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Herpetological Review 52(3), 2021
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© 2021 by Society for the Study of Amphibians and Reptiles
Does the Non-lethal Gastric Lavage Method Aect Subsequent
Feeding Behavior in Adult and Larval Plethodontid Stream
Salamanders?
Studies on the diet of vertebrates are critical to
understanding their importance to food webs and ecosystems.
While diverse approaches are regularly used to assess diets
that include lethal and non-lethal techniques, it is necessary
to evaluate how they can directly influence a study population,
particularly when the methods are relatively invasive. Although
diet studies can be lethal (i.e., stomach removal from museum
or recently euthanized specimens (Rodriguez-Robles et al.
1999), a variety of non-lethal techniques exist, including fecal
analysis (Crovetto et al. 2012), stable-isotope analysis (Fenolio
et al. 2007), emetics (Jernejcic 1969), fistulas (Krayukhin 1962),
forceps (Wales 1962), gastroscopes (Zweiacker 1972), insertion
of tubes (Den Avyle and Roussel 1980), intestinal flushing
(Baker and Fraser 1976), and gastric lavage stomach flushing
(Seaburg 1957). Non-lethal methods to collect diet samples are
preferable to lethal sampling, as they minimize the impacts on
local populations (Davic and Welsh 2004). Further, the ideal
method should remove all stomach contents, be easy, efficient,
and inexpensive, avoid long-term internal trauma, should not
JACOB M. HUTTON*
School of Biological Sciences, Southern Illinois University,
Carbondale, Illinois, 62901, USA;
Department of Forestry and Natural Resources,
University of Kentucky, Lexington, Kentucky, 40546, USA
ADRIAN D. MACEDO
School of Biological Sciences, Southern Illinois University,
Carbondale, Illinois 62901, USA
STEPHEN C. RICHTER
Division of Natural Areas and Department of Biological Sciences,
Eastern Kentucky University, Richmond, Kentucky 40475, USA
ROBIN W. WARNE
School of Biological Sciences, Southern Illinois University,
Carbondale, Illinois 62901, USA
STEVEN J. PRICE
Department of Forestry and Natural Resources,
University of Kentucky, Lexington, Kentucky 40546, USA
*Corresponding author; e-mail: jakemhutton@gmail.com
Herpetological Review 52(3), 2021
512 ARTICLES
impact subsequent feeding behavior, and be applicable to a
wide variety of species (Light 1983).
Gastric lavage is one of the oldest and frequently used
non-lethal techniques (Fraser 1976; Legler 1977; Romano et
al. 2012). The method of gastric lavage (i.e., stomach flushing)
is accomplished by pumping distilled or spring water into
the stomach via the insertion of a soft plastic tube. Stomach
contents are then flushed out and retained for identification.
Gastric lavage has been applied to fish (Foster 1977; Light 1983),
freshwater turtles (Legler 1977), squamate reptiles (Legler
and Sullivan 1979; Waddle et al. 2009; Nifong et al. 2012), birds
(Goldsworthy et al. 2016), small mammals (Kronfeld and Dayan
1998), and amphibians (Fraser 1976; Solé et al. 2005; Anthony
et al. 2008; Bondi 2015; Crovetto 2012; Costa 2014; Hutton et
al. 2017, 2018, 2019). Gastric lavage has been validated against
stomach dissection analysis in both salamanders (Salvidio 1992)
and frogs (Patto 1998; Wu et al. 2007). Further, Corvetto et al.
(2012) reported limitations of fecal analysis compared to gastric
lavage in salamanders, as prey digestion rate is taxa-dependent.
Despite wide support, it is uncertain how gastric lavage affects
individual health and subsequent feeding behavior after
returning to the wild.
Previous amphibian studies have documented mortality
and internal injury while attempting gastric lavage. Bondi et
al. (2015) observed damage to stomach mucosa in three of
124 flushed salamanders; the authors speculated the damage
was from the tubing entering the stomach lining. In an anuran
study, researchers reported mortality in eight Bufo ictericus and
one Scinax granulatus from tube-punctured gut or lung lining,
which then filled with water after attempted gastric lavage on
non-anesthetized individuals; however, mortality only occurred
in 9 of 583 animals (Solé et al. 2005). Interestingly, the authors
performed gastric lavage on 29 frogs, kept them in captivity, and
observed the same individuals consuming offered termites two
hours after the procedure (Solé et al. 2005).
