RESEARCH ARTICLE

Effects of prey, pitcher age, and microbes on

acid phosphatase activity in fluid from
pitchers of Sarracenia purpurea
(Sarraceniaceae)
Carl S. Luciano*, Sandra J. Newell

Department of Biology, Indiana University of Pennsylvania, Indiana, PA, United States of America

* luciano@iup.edu
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a1111111111 Abstract
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Carnivory in pitcher plants generally involves digestion of prey, by the plant itself, by symbi-
onts, or both. While symbionts appear to be important in the digestion of prey in Sarracenia
purpurea, the importance of pitcher-derived enzymes is less well documented. Our goal
was to reduce microbial numbers in pitcher fluid in order to measure the acid phosphatase
OPEN ACCESS
activity attributable to the pitchers themselves. Preliminary experiments indicated that vari-
Citation: Luciano CS, Newell SJ (2017) Effects of
ous antibiotics were minimally effective at reducing microbial populations and that antibiotic-
prey, pitcher age, and microbes on acid
phosphatase activity in fluid from pitchers of resistant microbes were easily cultured from pitcher fluid. Consequently, we measured the
Sarracenia purpurea (Sarraceniaceae). PLoS ONE abundance of culturable microbes in every sample taken for the measurement of acid phos-
12(7): e0181252. https://doi.org/10.1371/journal. phatase activity. Pitchers fed with one sterilized ant had higher levels of acid phosphatase
pone.0181252
activity than unfed pitchers. Older pitchers were more responsive to feeding than young
Editor: Dawn Sywassink Luthe, Pennsylvania State pitchers. Pitchers with high levels of microbes (on Day 5) had higher acid phosphatase activ-
University, UNITED STATES
ity than pitchers with low levels of microbes. However, fed pitchers were not more likely to
Received: March 17, 2017 have higher microbe levels and microbe levels were not related to pitcher age. When fluid
Accepted: June 28, 2017 samples from inside the pitcher were compared to appropriate controls incubated outside
Published: July 18, 2017 the pitcher, acid phosphatase activity was higher inside the pitcher. Results from the feeding
experiments are consistent with a primary role of microbes in the digestion of prey in pitchers
Copyright: © 2017 Luciano, Newell. This is an open
access article distributed under the terms of the of S. purpurea. However, the relationship between pitcher age and enzyme activity is not a
Creative Commons Attribution License, which function of microbes in the pitcher fluid and may depend on enzymes produced by the plant.
permits unrestricted use, distribution, and Our methods would not detect microbes embedded on the inner surface of the pitcher; and
reproduction in any medium, provided the original
author and source are credited.
if they survived the alcohol rinse and antibiotics, we cannot rule out microbes as the source
of the relationship between pitcher age and acid phosphatase activity.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.

Funding: The authors CSL and SJN received

funding from the IUP Senate (http://www.iup.edu/ Introduction
senate/committees/research/) and the IUP School
Carnivory evolved independently in several plant families resulting in a diversity of trapping
of Graduate Studies (http://www.iup.edu/
graduatestudies/) to support this work but no
mechanisms such as the snap trap of Dionaea muscipula, the suction trap of Utricularia, the
specific grant numbers were associated with the sticky-leaf trap of Drosera and Pinguicula, and the pitfall trap of the various genera of pitcher
funding. The funders had no role in study design, plants [1–4]. In all types of traps, carnivory generally involves digestion of prey. Digestion may

