urate samples.
A, air-dried urate sample (incomplete) from an adult ball
python (Python regius), captive held. B, air-dried urate
sample (complete) from an adult female Mojave rattlesnake
(Crotalus scutulatus), captive held. Scale bar: 1 cm.
Downloaded from https://academic.oup.com/biolinnean/advance-article/doi/10.1093/biolinnean/blab052/6261016 by Georgetown University user on 30 April 2021
4 A. M. THORNTON ET AL.
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, XX, 1–10
colubroids (Mexican hognose snake, desert kingsnake
and Trans-Pecos rat snake) had patterns that were
similar to the two species of rattlesnakes but occurred
earlier (i.e. faeces and urates were excreted in tandem
4–8 days after feeding).
powder X-ray diffraction
Representative PXRD data of urates from all snakes
tested are shown in Figure 2. All sample data shown
were collected ~2 months after excretion. Comparison
of the diffractograms obtained on urates from the three
ancient snake species (one boid and two pythonids)
showed that they were qualitatively similar. There
were no obvious differences between urate-1 and
urate-2 samples produced. Each sample exhibited
only one broad diffraction line of reasonable intensity
at 2θ = 27.8°. The peak position corresponds to an
average d-spacing of ~3.2 Å, which is consistent with
the expected separation distance between π-stacked
heteroaromatic units. With only one broad diffraction
line in the PXRD pattern, the sample is amorphous.
The PXRD patterns obtained on urates excreted by
all five modern snake species appeared qualitatively
similar to one another, but distinctly different from
those excreted by ancient snakes. Given the irregularity
of a second excretion, only the first voided urate in the
modern species was tested. All modern snake urates
exhibited diffraction peaks at 2θ = 9.1, 10.1, 15.6, 18.2,
19.1 and between 24.6 and 30.2° (±0.2°). The PXRD
patterns were compared against several known uric
acid and urate crystalline forms and found to match
most closely the pattern for ammonium acid urate
(Supporting Information, Fig. S2). During examination
of the Mojave rattlesnake urate under polarized light
microscopy, two individual optically transparent single
crystals in the sample were identified and isolated
from the larger sample (Supporting Information, Fig.
S3). Structure determination with single-crystal X-ray
diffraction confirmed that they were uric acid dihydrate.
Powder X-ray diffraction data were re-collected on
the same ball python and Mojave rattlesnake samples
~12 months after their initial excretion. The ball python
sample showed several additional diffraction lines,
indicating that the sample had partly crystallized during
this extended time period to a mixture of anhydrous uric
acid and uric acid dihydrate. There were no apparent
age-related changes in the PXRD pattern of the Mojave
rattlesnake sample (Supporting Information, Fig. S4).
infrared SpectroScopy analySiS
Comparison of infrared spectra of urates from
modern snakes showed that they were qualitatively
similar to each other, as in the PXRD analysis, but
different from those excreted by the ancient snakes
we examined (Fig. 3). Urates from ancient and
modern snakes have broad absorption in the region
Figure 2. Representative powder X-ray diffractograms obtained on snake urate samples excreted from eight different
ancient (boid and pythonid) and modern (colubroid) species. All individuals were fed the same controlled rodent diet and
maintained in common enclosure conditions.
Downloaded from https://academic.oup.com/biolinnean/advance-article/doi/10.1093/biolinnean/blab052/6261016 by Georgetown University user on 30 April 2021
URATE DIFFERENCES IN SNAKES 5
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, XX, 1–10
between 3500 and 2500 cm−1, and distinct vibrations
in the lower fingerprint region point toward different
compositions. Reference infrared spectra for many
crystalline constituents identified in renal deposits
have been reported previously (Modlin & Davies,
1981), including those of the most common forms of
uric acid/urate: ammonium acid urate, sodium urate
monohydrate, anhydrous uric acid and uric acid
dihydrate. Ammonium acid urate has a two sharp
peaks at 740 and 780 cm−1, whereas both anhydrous
and dihydrate forms of uric acid have three strong
absorption bands between 700 and 500 cm−1. The
infrared spectra of urates from all the colubroid taxa
(modern snakes) tested were entirely consistent with
ammonium acid urate, especially when taken in
combination with the PXRD data. In contrast, each of
the three urates from the boid and pythonids (ancient
snakes) had three absorptions in the region between
700 and 500 cm−1, which is consistent with uric acid in
its protonated form. Data are summarized in Table 1.