To our knowledge, no studies have assessed the potential
impacts of gastric lavage on subsequent feeding behavior in
the wild. As amphibian species continue to decline globally, it
is imperative to both consider and understand the possible
repercussions of research methods on a population. In this
study, we examined the potential effects of non-lethal gastric
lavage method on subsequent feeding behavior in adult and
larval plethodontid stream salamanders. We hypothesized that
there would be no effect of gastric lavage and anesthetization on
the subsequent ability of larval and adult stream salamanders to
obtain prey items.
Materials and Methods
Study Sites.—Our study sites consisted of ten first-order
streams in the Cumberland Plateau in Breathitt, Knott, and
Letcher counties in southeastern Kentucky, USA (Hutton et al.
2020). Seven streams were located at the University of Kentucky’s
experimental research station Robinson Forest (RF) and three
streams at Eastern Kentucky University’s Lilley Cornett Woods
Appalachian Ecological Research Station (LCW). All stream
sites were in old-growth and second-growth forests and had low
anthropogenic disturbance (i.e., specific conductivity values
ranging from 30–418 μS/cm), and the average forest cover at these
sites was 99.78% (Hutton et al. 2020). See Martin and Shepherd
(1973), Martin (1975), and Phillippi and Boebinger (1986) for
description of vegetative communities at our study sites.
Salamander Surveys and Diet.—We surveyed for adult and
larval salamanders in a single 10-meter stream reach at each site.
Stream reaches were selected to contain similar widths, depths,
and current velocities. All stream reaches contained a pool, run,
and riffle section to provide likely habitat to increase detections
of all possible species and life stages (Petranka 1998; Hutton et
al. 2020). Each stream reach was sampled four times (ca. every
29 days) from April to July 2017. Searches were conducted
during daylight hours (0800–1700 h) and in baseflow conditions.
Salamanders were captured using systematic dipnetting and
bank searches (Price et al. 2011). Dipnetting consisted of one
person, moving from downstream to upstream, searching for
salamanders around and under submerged rocks, logs, and other
cover within the 10-m reach. One person then conducted bank
searches, which included searching under rocks, logs, leaf litter,
and other material within 1-m of the wetted width of the stream.
Stream searches were limited to 0.5 hours and bank searches to
0.25 hours (Price et al. 2011). After sampling, we recorded the
species and life stage (larval or post-metamorphotic individuals).
Salamanders were anesthetized in the field, using a solution
of 1-g Maximum Strength Orajel® to 1-L distilled water (Cecala
et al. 2007). Once the salamanders failed to right themselves after
being flipped over, their stomach contents were obtained using
the current non-lethal gastric lavage method for amphibians
(Solé et al. 2005; Cecala et a. 2007; Bondi et al. 2015; Hutton et
al. 2017, 2018, 2019). Salamanders were placed on their dorsum
on a folded paper towel, and an ca. 6 cm long piece of water-
lubricated tubing was slowly inserted into the esophagus until
there was resistance. Distilled water was then pumped into the
tubing. Specifically, Nipro® 3-mL syringes with 22-gauge needles
and 0.8 mm and 1.3 mm OD PTFE tubing were used (Zeus Inc.,
#AWG24). As in previous studies, salamander stomachs were
pumped at least two additional times after the last prey item
was extracted to verify removal of all contents (Solé et al. 2005;
Cecala et a. 2007; Bondi et al. 2015; Hutton et al. 2017, 2018,
2019). The total amount of water pumped into each salamander
was dependent on salamander size, the amount of prey items,
and the size of prey and their resistance to removal. However,
in general, significantly less water was required for larval and
juvenile salamanders than adults.
