1. 1
Selectiveforagingby EasternRed-backedSalamanders, Plethodon cinereus,among3ant species;
Aphaenogasterpicea,Lasiusalienus,andLasiusnearticus.
NICOLASA.SPIES,SARAHBOLTON,and KYLE HOVEY
Departmentof Biology,John CarrollUniversity,University Heights,Ohio,44118
RunningHead:SPIES ET AL: SELECTIVEFORAGINGBY RED-BACKEDSALAMANDERS

2. 2
Abstract
Eastern Red-backed Salamanders, Plethodon cinereus, are one of the most abundant
organisms within the United States; because of this, many studies have been conducted on their
behavior, foraging tactics, and diet. P. cinereus has been described as a generalist predator of
invertebrates available in terrestrial environments. From all these studies, only a few have
managed to directly assess their dietary preferences to a species level. We examined foraging
tactics on 3 different ant species; Aphaenogaster picea, Lasius alienus, and L. nearticus, to
determine if P. cinereus prefers one species of ant over another within forests in northeastern
Ohio. All 3 ant species inhabit similar habitats of P. cinereus, but were eaten at different rates.
Our results indicate that P. cinereus forages on A. picea and L. alienus at about the same rate and
avoids foraging on L. nearticus. Plethodon cinereus avoid foraging on L. nearticus due to the
high abundance of volatile chemicals specifically the chemical compound, 2-tridecotone. P.
cinereus most likely prefer A. picea and L. alienus because of their high caloric values and the
energy they receive in return. Our results also demonstrate how salamanders rely on chemical
cues through vomeronasal sensory to access prey items. Since ants are an abundant species
within terrestrial environments, it is important to consider what the effects could be on forests
when red-backed salamanders prefer to forage on specific ant species and avoid foraging on
other ant species. More importantly this idea changes the way ecologists view tropic interactions
within complex communities in terrestrial environments.

3. 3
Introduction
One of the most widely distributed and commonly observed amphibian species in the
Eastern United States is the red-back salamander, Plethodon cinereus (Petranka, 1998). This
species is very important to food web structures in terrestrial ecosystems. According to Semiltch
et al’s (2014) study they believe that previous results have underestimated the importance of
salamander biomass, nutrient, and energy flux, and their functional role in regulating
invertebrates and carbon retention in forest ecosystems. This species can be found under logs,
leaf litter, and rocks in late successional deciduous forests of northeastern North America
(Petranka, 1998) and feed on a broad variety of invertebrates (Burton, 1976). Common prey of
P.cinereus are Acarina (mites), Araneida (spiders), Collembola (springtails), adult Coleoptera
(beetles, primarily Curculionidae), larval Coleoptera, adult Diptera (flies), larval Diptera,
formicid Hymenoptera (ants), non-formicid Hymenoptera (wasps), larval Lepidoptera(moths),
shelled Gastropoda (snails) and non-shelled Gastropoda (slugs) (Maerz et al, 2006).
P. cinereus forage exclusively at night (Alder, 1969, 1970) when visual cues would likely
not be available for prey detection under cover objects and the dense canopy of old growth
forests, so the primary detection of prey is through chemical cues (Placyk and Graves, 2001).
More accurately, David and Jaeger (1981) found that P. cinereus rely on visuals cues to detect
mobile prey but rely primarily on chemical cues to locate stationary prey. The olfactory system
of terrestrial salamanders is characterized by an elongate snout that houses paired and
cartilaginous olfactory capsules (Placyk and Graves, 2002). The nasal olfactory system is
primarily responsible for the detection of volatile odorants (airborne chemical cues), while the
vomeronasal system responds to primarily nonvolatile odorants (substrate-borne chemical cues)
(Burghardt, 1980, Bertmar, 1981, and Halpern, 1987).