DISCUSSION
In physiological solutions, uric acid (a weak acid, with
a pKa ~5.5; Finlayson, 1974) exists in equilibrium
with urate, its deprotonated anion. Studies of this
compound have shown that it can precipitate in many
different solid forms depending on the pH and ionic
strength of the fluid in which it is found. In mammals,
uric acid concentrations that exceed the solubility
limit can result in kidney stones and gout deposits. In
humans, the solid form identified in uric acid kidney
stones is most often the dihydrate or anhydrate
(Herring, 1962). In controlled laboratory conditions,
the former has been shown to be less stable, such that
it can convert to the latter over time (Zelellow et al.,
2010; Presores & Swift, 2014). The most common salt
forms identified in mammalian species include sodium
urate monohydrate, which is widely associated with
gout in humans (Mandel, 1976), and ammonium urate,
which is frequently identified in the kidney stones of
Dalmatian dogs (Bartges et al., 1994) and managed
bottlenose dolphins (Tursiops truncatus) (Venn-
Watson et al., 2010). The crystal structures of some of
these forms are shown in Figure 4.
Unlike mammals, uricotelic species within the
lineages Amphibia and Reptilia (avian and non-avian
reptiles) are thought not to suffer from such maladies,
owing to anatomical differences whereby excretory
products are released directly into the cloaca.
Nevertheless, to our knowledge at the outset of this
Figure 3. Experimental Fourier-transformed infrared spectra obtained on snake urate samples excreted by eight different
ancient and modern species snakes used in this study. All individuals were fed the same controlled rodent diet and
maintained in common conditions.
Downloaded from https://academic.oup.com/biolinnean/advance-article/doi/10.1093/biolinnean/blab052/6261016 by Georgetown University user on 30 April 2021
6 A. M. THORNTON ET AL.
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, XX, 1–10
study, there were no reports detailing the structural
or chemical composition of any postprandial snake
urates. Unlike previous work, which provided
incomplete descriptions of laboratory care (e.g. diet)
of test animals (e.g. Minnich, 1972), we intentionally
minimized several variables in our study by controlling
multiple environmental factors (i.e. enclosure type
and size, type and source of rodents, source of water
and temperature of the enclosure). Yet even in the
controlled conditions of this study, the postprandial
urates excreted by the boid and pythonids were
consistently different in several qualitative aspects
(e.g. production time after feeding, mechanical strength
and adhesion) compared with the urates produced by
Figure 4. (A) Ball python (Python regius). (B) Mojave rattlesnake (Crotalus scutulatus). Urates from the Mojave rattlesnake
consist primarily of (C) ammonium acid urate with small amounts of (D) uric acid dihydrate. Urates produced by the
Ball python, after extended ageing contain both (D) and (E) anhydrous uric acid. Crystal structure diagrams (C–E) were
generated in mercury from the corresponding cif files.
Table 1. Species of modern (colubrid and viperid) and ancient (boid and pythonid) snakes investigated in this study, with
a descriptive summary of general observations and powder X-ray diffraction data
Species Clade Ecology Consistency* Powder X-ray
diffraction
Major
component
Mexican hog-nosed snake Colubridae Semi-fossorial Powdery Multiple peaks NH4 urate
Desert kingsnake Colubridae Terrestrial Powdery Multiple peaks NH4 urate
Trans-Pecos rat snake Colubridae Terrestrial Powdery Multiple peaks NH4 urate
Western diamond-backed
rattlesnake
Viperidae Terrestrial Powdery Multiple peaks NH4 urate
Mojave rattlesnake Viperidae Terrestrial Powdery Multiple peaks NH4 urate
Madagascan tree boa Boidae Semi-arboreal Hard chunks Amorphous Uric acid
Angolan python Pythonidae Terrestrial Hard chunks Amorphous Uric acid
Ball python Pythonidae Terrestrial Hard chunks Amorphous Uric acid
*Refers to consistency of urates at the time of powder X-ray diffraction, which were dry when tested.
Downloaded from https://academic.oup.com/biolinnean/advance-article/doi/10.1093/biolinnean/blab052/6261016 by Georgetown University user on 30 April 2021
URATE DIFFERENCES IN SNAKES 7
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, XX, 1–10
the colubroids. Accordingly, it was not unexpected that
finer examination of the structural properties of these
urates would reveal important differences.