After lavage, each salamander was measured for snout–vent
length (SVL: from the tip of the snout to the posterior angle of
the vent) and total length (TL: from tip of the snout to the tail’s
terminus) to the nearest 0.01 mm with a digital caliper, and mass
(except larvae ≤ 30 mm TL) to the nearest 0.1 g with a digital scale.
We calculated body condition (mass/TL) on all salamanders ≥
30 mm TL; salamanders missing tails or parts of their tails were
excluded (Karraker and Welsh 2006). Salamanders ≥ 30 mm
TL were then marked with unique fluorescent visible implant
elastomers (VIE) to allow for capture-mark-recapture (CMR)
identification. Each salamander was then placed in a recovery
container of stream water until they could right themselves and
responded to tapping, which took ca. 15 min. Salamanders were
returned to their exact location of capture within 1.5 h. After the
first survey, each site was surveyed three additional times and
recaptured animals were stomach flushed again to examine
potential changes in diet.
Animals in the stomach contents were then identified to
lowest taxonomic level possible using a dissecting microscope
along with appropriate keys and guides (Peckarsky 1990;
Merritt and Cummins 1996; Wagner 2005; Fisher and Cover
2007; Bradley 2012; Evans 2014). Additionally, invertebrate life
Herpetological Review 52(3), 2021
ARTICLES 513
stage (larval or adult) was reported, if applicable. For Shannon
diversity calculations, largely different sized prey or prey with
unique characteristics in a single order, family, or genera were
considered to be separate morphospecies. The individual prey
items were then grouped into larger sections based on order/
class, life stage, and presumed origin (Hutton et al. 2018). Samples
were then placed into individually labeled vials containing 70%
ethanol. Samples are stored in the Branson Museum collection
at Eastern Kentucky University, Richmond, Kentucky.
Diet Analysis.—To calculate prey volume, we measured the
length and width of each prey item to the nearest 0.01 mm using
a digital caliper and estimated volume as a prolate spheroid
using the equation (Dunham 1983):
The number of prey items, number of prey types (i.e.,
morphospecies), average prey volume, total prey volume,
Shannon diversity, and body condition of individuals were
calculated for each capture event for adult, larvae, and combined
groups. We used t-tests to compare the above diet parameters
between first and second captures and between adults and
larvae. All parameters were log-transformed prior to analysis
to fit assumptions, averages and their standard deviations (SD)
were back log-transformed to the scale of the data. Number
of days since the previous capture was also examined for
correlation to the diet parameters. We found 17 out of 245
prey items as potential outliers (volume ≥ 50 mm3), only 6 of
which were in the first capture event, and 2 salamanders had
prey items ≥ 50 mm3 in both capture events. However, due to
the euryphagous behavior of stream salamanders (Jaeger 1981)
and the random sampling, we feel it was unfitting to remove
the larger prey items from the dataset. Further, the average
volumes of the large prey items from each capture event did not
differ (P = 0.93). All analyses were performed in the statistical
program R (Version 3.4.3).
results
We captured six stream salamander species during our
active searches (Desmognathus fuscus [DF], D. monticola [DM],
D. welteri [DW], Gyrinophilus porphyriticus [GP], Pseudotriton
ruber [PR], and Eurycea cirrigera [EC]). Larvae ≤ 30 mm TL were
excluded from this study because they were too small to mark
safely and uniquely with VIE. Overall, 260 salamanders, 134
adult and 126 larvae (39 DF, 78 DM, 4 DW, 12 EC, 105 GP, and
22 PR; Table 1) were stomach flushed and VIE marked across
our 10 stream sites. We recaptured 36 individuals (28 adult and
8 larvae; Table 1), 4 of which were recaptured at least twice (3
DM, 1 GP), for a total of 41 separate recapture observations.
Overall, all salamanders contained at least one prey item
in their stomachs, for a total of 245 identified and volume-
estimated prey items (Table 1). No anesthetization or lavage-
based mortality or signs of internal trauma occurred before
release.
When all adults and larvae were combined, we found no
differences between the first and second capture events in the
number of prey (P = 0.273), number of prey types (P = 0.241),
Shannon diversity (P = 0.513), or body condition (P = 0.562).