PLOS ONE | https://doi.org/10.1371/journal.pone.0181252 July 18, 2017 1 / 17

 Acid phosphatase in pitchers

data collection and analysis, decision to publish, or be accomplished by the plant, by its symbionts, or both [5,6]. Some studies of digestion in
preparation of the manuscript. pitcher plants have recognized the importance of distinguishing between enzymes produced
Competing interests: The authors have declared by plants and enzymes produced by microbial symbionts.
that no competing interests exist. There are pitcher plants that clearly produce digestive enzymes in the pitchers. Nepenthes
species are well studied in this regard. For example, Jentsch [7] purified the protease nepentha-
cin, and Tökés et al. [8] reported proteases and lipase in Nepenthes secretions taken from sterile
closed pitchers. More recently, Rotloff et al. [9] isolated a class III acid endochitinase from
closed pitchers of Nepenthes species. In the most extensive study to date, Buch et al. [10] mea-
sured protease activity in several Nepenthes species and the plant’s response to prey introduc-
tion. However, even in Nepenthes digestion may be facilitated by bacterial symbionts [11].
Other pitcher plants are less well studied, but there is evidence for plant-produced digestive
enzymes in other genera. Using enzyme labeled fluorescence (ELF), Płachno et al. [12] demon-
strated the activity of phosphatases on the walls of gland cells on the inner surface of pitchers
of Cephalotus follicularis and Nepenthes tobaica. However, they also noted phosphatase activity
associated with bacteria on the inner wall of Cephalotus pitchers and in the pitcher fluid of
both Cephalotus and Nepenthes. Closed pitchers of Heliamphora tatei contained fluid that
exhibited protein hydrolytic activity, but four other species of Heliamphora tested negative
[13]. Heliamphora and Sarracenia are closely related [14], and Darlingtonia californica, which
is sister to the Sarracenia-Heliamphora clade, apparently has no digestive glands and makes no
digestive enzymes [15]. The possible presence of plant-produced digestive enzymes in Sarrace-
nia is interesting from an evolutionary perspective since some related species appear to differ
in their digestive processes.
In Sarracenia much early work assayed for digestive activity in pitcher fluid of open pitchers
[16], although some studies distinguished between fluid taken from open or closed pitchers
[17]. Since unopened pitchers are sterile [18,19], this should effectively rule out a microbial
source of digestion. After pitchers open they are eventually colonized by a diverse assemblage of
bacteria [19]; and, depending on food web structure, bacteria increase in density and richness
when nutrients are added (e.g., [20–24]). Using a cytochemical stain, Stauffer [25] demonstrated
acid phosphatase activity in sessile glands on both the outside and inside surface of S. purpurea
pitchers as well as in the pitcher fluid, but he did not rule out the potential for bacterial enzymes.
Sarracenia purpurea pitchers also had measurable activities of protease, RNase, nuclease, and
phosphatase when sampled a week after adding antibiotics [26]. However, we suggest caution in
interpreting the source of this enzyme activity since bacteria may repopulate the pitcher after
the addition of antibiotics. Recently, Fukushima et al. [27] assayed pitcher fluid from four spe-
cies and found digestive proteins; but the potential for microbial sources was not investigated.
The extent to which S. purpurea pitchers produce digestive enzymes is still an open question
[28]. Acid phosphatase activity in pitcher fluid may be important for several reasons. Phospho-
rus is one of several limiting nutrients for S. purpurea, and insect prey provide phosphorus to
the plant [29–32]. Pitcher fluid is often slightly acidic [28], and Stauffer [25] found glands on
the surface of S. purpurea leaves that stained positive for acid phosphatase activity. Extracellu-
lar acid phosphatase hydrolyzes phosphate from phosphoesters, making phosphorus from
organic sources more readily available to the plant; and extracellular acid phosphatase may
derive from plants or microbes [33–35].
Since S. purpurea pitchers are open to rainfall, a diverse aquatic community forms within
the confines of a pitcher. Inquiline communities of S. purpurea pitchers may include bacteria,
archaea, algae, fungi, protists, rotifers, cladocerans, copepods, mites, mosquito larvae (Wyeo-
myia smithii), midge larvae (Metriocnemus knabi), and sarcophagid fly larvae (Fletcheromyia
fletcheri); and the composition and relative abundances of these organisms is highly variable
among pitchers and among geographical locations [19,36–38]. Much research has focused on

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 Acid phosphatase in pitchers