The PXRD analyses of the urates from the five
colubroid species we tested had multiple diffraction
lines, which, although broad, had a pattern consistent
with what is expected for ammonium urate. This
particular assignment is also in agreement with
infrared spectral bands in the fingerprint region
for this phase. However, neither piece of evidence
precludes the existence of minor amounts of other
forms, as demonstrated by the presence of a few
small, isolatable uric acid dihydrate crystals from
the Mojave rattlesnake urate sample. In contrast,
the urates of the boid and pythonid species were
amorphous and had infrared spectra consistent with
uric acid in its protonated state. The time between
sample excretion and testing also appears to be a
crucial variable, because at least in the boid and
pythonid species, changes in crystallinity in the
samples were found to occur over extended time
periods.
To the best of our knowledge, the qualitative
differences in ‘adhesiveness’ and mechanical strength
in the urates produced by ancient and modern snakes
have not been discussed in the scientific literature. The
closest to commenting on this aspect are Minnich &
Piehl (1972), who indicate that the urates of geckos and
other lizards dry to a harder material than those of most
snakes, which are much less compact. Their reasoning
for the difference in this property was the presence
of more ammonium urate and sodium chloride that
minimize aggregation of snake urates. The mechanical
properties of some, but not all, forms of solid uric
acid have been reported previously (Liu et al., 2018),
although rigorous mechanical measurements on pure
laboratory samples and biologically derived materials
are likely to be different owing to vast differences in
purity. Additional testing would be required to gain a
better understanding of this aspect.
Although the number of snake taxa we studied here
is a tiny fraction of the 3800 or so extant species (THE
REPTILE DATABASE, www.reptile-database.org),
the snakes included in this study allowed for the same
dietary input. In these controlled feeding conditions,
the analyses performed confirm that fundamental
differences exist in the urates across lineages.
Additional testing of urate samples from other species
in the clades we studied and species from other clades,
both ancient (e.g. scolecophidians, cylindrophiids,
uropeltids, loxocemids and xenopeltids) and modern
(e.g. acrochordids, elapids, lamprophiids and natricids),
would help to confirm whether the trends we report
here are substantiated across the broader phylogeny
of snakes. Also, we do not know to what extent varying
the dietary input would influence the production
and physicochemical attributes of the snake urates,
although diet might be significant (Greene & Cundall,
2000). This question might be addressed easily in
future studies in snake species where their diet is
broad (e.g. earthworms, insects and rodents) and can
be manipulated easily.
Of course, why snakes fed the same diets produce
urates with different chemical compositions in the
first place remains an open question. It is certainly
possible that the results derive from fundamental
physiological or metabolic differences. For example,
there might be differences in the levels of various
enzymes that regulate nitrogen metabolism across
snake species. At a minimum, it must be the case
that the colubroids examined here have higher
ammonium levels in their urate wastes than do boas
and pythons. However, it is not clear whether this is
because colubroids have a decreased ability to convert
ammonia to glutamine and other higher nucleotides
or if they are simply better able to handle the
ecological consequences of direct ammonia excretion.
Addressing these hypotheses would require rigorous
enzyme assays. Fundamental differences in the
microbiome in different species might also be relevant.
For example, if nitrogen-fixing bacteria that convert
ammonia to nitrogen gas are abundant in the guts
of boas and pythons, efficient ammonia conversion to
N2 might simply reduce ammonium concentrations
to levels too low to result in the crystallization of
ammonium urate.
At this time we are not able to provide experimental
evidence for the ecological or evolutionary significance
of the different types of urates produced by these
snakes. Unquestionably, additional research is
required. Owing to a paucity of comparative data on
this topic, especially for snakes and other reptiles
(Danzler 1996, 2005; H. Lillywhite, pers. com), new
research on the anatomy and physiology of snake
kidneys (and associated renal structures) and
metabolic processes would be needed to explore
potential differences in snakes from ancient and
modern lineages. Other research needs to be directed
at the potential functional role(s) of deposited urates
in nature. In many vertebrates, including squamate
reptiles such as lizards, urine (urates) and faeces
serve communicative and social functions (Müller-
Schwarze, 2006; Fenner & Bull, 2010; Apps et al., 2015;
Marneweck et al., 2017; Baeckens, 2019). Some of the
properties of urates from boids and pythonids, for
example, make them good candidates for behavioural
and physiological assays in the laboratory and the
field. Accordingly, important next steps based on the
new observations made in the present study might
include analysis of chemical profiles, such as hormones,
pheromones and signature mixtures (Halpern &
Martínez-Marcos, 2003; Mason & Parker, 2010; Wyatt,
Downloaded from https://academic.oup.com/biolinnean/advance-article/doi/10.1093/biolinnean/blab052/6261016 by Georgetown University user on 30 April 2021
8 A. M. THORNTON ET AL.
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, XX, 1–10
2010), coupled with behavioural experiments (Hebets
& Papaj, 2005; Hebets et al., 2016).