4. 4
Red-backed salamanders are generalist predators with ants being an important part of
their diet, making up 33% of prey taken in northeastern Ohio (Ivanov et al, 2011). Ants are very
abundant organisms in terrestrial environments, having the ability to physically and chemically
alter the soil environment which in turn indirectly affects plants and other organisms inhabiting
the leaf litter (Holldobler and Wilson, 1990; Folgarait, 1998). Recent studies suggest that P.
cinereus exhibit selective foraging behavior within their territories and ant diversity observed in
the diet represents a subset of that available in the surrounding leaf litter habitat (Paluh et al,
under review). Therefore, P. cinereus preyed on a subset of the available ants present within
their territories (Paluh et al, under review). Paluh (under review), indicated that in northeastern
Ohio the ant species, Aphaenogaster picea, were among the most eaten ant species within P.
cinereus territories, while Lasius aliens tended to be avoided.
For this current study the goal is to determine if P. cinereus is a specialist on one
particular ant species over another. We chose to use 3 different species of ants for this
experiment that are common within leaf litter in northeastern Ohio. Two of the 3 ant species
were selected from data previously collected, A. picea and L. alienus, and the third ant species
selected was L. nearticus. A. picea is a very common species on the edge of woods in moist or
dense woods (Wesson and Wesson Jr., 1940), L. alienus is a non parasitic ant found throughout
North Temperate Zone (Regnier and Wilson 1968), and Lasius nearticus is a moderately
abundant species found under stones and logs in upland woods (Wesson and Wesson Jr., 1940).
Maerz et al (2005) found one introduced ant species, Lasius alienus, among seven native ant
species. L. alienus was the fourth most frequent and volumetrically important ant in salamander
diets, occurring in 13% of samples with ants and accounting for an average of 10% of ant
volume. L. alienus was significantly more common than its native congener, L. nearticus, which

5. 5
occurred in only 2% of diet samples with ants (Anthony and Pfingsten, 2013). The hypothesis is
that P. cinereus would prefer A. picea over L. alienus and L. nearticus, because of the defensive
compounds found in L. alienus, which could be also present in the congener, L. nearticus.
Methods
Specimens collected and housing
Ants were collected from the beginning of September 2014 to the beginning of
November 2014 at Doan Brook Watershed, Cuyahoga County, Ohio (N 41° 27.779 W 81°
18.252) and The West Woods, Geauga County, Ohio (N 41° 29’ 0.76” W 81° 34’ 19.85”).
During this time 3 different colonies of each ant species (A. picea, L. alienus, and L. nearticus)
were collected from underneath cover objects (rocks, logs, etc.). When collecting ants the
observations was made that the species, L. nearticus was never found under a cover object
cohabitating with P. cinereus. Ants were collected using a garden shovel and then placed into
plastic Ziploc containers with soil and leaf litter substrate. These ants were then housed in a
temperature controlled lab room at 17 C under a natural photo period.
P. cinereus were collected from the same locality as Paluh et al in April 2014. These
individuals were used during the summer 2014 in a separate experiment. Salamanders were kept
in the same lab room, under the same conditions. They were housed in 480 cc glass bowl
containing leaf litter prior to experiment.
Feeding Experiment
The feeding experiment was conducted over a consecutive 3 week time span from
October 25, 2014 – November 9, 2014. Twenty-four striped adult female P. cinereus (snout-vent
length (SVL) of 32-34 mm) (Anthony and Pfingsten, 2013). Anthony et al (2008) indicated that
unstriped P. cinereus would consume more ants as prey rather than striped P. cinereus, however,

6. 6
Paluh et al (under review) found that striped P. cinereus consumed 2.5 times more ants than
unstriped P. cinereus. Because of this recent study, we chose to use the striped morph of P.
cinereus in this feeding experiment. In order to control for the level of hunger at the start of each
trial, a rigid feeding schedule was maintained (Jaeger and Barnard, 1981) and individual P.
cinereus received ants to eat once every 7-8 days during the 3 week period.
Individual P. cinereus were placed into petri dishes (65mm X 15mm) containing a lid
covered with white opaque masking tape while the bottom of the petri dish lined with damp filter
paper (Jaeger and Barnard, 1981). The cover lids were wrapped with white opaque masking tape
to replicate the natural microhabitats of a cover object, such as a rock or log, where P. cinereus
find refuge (Petranka, 1998). Salamanders were placed into these petri dishes 7 days prior to the
feeding experiment allowing the substrate to be marked with their own pheromones so they
would exhibit little escape behavior (Tristram, 1977).
During the first week trial, 8 individual P. cinereus were fed A. picea, 8 additional
individual P. cinereus were fed L. alienus, and the remaining 8 individual P. cinereus were fed
L. nearticus. For the second week of trials, individual P. cinereus were assigned ant species by
the flipping of a coin. Prior week data were used to determine which individuals ate which ant
species. After that was determined, the heads face value on the coin was assigned one ant
species which an individual was not yet fed and the tails value on the coin was assigned the other
ant species which an individual was not yet fed. In the third week of trials, the previous 2
weeks’ data were used to determine which individuals had been fed which ant species. Based on
that information, individuals were fed an ant species they that had not yet been fed in the prior 2
weeks of the experiment.