However, there were differences in the average prey volumes
(mean = 4.15 ± SD 4.79 mm3 and 8.01 ± SD 3.83 mm3; first and
second capture, respectively; Table 2) and total prey volumes
(mean = 9.33 ± SD 5.16 mm3 and 20.42 ± SD 3.89 mm3; Table
2), with larger volumes in the second capture events (P = 0.045
and 0.021, respectively). In combined groups, we found no
correlation between the days since the last capture and the
following: number of prey (r = -0.086), number of prey types (r
= -0.058), average prey volume (r = -0.016), total prey volume
(r = -0.069), Shannon diversity (r = -0.181), or body condition
(r = 0.149).
When we examined just the adult salamanders, the results
were similar to all salamanders combined group. Among the
adults, we found no differences between the first and second
capture events in the number of prey (P = 0.155), number of
prey types (P = 0.234), Shannon diversity (P = 0.589), or body
condition (P = 0.616). However, there were differences in
average prey volumes (mean = 3.67 ± SD 4.85 mm3 and 8.58
± SD 3.42 mm3; Table 2) and total prey volumes (mean = 8.49
± SD 5.50 mm3 and 23.19 ± SD 3.59 mm3; Table 2), with larger
volumes in the second capture events (P = 0.019 and 0.010,
respectively). We found no correlation between the days since
the last capture and the number of prey (r = 0.029), number of
prey types (r = 0.051), average prey volume (r = -0.221), total
prey volume (r = -0.205), Shannon diversity (r = -0.143), or body
condition (r = 0.079).
Lastly, when we examined the larval salamanders, we found
no differences between the first and second capture events in
the number of prey (P = 0.891), number of prey types (P = 0.494),
average prey volume (P = 0.838), total prey volume (P = 0.411),
Shannon diversity (P = 0.633), or body condition (P = 0.557). For
the larvae, we were unable to analyze correlation of the days
since first capture to the diet parameters due to sample size
restraints. Among all salamanders, the least amount of time
between capture events was 18 days and the greatest was 65.
A disproportionate volume of large (i.e., ≥ 50 mm3) individual
prey items were found in the second capture event compared
to the first for the combined (all) and adult groups. Six large
individual prey items from the first capture event had a total
volume of 540.13 mm3, whereas the 11 large items in the second
capture event totaled 934.02 mm3, despite the average volumes
of the large prey items from each event (83.92 ± 1.49 and 85.53
± 1.52 mm3, respectively) not being statistically different (P =
0.93). Thus, the statistically observed increases in the average
and total volumes in the second capture event are related to
just a few large prey items.
table 1. Salamander species and life stage capture and recapture for
Desmognathus fuscus, D. monticola, D. welteri, Eurycea cirrigera,
Gyrinophilus porphyriticus, and Pseudotriton ruber over four
sampling periods at 10 stream reaches in southeastern Kentucky,
USA. Numbers in parentheses represent the number of recaptured
individuals.
Species Adult Larvae
Desmognathus fuscus 39 (4) –
Desmognathus monticola 78 (19) –
Desmognathus welteri 4 (4) –
Eurycea cirrigera 12 –
Gyrinophilus porphyriticus 1 (1) 104 (8)
Pseudotriton ruber – 22
TOTAL 134 126
Herpetological Review 52(3), 2021
514 ARTICLES
discussion
It is important for researchers to understand how various
study methods and techniques may directly influence the
study population, particularly when the methods are relatively
invasive. In this study, our data provide the first field-based
assessment of gastric lavage stomach flushing on subsequent
feeding behavior of stream salamanders. Overall, we found no
negative effects on the future ability of either larval or adult
salamanders to obtain prey. In our study, we surveyed streams
four times over a 3-mo period and had 41 recapture events from
260 marked salamanders (15.77% recapture rate). This recapture
rate is within the range expected in comparison to previous
studies and suggests our study and approach did not impact
survival. Cecala et al. (2009) for example, reported a recapture
rate of 29.04% for larval PR however the streams were sampled
14 times from May 2006 to April 2007, which likely explains
the higher recapture rate. Additionally, Bailey et al. (2004)
reported that plethodontid salamander recapture probabilities
in southern Appalachia ranged between 0.20–0.30 after 3 years
of sampling, illustrating a relationship between sampling effort
and recapture rates and probabilities. Beyond survival effects,
our study also demonstrates that gastric lavage is a safe assay
that does not impose long term costs on foraging or digestion
and assimilation of prey.