ACKNOWLEDGEMENTS
The Chiricahua Desert Museum kindly supplied the
urate samples. We thank Rachel Tevis, Richard Ihle and
Alexander Hall for their interest and assistance. Harvey
Lillywhite provided important literature and helpful
insights. We thank the three anonymous reviewers
for their valuable insights and recommendations that
greatly improved an earlier version of this manuscript.
None of the procedures involved institutional approval.
No animal subject was submitted to a testing
(manipulation) protocol. G.W.S. devised the study and
developed the testing procedures with J.A.S. and A.M.T.
All authors collected and analysed the data. G.W.S.,
J.A.S. and A.M.T. drafted and revised the manuscript.
All authors approved the final version of the manuscript
and agree to be held accountable for the content therein.
We declare we have no competing interests. We are
grateful for financial support provided by the National
Science Foundation (DMR 2004435), Georgetown
University and the Chiricahua Desert Museum.
REFERENCES
Apps PJ, Weldon PJ, Kramer M. 2015. Chemical signals in
terrestrial vertebrates: search for design features. Natural
Product Reports 32: 1131–1153.
Baeckens S. 2019. Evolution of animal chemical
communication: insights from non-model species and
phylogenetic comparative methods. Belgian Journal of
Zoology 149: 63–93.
Baeckens S, De Meester W, Tadić Z, Van Damme R. 2019.
Where to do number two: Lizards prefer to defecate on the
largest rock in the territory. Behavioural Processes 167: 103937.
Balinsky JB. 1972. Phylogenetic aspects of purine metabolism.
South African Medical Journal 46: 993–997.
Balinsky JB, Chemaly SM, Currin AE, Lee AR,
Thompson RL, Van der Westhuizen DR. 1976. A
comparative study of enzymes of urea and uric acid
metabolism in different species of Amphibia, and the
adaptation to the environment of the tree from Chiromantis
xerampelina Peters. Comparative Biochemistry and
Physiology Part B: Comparative Biochemistry 54: 549–555.
Bartges JW, Osborne CA, Lulich JP, Unger LK,
Koehler LA, Bird KA, Clinton CW, Davenport MP.
1994. Prevalence of cystine and urate uroliths in bulldogs
and urate uroliths in dalmatians. Journal of the American
Veterinary Medical Association 204: 1914–1918.
Bazin D, Daudon M, Elkaim E, Le Bail A, Smrčok Ĺ. 2016.
Ab initio structure determination of kidney stone potassium
quadriurate from synchrotron powder diffraction data, a 150 year
problem solved. Comptes Rendus Chimie 19: 1535–1541.
Booth W, Schuett GW. 2016. The emerging phylogenetic
pattern of parthenogenesis in snakes. Biological Journal of
the Linnean Society 118: 172–186.
Bull CM, Griffin CL, Johnston GR. 1999a. Olfactory
discrimination in scat-piling lizards. Behavioral Ecology 10:
136–140.
Bull CM, Griffin CL, Perkins MV. 1999b. Some properties of
a pheromone allowing individual recognition from the scats
of an Australian lizard, Egernia striolata. Acta Ethologica 2:
35–42.
Burbrink FT, Grazziotin FG, Pyron RA, Cundall D,
Donnellan S, Irish F, Keogh JS, Kraus F, Murphy RW,
Noonan B, Raxworthy CJ, Ruane S, Lemmon AR,
Lemmon EM, Zaher H. 2020. Interrogating genomic-scale
data for Squamata (lizards, snakes, and amphisbaenians)
shows no support for key traditional morphological
relationships. Systematic Biology 69: 502–520.
Castoe TA, de Koning AP, Hall KT, Card DC, Schield DR,
Fujita MK, Ruggiero RP, Degner JF, Daza JM, Gu W,
Reyes-Velasco J, Shaney KJ, Castoe JM, Fox SE,
Poole AW, Polanco D, Dobry J, Vandewege MW,
Li Q, Schott RK, Kapusta A, Minx P, Feschotte C,
Uetz P, Ray DA, Hoffmann FG, Bogden R, Smith EN,
Chang BS, Vonk FJ, Casewell NR, Henkel CV,
Richardson MK, Mackessy SP, Bronikowski AM,
Yandell M, Warren WC, Secor SM, Pollock DD. 2013.