7. 7
Every feeding trial was ran for a duration of 1 hour. The hour feeding duration was
initiated once an individual P. cinereus attacked and ate its first ant. Latency to attack was not
collect if the time recorded was longer than 15 minutes. For each feeding trial, 10 individuals of
each species were presented in each individual salamander’s petri dish.
Chemical Evaluation
When each colony of the 3 species of ants were collected, 5 individuals from each colony
were immediately collected and placed into a vial filled with a 2 ml of a 70% methanol solution.
Three vials of 70% methanol extract of each species were analyzed by using gas chromatography
mass spectral analysis (GC-MS) (Saporito et al, 2004) to determine the chemical compounds
present in the 3 ant species; A. picea, L. alienus, and L. nearticus. Each 70% methanol extract
sample took 29 minutes to run though the GC-MS.
Statistical Analysis
We used a repeated measures analysis (SPSS Version 21) to compare the total number of
ants consumed by each 1 of the individual 24 P. cinereus during the feeding experiment and the
total number of each of the 3 individual ant species consumed during the feeding experiment.
Repeated measures analysis (SPSS Version 21) was also used to compare latency to first attack
between each 1 of the individual 24 P. cinereus used in the feeding experiment and the latency to
first attack between each of the 3 individual ant species during the feeding experiment.
Results
Feeding Experiment
Twenty-four individual striped female P. cinereus were fed 3 different species of ants; A.
picea, L. alienus, and L. nearticus over a consecutive 3 week time frame. Latency to first attack
was collected for most P. cinereus individuals except for those individuals that failed to attack

8. 8
within 15 minutes. Individuals who did not attack within the first 15 minutes were considered to
be “unresponsive” (Jaeger and Bernard, 1981), however, these individuals were still allowed to
feed on ants for 1 hour and were assigned a latency to first attack of 900 seconds (15 minutes).
We found significant differences among the number of ants eaten (df Hypothesis=2, df Error=46,
F=15.439, and p<0.001) and no significance among the number of ants each individual P.
cinereus ate (df Hypothesis=23, df Error=46, F=2.160, p=0.013). After analyzing the latency
results, we detected significant differences in latency to first attack among individual
salamanders (df Hypothesis=23, df Error=46, F=1.878, p=0.034), and a significant effect of ant
species on latency to first attack among individual salamanders (df Hypothesis=2, df Error=23,
F=2.808, p=0.071).
We also recorded if any P. cinereus were attacked by ants during the feeding experiment.
There was a slightly significant effect indicated by 1 x 3 chi square analysis on the mean number
of ants eaten of each of the three species when compared to the total number of times an
individual P. cinereus was attacked (x2=5.12, df=2, P=0.077) (Figure 4). However, there was a
weak correlation present to whether P. cinereus were deterred from eating ants after being
attacked.
Chemical Evaluation
Three vials containing 5 individuals from 3 separate colonies of A. picea, L. alienus, and
L. nearticus were ran through the GC-MS for 29 minutes. During the GC-MS analysis a graph
was produced with peaks of compounds retentions times, representing chemical compounds
found within the ants. This data were then analyzed to determine if any of these chemical
compounds were chemical defenses present in any of the three species of ants. The GC-MS
analysis indicated that the most abundant chemical compound within the ant species, A. picea

9. 9
and L. alienus, was a fatty acid with a retention time of about 15 minutes (Figures 5 & 6). The
GC-MS analysis indicated that the 2 most abundant chemical compounds within the ant species,
L. nearticus, were 2-pentadrcanone and 2-tridecanone; which is a volatile chemical compound
used as defense chemical against predators and other ants (Regnier and Wilson, 1969). L.
alienus also exhibited this compound, but only in trace amounts.
Discussion
Our results suggest that P. cinereus forage selectively between the three ant species; A.
picea, L. alienus, and L. nearticus (Figures 1, 2, 3, & 4). Recent studies indicated that A. picea is
commonly consumed in the spring and summer and became rare in the environment during the
fall. However it was preferentially sought out by P. cinereus remaining abundant in their diet,
making up the largest portion of the formicid diet of salamanders (37.2%) (Paluh et al, under
review). L. alienus was suggested to be avoided in the fall season, but was prevalent in P.
cinereus diets in the spring and summer (5.8% of diet) (Paluh et al, under review). However, our
data shows differently, salamanders tended to prefer both A. picea and L. alienus at about the
same rate (Figures 1 & 2) and avoided L. nearticus (Figure 4).
A. picea most commonly nest under rocks, logs, limbs, barks of rotten logs, and under
barks of logs in often moderately large colonies (Coover, 2005), L. alienus most commonly nest
under rocks, logs and bark in typically large and vigorous colonies (Coover, pgs. 120-121), and
L. nearticus most commonly nest in soil and under rocks and logs in small colonies (Coover,
pgs. 124-125). All three of these species colonies are located in similar habitats to that of P.
cinereus; however, they were preferred at different rates. This idea of similar habitats between
P. cinereus and the ant species; A. picea and L. alienus, indicates why salamanders may have
preferred this species of ant, but does not explain why salamanders would not prefer the ant