We observed no negative effects of recapture and flushing on
any of the diet parameters, suggesting foraging behavior was not
impacted. In this study, we recaptured and flushed individuals
approximately every 29 days and found no relationship between
the days since first capture and the subsequent number of prey,
number of prey types, average prey volume, total prey volume,
Shannon diversity, or body condition. However, it is unknown
how quickly (following full recovery from anesthesia and
release) salamanders will begin to forage again, especially due
to their primarily nocturnal behavior. In our study, the shortest
time between the second stomach flushing was 18 days. By
comparison, Patto (1998) captured, obtained stomach contents
via gastric lavage, and uniquely toe clipped 97 Hylodes asper
(Anura: Leptodactylidae) before release. Twenty animals were
successfully recaptured and stomach flushed a second time over
an 18-d period. Patto (1998) reported prey items in 88% of the
frogs, suggesting no significant effects of stomach flushing on
the recapture and subsequent prey consumption on the study
species. Solé et al. (2005) reported the consumption of offered
termite prey by captive anurans just two hours after lavage, the
authors then kept the anurans in captivity for a month before
releasing. Taken together, these results suggest amphibian
foraging behavior is not unduly affected by gastric lavage.
Although some amphibians in captivity appear to be able
to accept food shortly after gastric lavage recovery, there are
still implications of the immediate loss of captured prey items
from stomach flushing in the wild sample population. The
minimum amount of time suggested to wait before flushing
again or the absolute maximum number of times researchers
should flush an individual in a season are not well tested and
are very likely dependent on the season, species, and age class.
For example, Maiorana (1978) reported complete digestion
of prey in terrestrial salamanders to take approximately four
days, therefore, prey items found via gastric lavage potentially
represent prey that would have been assimilated over several
days after feeding. However, salamander prey digestion has also
been shown to be temperature dependent (Fontaine et al. 2018);
therefore, researchers have an obligation to consider how their
methods influence the foraging behavior and energy balance
of their study species. This concern is of particular importance
during differing seasons and life stages when energy demand
is highest (i.e., breeding season or times of reduced resources).
While our study does not necessarily provide clarity on this
concern because the study took place over a single season, our
results suggest both adults and larvae foraging behavior are not
unduly affected.
In our study, there was a disproportionate number of large
volume prey items found in the second capture events of
salamanders, which may be due to differences in the abundance
or emergence of large prey types later in the season. Larval
EC (68.74–83.28 mm3) were found in the stomach contents of
two larval GP and an adult DF during second capture events.
Additionally, during second capture events, an adult stonefly
species (Plecoptera: Perlidae; 110.06 mm3) and an adult Bark
Centipede (Chilopoda: Scolopocryptopidae: Scolopocryptops
sexspinosus; 127.30 mm3) were found in stomach contents of
two separate adult DW. At our study sites, the eggs of EC did
table 2. Mean (± SD) diet parameters among stream salamanders after repeated recapture and gastric
lavage stomach flushing, results were back-transformed to scale of the data.