The Burmese python genome reveals the molecular
basis for extreme adaptation in snakes. Proceedings of
the National Academy of Sciences of the United States of
America 110: 20645–20650.
Cundall D, Greene HW. 2000. Feeding in snakes. In:
Schwenk K., ed. Feeding: form, function and evolution in
tetrapod vertebrates. San Diego: Academic Press, 293–333.
Dantzler WH. 1996. Comparative aspects of renal urate
transport. Kidney International 49: 1549–1551.
Dantzler WH. 2005. Challenges and intriguing problems in
comparative renal physiology. The Journal of Experimental
Biology 208: 587–594.
Dantzler WH, Bradshaw SD. 2008. Osmotic and ionic
regulation in reptiles. In: Evans DH ed. Osmotic and ionic
regulation: cells and animals. Boca Raton: CRC Press,
443–503.
Ditrich H. 1996. A comparison of the renal structures of the
anaconda and the ball python. Scanning Microscopy 10:
1163–1172.
Dubler E, Jameson GB, Kopajtic Z. 1986. Uric acid salts of
magnesium: crystal and molecular structures and thermal
analysis of two phases of Mg(C5H3N4O3)2 · 8H2O. Journal of
Inorganic Biochemistry 26: 1–21.
Duvall D, Graves BM, Carpenter GC. 1987. Visual and
chemical composite signaling effects of Sceloporus lizard
fecal boli. Copeia 1987: 1028–1031.
Fenner AL, Bull CM. 2010. The use of scats as social signals
in a solitary, endangered scincid lizard, Tiliqua adelaidensis.
Wildlife Research 37: 582–587.
Downloaded from https://academic.oup.com/biolinnean/advance-article/doi/10.1093/biolinnean/blab052/6261016 by Georgetown University user on 30 April 2021
URATE DIFFERENCES IN SNAKES 9
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, XX, 1–10
Finlayson B, Smith A. 1974. Stability of first dissociable
proton of uric acid. Journal of Chemical & Engineering Data
19: 94–97.
Friedel P, Bergmann J, Kleeberg R, Schubert G. 2006.
A proposition for the structure of ammonium hydrogen
(acid) urate from uroliths. Zeitschrift für Kristallographie
Supplemente 23: 517–522.
Gamble T, Castoe TA, Nielsen SV, Banks JL, Card DC,
Schield DR, Schuett GW, Booth W. 2017. The discovery of
XY sex chromosomes in a Boa and Python. Current Biology:
CB 27: 2148–2153.e4.
Greene HW. 1997. Snakes: the evolution of mystery in nature.
Berkeley: University of California Press.
Greene HW, Cundall D. 2000. Perspectives: evolutionary
biology. Limbless tetrapods and snakes with legs. Science
287: 1939–1941.
Groom CR, Bruno IJ, Lightfoot MP, Ward SC. 2016. The
Cambridge structural database. Acta Crystallographica
Section B, Structural Science, Crystal Engineering and
Materials 72: 171–179.
Halpern M, Martínez-Marcos A. 2003. Structure and
function of the vomeronasal system: an update. Progress in
Neurobiology 70: 245–318.
Hebets EA, Barron AB, Balakrishnan CN, Hauber ME,
Mason PH, Hoke KL. 2016. A systems approach to animal
communication. Proceedings of the Royal Society B: Biological
Sciences 283: 20152889.
Hebets EA, Papaj DR. 2005. Complex signal function:
developing a framework of testable hypotheses. Behavioral
Ecology and Sociobiology 57: 197–214.
Herring LC. 1962. Observations on the analysis of ten
thousand urinary calculi. The Journal of Urology 88:
545–562.
Khalil F. 1948a. Excretion in reptiles; nitrogen constituents of the
urinary concretions of the oviparous snake Zamenis diadema,
Schlegel. Journal of Biological Chemistry 172: 101–103.
Khalil F. 1948b. Excretion in reptiles; nitrogen constituents
of the urinary concretions of the viviparous snake Eryx
thebaicus, Reuss. Journal of Biological Chemistry 172:
105–106.
King PA, Goldstein L. 1985. Renal excretion of nitrogenous
compounds in vertebrates. Renal Physiology 8: 261–278.
Lillywhite HB. 2014. How snakes work. Structure, function and
behavior of the World’s snakes. New York: Oxford University
Press.
Liu F, Hooks DE, Li N, Mara NA, Swift JA. 2018. Mechanical
properties of anhydrous and hydrated uric acid crystals.