10. 10
species, L. nearticus, as well especially given that they have small colonies that would be less
able to mount a defense against an intruding salamander. Therefore, there must be something
chemically different between the three ant species indicating why P. cinereus would avoid one
species and prefer the two other species.
There have been at least 12 volatile compounds described in the ant species, L. alienus,
which are used in alarm communication within the colonies and for combat defense of predators
and other ants (Regnier and Wilson, 1969). Paluh et al (under review) hypothesized this to be
one reason why L. alienus was avoided by P. cinereus, however, our data our data suggest
otherwise, indicating that even with these volatile defense chemicals, P. cinereus preferred L.
alienus at about the same rate as A. picea. This is probably due to the small trace amounts that
were detected through chemical analysis. These results support the data collected by Maerz et al
(2005) indicating that L. alienus was the fourth most common ant species in P. cinereus diet
from data collected in Pennsylvania and New York.
The least eaten species of ant, L. nearticus, contains the same 12 volatile compounds as
the ant species, Lasius alienus, however, the presence of these compounds were found in L.
nearticus at a much higher rate though GC-MS (Figures 6 & 7). During the feeding experiment,
Plethodon cinereus was observed multiple times attacking L. nearticus, but soon after spat out
the individual that was attacked. Following the rejection of eating this species the individual
salamanders were observed rubbing their nose against the most paper substrate at the bottom of
the petri dish.
Taste-rejection, the assessment of palatability or toxicity of food after an attack that
involves oral contact and sometimes ingestion of a small portion of the food item, is a method
used by predators to evaluate chemical prey defenses (Lindquist, 1996; Sillen-Tullberg, 1985;

11. 11
Skelhorn and Rowe, 2006; Wiklund and Jarvi 1982). Through taste-rejection a predator is able
to determine, either through taste specifically or through minor intoxication, the relative toxicity
of a prey item (Avila at el, 2011). Taste-rejection occurs well after prey have been captured,
thus requiring direct contact between the predator and prey (Avila at el, 2011). All of these
taste-rejection observations were only made during the feeding experiment with the ant species,
L. nearticus, indicating that their volatile compounds work as an anti-predatory defense against
P. cinereus. The most abundant volatile compound found in this species was the compound, 2-
tridecanone (Figure 7). Morgan et al (2005) describes 2-tridecanone as a long hydrocarbon
chain with a ketone, which is used as a defensive chemical against other ants and predators
(Regnier and Wilson, 1969).
Optimal foraging theory predicts that predators will rank all potential types of prey by
their profitability and then specialize on the most profitable type when it is abundant (Jaeger and
Rubin, 1982). Our findings show correlation with this theory through the foraging behavior
exhibited by P. cinereus during the feeding trials.
The idea that P. cinereus foraged on A. picea and L. alienus at about the same rate
indicates that they specialized on those prey types that are most profitable, because of their high
caloric values exhibited through the GC-MS analysis (Figures 5 & 6). There were selective
differences present between these two species that were preferred in terms of latency to first
attack. Latency to first attack on L. alienus occurred in a shorter time span than latency to first
attack on A. picea. Also L. alienus was eaten at a steadier rate when compared to the number of
ants eaten and latency to first attack when compared to the rate at which A. picea was eaten when
compared to latency to first attack (Figures 1 & 2). The highest average latency to first attack
(526 seconds) was calculated within A. picea and the lowest average latency to first attack (319

12. 12
seconds) was observed in L. alienus. This may indicate that P. cinereus prefers to forage on L.
alienus more so than A. picea. Another reason why salamanders may prefer L. alienus slightly
more over A. picea could be because of the more aggressive behavior exhibited by A. picea
(Figure 4). In nature, L. alienus benefits from occurring in large colonies, but in our experiment
that component was removed, only providing 10 ants. This may explain the differences between
our results and Paluh et al’s results.
Plethodon cinereus tended to avoid L. nearticus because of their aggressive behavior that
was exhibited and volatile chemicals when ingested (Figure 7). However, our results indicated
that P.cinereus had the second lowest average latency (385 seconds) to first attack on this ant
species. The species within the genus Lasius could have been more readily detected by
salamanders, because of the volatile chemical compound, 2-tridecanone. This compound could
be both good and bad for ant prey. It seems that P. cinereus was able to detect this volatile
compound in L. alienus and L. nearticus though their vomernasal sensory organ making it more
easy for salamanders to forage for these species (Figures 2, 3, 6, & 7). If this chemical
compound was found in high concentration within an ant species then it was an effective
defensive component against predators (i.e. L. nearticus) (Figure 7).
This study demonstrates that P. cinereus, a known generalist that feeds on a broad variety
of invertebrates (Burton, 1976) may exhibit selective foraging on certain invertebrates over
others, specifically ants. Ants make up a large portion of salamander’s diets (33% of prey taken)
within northeastern Ohio (Ivanov et al, 2011). The most important finding within this study is
that P. cinereus was able to accurately determine that L. nearticus was an unprofitable prey
through taste-rejection. More importantly salamanders may prefer one species over another for
caloric value or avoid a species, because of high concentrations of volatile chemicals. Also P.