the dipterans. For example, Heard [39] described the relationship between dipteran larvae, W.
smithii and M. knabi, and S. purpurea as mutualistic, demonstrating that the M. knabi larvae
initiate the breakdown of prey by physically tearing the carcass, making more nutrients avail-
able to other inquilines and to the pitcher.
However, microbes appear to play a pivotal role in the digestive capacity of S. purpurea. Sar-
racenia purpurea pitchers with and without complete inquiline communities did not differ in
their nitrogen uptake [40]. And, Butler et al. [40] demonstrated that nitrogen uptake by S. pur-
purea pitchers was similar to nitrogen uptake by pitchers of Sarracenia alata and Sarracenia
flava. Unlike S. purpurea, neither S. alata nor S. flava supports a complex aquatic community,
but like S. purpurea both are exposed to bacterial colonization. Koopman et al. [41] and Koop-
man and Carstens [42] found a rich bacterial community within S. alata pitchers, one that was
quite different from the microbial community of the surrounding soil yet mirroring the popu-
lation genetic structure of the plant species. Mouquet et al. [43] modeled the inquiline ecosys-
tem within S. purpurea pitchers and concluded that bacteria alone would maximize the
availability of nitrogen to the plant and that additional components of the food web would
likely influence the plant via their effect on the bacteria. Clearly, the bacterial community is
important for digestion of prey in Sarracenia pitchers.
Does S. purpurea rely on symbiotic organisms such as bacteria for the production of diges-
tive enzymes? What is the plant’s capacity for digestion of prey? Initially our goal was to quan-
tify the amount of enzyme activity deriving from the plant itself by preventing the colonization
of new pitchers by bacteria or eliminating bacteria from older pitchers. We experimented with
several antibiotics to determine their effectiveness. After it became clear that antibiotics were
not necessarily effective in eliminating microbes in the fluid of the pitcher, we used an ethyl
alcohol rinse and antibiotics to try to enhance the inhibition of microbes in the pitcher. Our
initial goal of eliminating the bacteria was unrealistic, as we document below. Our modified
goal was to sufficiently reduce the bacterial numbers for a short period of time to be able to
quantify the enzyme activity of the pitcher.
More specifically, we ask the following questions:
1. How best can we eliminate or reduce the impact of microbes on enzyme activity so that
measurements reflect the enzyme activity of the pitchers themselves?
2. Does feeding the pitcher increase the enzyme activity within the pitcher?
3. Is enzyme activity related to pitcher age? Are older pitchers more responsive to feeding
than younger pitchers?
4. Within the context of the experiment (where we have attempted to reduce microbes), are
microbe levels correlated with either initial or final enzyme activity levels? Do samples with
higher microbe levels have higher levels of enzyme activity? Do older pitchers or fed pitch-
ers have higher levels of microbes?
5. Can we distinguish the relative importance of microbes vs. pitchers for the acid phosphatase
activity? Do outside controls exhibit enzyme activity? In other words, when samples are
removed from the pitcher on Day 0 and incubated for 5 d outside the pitcher, do they
exhibit the same patterns of enzyme activity as samples incubated inside the pitchers?

Methods
Sarracenia purpurea L. [44] were obtained from a commercial nursery as adult plants and main-
tained in a growth chamber under controlled conditions for several years prior to the experiments.

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 Acid phosphatase in pitchers

Growth medium was an unsterilized soil mixture of 6 parts commercial peat, 2 parts perlite, and 1
part sand. Day:night schedule in the chamber was 14 h:10 h photoperiod and 23 C:15 C tempera-
ture regime. Relative humidity was at or above 60%. Plants were routinely monitored for pitcher
formation and pitcher age, and pitchers were used in experiments as they became available. Mar-
gins of immature pitchers are sealed and thus the interiors are microbiologically sterile until the
margins separate and the pitchers open [19]. To prevent aerial colonization by microorganisms
following opening, individual pitchers were covered with sterile plastic Whirl-Pak bags prior to
opening. Bags were suspended over pitchers by attaching the bag to two slender stakes, forming a
tent over the entire pitcher. The bag was placed over the pitcher shortly before it opened and
remained on the pitcher for the duration of the experiment. Raising the bag on the stakes subse-
quently allowed access to the pitcher during experiments. The bag was not sealed around the base
of the pitcher, and no condensation occurred in the bags.
Ants were used as simulated prey animals. Ants for prey were purchased from Carolina Bio-
logical Supply Company (“black wood ants, species not identified”) and stored at -20 C until
used. Ant carcasses were sterilized by autoclaving for 15 min at 121 C.
All chemicals, substrates, buffers and antibiotics were purchased from Sigma-Aldrich.
Microbiological media included Nutrient Agar (NA), Corn Meal Agar (CMA), Luria-Bertani
Agar (LB), Potato Dextrose Agar (PDA), and Trypticase Soy Agar (TSA). All microbiological
media were purchased from Becton and Dickinson Company (BD) under a variety of brand
names and prepared according to the supplier’s instructions.
The acid phosphatase assay was standard, following the directions provided with the
substrate from Sigma-Aldrich. Acid phosphatase activity was assayed in 0.01 M MES (2[N-
morpholino)ethanesulfonic acid), at pH 6.0 with 0.01 M 4-nitrophenylphosphate (Paranitro-
phenylphosphate or PNPP) as substrate. Assays were incubated at room temperature for at
least 1.5 h. The reaction was then stopped by the addition of 1.0M NaOH. In qualitative assays
results were assessed visually by the presence of yellow color in the sample, but in quantitative
assays A405 nm was measured in a 96-well microplate format using a microplate reader with
readings in μg/mL accurate to three decimal places. Assays were done in duplicate. Each sam-
ple was assayed twice in adjacent wells of a microtiter plate, and the two acid phosphatase
measurements were averaged to provide one measurement per sample. Wheat germ acid phos-
phatase was used as a standard. Pitcher fluid was assayed for a small number of pitchers as the
pitchers became available; fresh standards were used for every set of assays. Beckman Coulter
Phi Series pH meters and combination electrodes were used for pH measurements.