Chemistry of Materials 30: 3798–3805.
Mandel NS, Mandel GS. 1976. Monosodium urate
monohydrate, the gout culprit. Journal of the American
Chemical Society 98: 2319–2323.
Marneweck C, Jürgens A, Shrader AM. 2017. Dung odours
signal sex, age, territorial and oestrous state in white rhinos.
Proceedings of the Royal Society B: Biological Sciences 284:
20162376.
Mason RT, Parker MR. 2010. Social behavior and
pheromonal communication in reptiles. Journal of
Comparative Physiology. A, Neuroethology, Sensory, Neural,
and Behavioral Physiology 196: 729–749.
Minnich JE. 1972. Excretion of urate salts by reptiles.
Comparative Biochemistry and Physiology. A, Comparative
Physiology 41: 535–549.
Minnich JE, Piehl PA. 1972. Spherical precipitates in the
urine of reptiles. Comparative Biochemistry and Physiology.
A, Comparative Physiology 41: 551–554.
Modlin M, Davies PJ. 1981. The composition of renal stones
analysed by infrared spectroscopy. South African Medical
Journal = Suid-Afrikaanse tydskrif vir geneeskunde 59:
337–341.
Müller-Schwarze D. 2006. Chemical signals in vertebrates.
Cambridge: Cambridge University Press.
Parkin S, Hope H. 1998. Uric acid dihydrate revisited. Acta
Crystallographica 54: 339–344.
Perry BW, Andrew AL, Kamal AHM, Card DC, Schield DR,
Pasquesi GIM, Pellegrino MW, Mackessy SP,
Chowdhury SM, Secor SM, Castoe TA. 2019. Multi-
species comparisons of snakes identify coordinated signalling
networks underlying post-feeding intestinal regeneration.
Proceedings of the Royal Society B: Biological Sciences 286:
14–17.
Presores JB, Cromer KE, Capacci-Daniel C, Swift JA.
2013. Calcium urate hexahydrate. Crystal Growth and
Design 13: 5162–5164.
Presores JB, Swift JA. 2014. Solution-mediated phase
transformation of uric acid dihydrate. CrystEngComm 16:
7278–7284.
Ringertz H. 1966. The molecular and crystal structure of uric
acid. Acta Crystallographica 20: 397–403.
Schmidt-Nielsen K. 1997. Animal physiology: adaptation
and environment. Cambridge: Cambridge University
Press.
Schubert G, Reck G, Jancke H, Kraus W, Patzelt C. 2005.
Uric acid monohydrate—a new urinary calculus phase.
Urological Research 33: 231–238.
Secor SM, Diamond J. 1998. A vertebrate model of extreme
physiological regulation. Nature 395: 659–662.
Shah B, Hudson S, Shine R. 2006. Social aggregation by
thick-tailed geckos (Nephrurus milii, Gekkonidae): Does scat
piling play a role? Australian Journal of Zoology 54: 271–275.
Urity VB, Issaian T, Braun EJ, Dantzler WH,
Pannabecker TL. 2012. Architecture of kangaroo rat inner
medulla: segmentation of descending thin limb of Henle’s
loop. American Journal of Physiology. Regulatory, Integrative
and Comparative Physiology 302: 720–726.
Venn-Watson SK, Townsend FI, Daniels RL, Sweeney JC,
McBain JW, Klatsky LJ, Hicks CL, Staggs LA,
Rowles TK, Schwacke LH, Wells RS, Smith CR. 2010.
Hypocitraturia in common bottlenose dolphins (Tursiops
truncatus): assessing a potential risk factor for urate
nephrolithiasis. Comparative Medicine 60: 149–153.
Walsh PJ, Wright PA. 1995. Nitrogen metabolism and
excretion. New York: CRC Press.
Wyatt TD. 2010. Pheromones and signature mixtures:
defining species-wide signals and variable cues for
Downloaded from https://academic.oup.com/biolinnean/advance-article/doi/10.1093/biolinnean/blab052/6261016 by Georgetown University user on 30 April 2021
10 A. M. THORNTON ET AL.
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, XX, 1–10
identity in both invertebrates and vertebrates. Journal of
Comparative Physiology. A, Neuroethology, Sensory, Neural,
and Behavioral Physiology 196: 685–700.