All Adults Larvae
Days Since 1st Capture 29.47 (± 1.57) 29.12 (± 1.57) 30.74 (± 1.58)
# Prey Items 1 2.42 (± 1.83) 2.47 (± 1.87) 2.27 (± 1.71)
# Prey Items 2 2.79 (± 1.73) 3.03 (± 1.66) 2.07 (± 1.86)
# Prey Types 1 2.12 (± 1.77) 2.26 (± 1.76) 1.66 (± 1.70)
# Prey Types 2 2.44 (± 1.68) 2.66 (± 1.65) 1.79 (± 1.64)
Average Prey Vol 1 4.15 (± 4.79) 3.67 (± 4.85) 6.45 (± 4.61)
Average Prey Vol 2 8.01 (± 3.83) 8.58 (± 3.42) 6.28 (± 5.74)
Total Prey Vol 1 9.33 (± 5.16) 8.49 (± 5.50) 13.07 (± 4.19)
Total Prey Vol 2 20.42 (± 3.89) 23.19 (± 3.59) 13.13 (± 5.07)
Shannon Diversity 1 0.952 (± 0.33) 0.983 (± 0.34) 0.842 (± 0.25)
Shannon Diversity 2 1.002 (± 0.35) 1.032 (± 0.37) 0.899 (± 0.24)
Body Condition 1 0.0216 (± 0.015) 0.0255 (± 0.016) 0.0171 (± 0.005)
Body Condition 2 0.0234 (± 0.015) 0.0275 (± 0.016) 0.0186 (± 0.005)
Herpetological Review 52(3), 2021
ARTICLES 515
not hatch until early June, and stonefly metamorphosis was
noted in June–July. Further, these three prey types were only
found in stomach contents during second capture events.
Since plethodontid salamanders display a euryphagous feeding
strategy (Jaeger 1981), differences in prey availability are more
likely to contribute to observed differences in diet composition
between sampling periods. However, our results highlight the
importance for the inclusion of additional diet parameters such
as Shannon diversity, number of prey items, and number of prey
types consumed. In this study, despite differences in the average
and total consumed prey volumes, the overall Shannon diversity,
number of prey items, and number of prey types were not
found to significantly change between capture events as those
differences were driven by just a few individual prey items.
Overall, this study illustrates the efficacy of gastric lavage
stomach flushing, in combination with a Maximum Strength
Orajel® anesthetic solution, on stream salamanders. Diet
studies are critical to understanding the roles of salamanders
in ecosystem processes and community dynamics (Davic and
Welsh 2004; Jouquet et al. 2006; Lavelle et al. 2006; Walton 2013).
As amphibians continue to decline globally, it is imperative
to consider and understand the possible method-based
repercussions of each study on a population. Gastric lavage
stomach flushing is a popular method which can provide reliable
stomach content data with relative ease, requires easily obtained
and inexpensive materials, and can be used on numerous species
in the same region. Future studies should focus on evaluating
the method on previously unreported species as well as species
lacking dietary information.
Acknowledgments.—This is contribution No. 58 of Lilley Cornett
Woods Appalachian Ecological Research Station, Eastern Kentucky
University. Funding for this project was provided by the following
organizations: Kentucky Academy of Science (Marcia Athey Grant),
Tracy Farmer Institute for Sustainability and the Environment at the
University of Kentucky (Karri Casner Environmental Sciences Fel-
lowship), Appalachian Center at the University of Kentucky (Eller
Billings Summer Research Mini-Grant), Eastern Kentucky University
Division of Natural Areas Student Grant-in-Aid of Research, the So-
ciety for the Study of Amphibians and Reptiles (Roger Conant Grants
in Herpetology Program, Conservation of Amphibians and Reptiles),
McIntire-Stennis Research Program (Accession Number 1001968),
Foundation for the Conservation of Salamanders (Daniel M. Digia-
como Grant), and the Society of Freshwater Science (Graduate Stu-
dent Conservation Award). We thank The Department of Forestry
and Natural Resources at the University of Kentucky for provided
resources, facilities, and permission for usage of Robinson Forest as
well as The Department of Natural Areas at the University of Eastern
Kentucky for permission for usage at Lilley Cornett Woods. We thank
Dan Dourson for assistance on micro-gastropod identification and
John W. Reynolds for assistance with identifying oligochaetes and
Andrea N. Drayer, Wendy Leuenberger, Allison Davis, Rebecca Le-
loudis, and Millie E. Hamilton for assistance with data collection and
analysis. Research was performed under the University of Kentucky
Institutional Animal Care and Use Committee protocol No. 05-2015
and Kentucky Department of Fish and Wildlife Resources permit No.
SC1711117.
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