Preliminary experiments
In order to attribute enzyme activity to the pitcher, we needed to eliminate the possibility of
enzyme activity by bacteria. Hence, we needed to establish the best methods for removal of
microbes from pitchers and best possible conditions for microbial growth in order to be confi-
dent bacteria were not present in the samples. Initially, we tested various agars and incubation
temperatures to determine the optimal conditions for growth of microbes derived from pitch-
ers. Microbial cultures from two older pitchers, freely exposed to the air in the growth chamber
for an extended time, were plated onto nutrient agar (NA) at pH 6.8, potato dextrose agar
(PDA) at pH 5.8, Luria broth (LB) agar at pH 7.0, corn meal agar (CMA) at pH 6.0 and trypti-
case soy agar (TSA) at pH 7.3. These pHs result with standard preparation of these agars. Plates
were incubated at 15 C (the low temperature setting of the growth chamber), 25 C (similar to
the high temperature setting of the growth chamber), or 35 C (near the standard of 37 C for
incubating bacteria) for four days. Numbers of colony forming units per plate (CFU/plate)
were counted daily and averaged for each medium at each temperature.

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 Acid phosphatase in pitchers

Microbes were abundant in pitchers. In order to ascribe enzyme activity to the pitcher, we
began a series of experiments to determine the effectiveness of antibiotics at reducing micro-
bial populations. At the start of the experiments (n = 13 pitchers), endogenous pitcher fluid
was removed and each pitcher received 8 mL of sterile water containing 250 μg/mL cefotax-
ime, 100 μg/mL carbenicillin, and 100 μg/mL ampicillin (CCA cocktail). Two mL samples
were removed from each pitcher, starting on Day 0 and every other day through Day 8. Sam-
ples were assayed for acid phosphatase as described above. Also, for each sample pH was mea-
sured, and each sample was plated on NA and LB and incubated at 25 C for 4 d to check for
microbial growth. Some pitchers received additional CCA cocktail on Days 2, 4, and 6. For
these pitchers, all pitcher fluid was removed, and 2.5 mL of fresh antibiotic solution were
placed in the pitcher. pH was measured and a sample removed (prior to replacing the antibiot-
ics) for the acid phosphatase assay. Replacement of antibiotics did not produce different
results, so data from this series of experiments have been combined.
When these antibiotics proved relatively ineffective, bacteria derived from 12 pitchers were
plated in duplicate samples (i.e., from one loop) onto two additional plates of NA, one with
and one without CCA cocktail to test for antibiotic resistance. Escherichia coli served as a
control.
A second series of preliminary experiments (n = 31 pitchers) tested a different set of antibi-
otics. At the start of each experiment, each pitcher received PSA cocktail, a mixture of 10,000
units per mL of penicillin, 10 μg/mL of streptomycin and 25 μg/mL of the antimycotic ampho-
tericin B (Sigma antibiotic/antimycotic lyophilized powder, A7292). The buffer was 10 mM
solution of MES, calibrated to pH 6.0. This buffer has a useful pH range of 5.5–6.7 with a pKa
of 6.1. The PSA cocktail (5–6 mL) was added to each pitcher within a couple days after the
pitcher opened, as soon as the width of the pitcher opening allowed access. To determine
whether bacteria were present in a pitcher, each sample was plated onto nutrient agar (NA)
and incubated at 25 C for 3–4 d. Four of the 31 pitchers were also fed sterilized prey. To feed a
pitcher one ant was included with the buffered antibiotics. Some pitchers were also subjected
to repeated antibiotics after the start of the experiment. Experiments in this second series had
similar results, so data from these preliminary experiments were combined.
Bacteria were isolated from 17 different pitchers and were tested for resistance to several
antibiotics. Antibiotics included 1) a mixture of 20,000 units/mL penicillin + 20 μg/mL strep-
tomycin + 50 μg/mL amphotericin B (which is 2x standard concentration), 2) 50 μg/mL tetra-
cycline, 3) 150 μg/mL chloramphenicol, 4) 50 μg/mL gentamicin. Bacterial isolates were plated
onto NA containing antibiotics and incubated at 25 C for 3 d. A control plate did not contain
antibiotics.
The final set of preliminary experiments tested the combination of an ethanol rinse and
PSA cocktail. The pitcher was filled with 70% (v/v) ethanol. The surface sterilant was removed
after 5 minutes of exposure. The pitcher was filled with PSA antibiotic mixture in 10mM MES
pH6. The pitcher was immediately plugged with sterile cotton to prevent microbial contamina-
tion. These experiments proceeded as in previous experiments, measuring acid phosphatase
activity, pH, and microbial growth. This procedure produced minimum microbial growth and
was used for the subsequent experiment. In the preliminary experiments results were assessed
qualitatively; no statistical analyses were performed.