Zellelow AZ, Kim H-K, Sours RE, Swift JA. 2010. Solid
state dehydration of uric acid dihydrate. Crystal Growth &
Design 10: 418–425.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:
Figure S1. Samples of the snake urates used in this study. A, ball python (Python regius). B, Angolan python
(Python anchietae). C, Madagascan tree boa (Sanzinia madagascariensis). D, Mojave rattlesnake (Crotalus
scutulatus). E, western diamond-backed rattlesnake (Crotalus atrox). F, Trans-Pecos rat snake (Bogertophis
subocularis). G, desert kingsnake (Lampropeltis splendida). H, Mexican hog-nosed snake (Heterodon kennerlyi).
Scale bar: 1 cm.
Figure S2. Experimental powder X-ray diffraction (PXRD) diffractograms obtained on urate samples excreted
from five different species of ancient snakes compared with simulated PXRD patterns of known crystalline
forms of uric acid (anhydrous uric acid, uric acid monohydrate and uric acid dihydrate) and urate salts (sodium
urate monohydrate, calcium urate hexahydrate, magnesium urate, potassium quadriurate and ammonium acid
urate). Simulated PXRD diffractograms were generated in mercury from cif files obtained from the Cambridge
Structure Database.
Figure S3. Sharp diffraction lines at 2θ = 10.1, 27.8 and 28.3° in the Mojave rattlesnake urate correspond to
intense peaks expected for uric acid dihydrate. Peaks at the higher 2θ values appear shifted owing to effects of
temperature. The inset is an optical micrograph, which shows individual crystals (red arrows) present in the
sample. Scale bar: 100 µm. Single-crystal X-ray diffraction confirmed them to be uric acid dihydrate.
Figure S4. Experimental powder X-ray diffraction (PXRD) diffractograms obtained on the same ball python and
Mojave rattlesnake urate samples collected ~2 and 12 months after excretion.
Downloaded from https://academic.oup.com/biolinnean/advance-article/doi/10.1093/biolinnean/blab052/6261016 by Georgetown University user on 30 April 2021
Hematology and biochemistry testing of boas and pythons is a valuable topic for practicing clinicians and researchers alike. This article reviews blood cell morphology (with accompanying images) and reviews the literature for hematologic and biochemical material clinically relevant to the families Boidae and Pythonidae.
Article
Full-text available
Genomics is narrowing uncertainty in the phylogenetic structure for many amniote groups. For one of the most diverse and species-rich groups, the squamate reptiles (lizards and snakes, amphisbaenians), an inverse correlation between the number of taxa and loci sampled still persists across all publications using DNA sequence data and reaching a consensus on the relationships among them has been highly problematic. Here, we use high-throughput sequence data from 289 samples covering 75 families of squamates to address phylogenetic affinities, estimate divergence times, and characterize residual topological uncertainty in the presence of genome scale data. Importantly, we address genomic support for the traditional taxonomic groupings Scleroglossa and Macrostomata using novel machine-learning techniques. We interrogate genes using various metrics inherent to these loci, including parsimony-informative sites, phylogenetic informativeness, length, gaps, number of substitutions, and site concordance to understand why certain loci fail to find previously well-supported molecular clades and how they fail to support species-tree estimates. We show that both incomplete lineage sorting and poor gene-tree estimation (due to a few undesirable gene properties, such as an insufficient number of parsimony informative sites), may account for most gene and species-tree discordance. We find overwhelming signal for Toxicofera, and also show that none of the loci included in this study supports Scleroglossa or Macrostomata. We comment on the origins and diversification of Squamata throughout the Mesozoic and underscore remaining uncertainties that persist in both deeper parts of the tree (e.g., relationships between Dibamia, Gekkota, and remaining squamates; and between the three toxiferan clades Iguania, Serpentes, and Anguiformes) and within specific clades (e.g., affinities among gekkotan, pleurodont iguanians, and colubroid families).
Article
Full-text available
Chemical communication is probably the oldest, most ubiquitous form of information exchange in the natural world, spanning all three domains of life. While excellent sociobiological and behavioral ecological research has been conducted on the form and function of chemical signals in animals, we still know remarkably little on their evolution. Besides, much of our understanding of chemical signal diversity is restricted to insects, since studies on chemical communication in vertebrates are relatively scarce. In this review, I introduce the key concepts of animal communication and expand on the past, present, and future of research in chemical communication. When doing so, I highlight the current gaps in our knowledge on the evolution of the chemical communication system in animals, whilst emphasizing the heavy research bias towards lepidopterans. Here, I detail the benefits of using phylogenetic comparative methods to identify the motors and brakes that guide the evolution of chemical signals and chemical sensory systems. Moreover, I point out that focusing on non-model species in chemical ecology, specifically lizards, can provide valuable insights into how vertebrate chemical signals evolve, and how biological systems responsible for sending and receiving signals co-evolve with signal design. Lastly, I present a case study on lacertid lizards, demonstrating the possibilities of the phylogenetic comparative approach and the use of non-model species to study the evolution of animal chemical communication systems.