13. 13
cinereus exhibited that the use of chemical cues through vomernasal sensory is an important
adaptation for detecting prey during foraging. This data then changes the understanding of leaf
litter tropic interactions between guilds and tropic level interactions. Since ants have the ability
to physically and chemically alter the soil environment which in turn indirectly affects plants and
other organisms inhabiting the leaf litter (Holldobler and Wilson, 1990), be selectively preyed on
or avoid could drastically change the biodiversity of forests from one region to another.
Acknowledgments
We thank both Doan Brook Watershed and The West Woods for allowing use to use their
properties to obtain ants. We also thank John Carroll Department of Chemistry for allowing us
to use the GC-MS for data analysis and Dr. Ralph Saporito for helping in determining the
chemical compounds found within each ant species.

14. 14
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FIGURE LEGENDS
Figure 1: Latency in seconds to first attack on ant species Aphaenogaster picea as a function of
Aphaenogaster picea eaten in an hour feeding time period by Plethodon cinereus (n=24). As
latency to first attack increased, the number of Aphaenogaster picea eaten decreased at a
relatively rapid rate. Data were collected on October 25, 2014, November 2, 2014, and
November 9, 2014.
Figure 2: Latency in seconds to first attack on ant species Lasius alienus as a function of Lasius
alienus eaten in an hour feeding time period by Plethodon cinereus (n=24). As latency to first
attack increased, the number of Lasius alienus eaten slightly increased. Data were collected on
October 25, 2014, November 2, 2014, and November 9, 2014.
Figure 3: Latency in seconds to first attack on ant species Lasius nearticus as a function of
Lasius nearticus eaten in an hour feeding time period by Plethodon cinereus (n=24). As latency
to first attack increased, the number of Lasius nearticus eaten slightly increased. Data were
collected on October 25, 2014, November 2, 2014, and November 9, 2014.
Figure 4: Mean number of ants eaten (Aphaenogaster picea, Lasius alienus, and Lasius
nearticus) by Plethodon cinereus (n=24) compared to the number of times each individual ant
species attacked an individual P. cinereus during the feeding experiment.
Figure 5: Gas chromatography mass spectral analysis (GC-MS) graph produced indicating the
compounds found in the ant species, Aphaenogaster picea. The largest peak produced with the
retention time of 15 minutes indicates fatty acids as the most abundant chemical compound
within A. picea.
Figure 6: Gas chromatography mass spectral analysis (GC-MS) graph produced indicating the
compounds found in the ant species, Lasius alienus. The largest peak produced with the

18. 18
retention time of 15 minutes indicates fatty acids as the most abundant chemical compound
within L. alienus. This sample also contained the chemical compound, 2-tridacanone (a
defensive (volatile) chemical compound), which had a retention time of about 8 minutes and 11
minutes, however, it was present in small amounts.
Figure 7: Gas chromatography mass spectral analysis (GC-MS) graph produced indicating the
compounds found in the ant species, Lasius nearticus. The 2 largest peaks produced with the
retention times at about 8 minutes and 11 minutes represent an isomer known as, 2-tridecanone
(a defensive (volatile) chemical compound).

19. 19
0
1
2
3
4
5
6
7
8
9
0 100 200 300 400 500 600 700 800 900 1000
NumberofAphaeanogasterpiceaeaten
Latency(seconds)to firstattack

20. 20
0
1
2
3
4
5
6
7
8
9
10
0 100 200 300 400 500 600 700 800 900 1000
NumberofLasiusalienuseaten
Latency (seconds) to fistattack

21. 21
0
1
2
3
4
5
6
0 100 200 300 400 500 600 700 800 900 1000
NumberofLasiusnearticuseaten
Latency (seconds) to firstattack

22. 22
0
2
4
6
8
10
12
14
16
Apeanogaster picea Lasius alienus Lasius nearcticus
Mean number of ants eaten Times attacked by ants

23. 23

24. 24

25. 25