Article
Many animals use their excrements to communicate with others. In order to increase signal efficacy, animals often behaviourally select for specific defecation sites that maximize the detectability of their faecal deposits, such as the tip of rocks by some lizard species. However, the field conditions in which these observations are made make it difficult to reject alternative explanations of defecation site preference; rock tips may also provide better opportunities for thermoregulation, foraging, or escaping predators, and not solely for increasing the detectability of excrements. In addition, we still know little on whether lizard defecation behaviour varies within-species. In this laboratory study, we take an experimental approach to test defecation site preference of Podarcis melisellensis lizards in a standardized setting, and assess whether preferences differ between sexes, and among populations. Our findings show that in an environment where all stones provide equal thermoregulatory advantage, prey availability, and predator pressure, lizards still select for the largest stone in their territory as preferred defecation site. Moreover, we demonstrate that lizards' defecation preference is a strong conservative behaviour, showing no significant intraspecific variation. Together, these findings corroborate the idea that lizards may defecate on prominent rocky substrates in order to increase (visual) detectability of the deposited faecal pellets.
Article
Several snake species that feed infrequently in nature have evolved the ability to massively upregulate intestinal form and function with each meal. While fasting, these snakes downregulate intestinal form and function, and upon feeding restore intestinal structure and function through major increases in cell growth and proliferation, metabolism and upregulation of digestive function. Previous studies have identified changes in gene expression that underlie this regenerative growth of the python intestine, but the unique features that differentiate this extreme regenerative growth from non-regenerative post-feeding responses exhibited by snakes that feed more frequently remain unclear. Here, we leveraged variation in regenerative capacity across three snake species-two distantly related lineages ( Crotalus and Python) that experience regenerative growth, and one ( Nerodia) that does not-to infer molecular mechanisms underlying intestinal regeneration using transcriptomic and proteomic approaches. Using a comparative approach, we identify a suite of growth, stress response and DNA damage response signalling pathways with inferred activity specifically in regenerating species, and propose a hypothesis model of interactivity between these pathways that may drive regenerative intestinal growth in snakes.
Book
It is generally accepted that the recent progress in molecular and cellular biology would not have been possible without an understanding of the mechanisms and signaling pathways of communication inside the cell and between various cells of the animal organism. In fact a similar progress occurred in the field of chemical communication between individual organisms of vertebrate species, and this volume is aimed at presenting the current state of the art on this subject. The reader can find here both original results obtained in the laboratory or field studies and comprehensive reviews summarizing many years of research. The presentations of over 60 scientists have been grouped according to their approach into nine parts covering such fields as ecological and evolutionary aspects of chemical communication, structure and neuronal mechanisms of chemosensory systems, chemical structure of pheromones and binding proteins, kin, individual and sexual recognition, predator-prey relationships, purpose and consequences of marking behavior, scent signals and reproductive processes. Expanding on former volumes of this series, entirely new chapters have been added on prenatal chemical communication describing specific effects of the intrauterine environment. In many cases a truly multidisciplinary approach was required, such as with the population analysis of polymorphic variants of the mouse's major urinary proteins that function in carrying pheromones.
Article
For over 50 years, biologists have accepted that all extant snakes share the same ZW sex chromosomes derived from a common ancestor [1–3], with different species exhibiting sex chromosomes at varying stages of differentiation. Accordingly, snakes have been a well-studied model for sex chromosome evolution in animals [1, 4]. A review of the literature, however, reveals no compelling support that boas and pythons possess ZW sex chromosomes [2, 5]. Furthermore, phylogenetic patterns of facultative parthenogenesis in snakes and a sex-linked color mutation in the ball python (Python regius) are best explained by boas and pythons possessing an XY sex chromosome system [6, 7]. Here we demonstrate that a boa (Boa imperator) and python (Python bivittatus) indeed possess XY sex chromosomes, based on the discovery of male-specific genetic markers in both species. We use these markers, along with transcriptomic and genomic data, to identify distinct sex chromosomes in boas and pythons, demonstrating that XY systems evolved independently in each lineage. This discovery highlights the dynamic evolution of vertebrate sex chromosomes and further enhances the value of snakes as a model for studying sex chromosome evolution.