Effect of prey, pitcher age, and microbes on acid phosphatase activity

In this experiment, endogenous pitcher fluid was removed from the pitcher and stored frozen.
Based on preliminary experiments each pitcher was treated with ethanol followed by the PSA
cocktail. Forty-six pitchers, ranging in age from 2–43 d since opening, were included in the

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 Acid phosphatase in pitchers

unfed treatment. Fifty-six pitchers, ranging in age from 1–43 d since opening, were included
in the fed treatment (i.e., fed prey of one sterile ant in sterile water). Pitchers were on different
plants, generally similar in size, and large enough to contain the antibiotic cocktail. Pitchers
were haphazardly assigned to the fed or unfed treatment, taking care to include a wide spread
of pitcher ages in each group.
For each pitcher, acid phosphatase (AcP) activity was assayed several times, using the proto-
col described above for: 1) pitcher fluid sampled immediately after the fluid was placed in the
pitcher on Day 0 (AcP(0)); 2) pitcher fluid sampled after 5 d inside the pitcher (AcP(5)); 3) fluid
placed in the pitcher, immediately removed, and incubated in a test tube in the growth chamber
for 5 d (AcP(o-5)); 4) fluid not placed in the pitcher, sampled at the start of the experiment (Ini-
tial buffer control); 5) fluid not placed in the pitcher, incubated in a test tube in the growth
chamber for 5 d (Final buffer control). Fed pitchers had controls with and without an ant. Each
sample was plated onto nutrient agar (NA) and incubated at 25 C for 3–4 d. Microbes(0) is the
count of colony-forming units per plate (CFUs) in the Day 0 or initial sample. Microbes(5) is
the count of CFUs in the Day 5 sample take from inside the pitcher. Microbes(o-5) is the count
of CFUs in the Day 5 sample taken from the test tube outside the pitcher.
Simple statistical analyses, including descriptive statistics, regressions, and t-tests, were per-
formed in Excel (Microsoft Office 2007, Microsoft Corporation, Redmond, WA). ANOVAs
and nonparametric tests were performed in SPSS (IBM SPSS Statistics Version 23, Release
23.0.0.0, 2015, IBM Corporation, Armonk, NY).

Results
Preliminary experiments
Even with no opportunity to collect rainfall, growth chamber pitchers produced an endoge-
nous fluid within the confines of the pitcher trap (Range = 0–2.2 mL). All samples of pitcher
fluid contained culturable microorganisms though their number and colony morphology var-
ied from pitcher to pitcher. Pitcher fluid microbes had diverse growth patterns in culture but
we did not attempt to identify microbial species. In preliminary experiments we examined
growth of pitcher fluid microbes on various standard growth media and at different incubation
temperatures. Table 1 presents data from one pitcher in a representative experiment.
Microbes from endogenous pitcher fluid grew most rapidly at 25 C. On all five media tested
at 25 C, colony numbers increased to a quantity too numerous to count (TNTC) by 72 h of
incubation. In contrast, at 35 C only organisms plated on NA reached comparable levels,
whereas at 15 C none of the samples on any medium tested reached levels too numerous to
count even after 96 h of incubation.
Microbes from endogenous pitcher fluid had different growth rates on the five different
media tested. At all three incubation temperatures tested, microbial growth was slower on
CMA and PDA than on other media. At 25 C and 35 C growth was most rapid on NA, and at
15 C the growth rate on NA was approximately the same as the growth rate on TSA. Overall,
NA at 25 C supported the most rapid rate of microbial growth. Based on this experiment, we
selected nutrient agar (NA) and an incubation temperature of 25 C for additional experiments.
LB agar was included for comparison with other studies using this medium [20,22,45].
In additional preliminary experiments we evaluated the effect of two antimicrobial cocktails
on the growth of microorganisms in pitcher fluid. In some of these experiments, we removed
endogenous fluid from the pitcher and replaced it with a CCA cocktail (as in [26]). Of thirteen
samples removed from different pitchers 4 d after receiving CCA cocktail, 5 (38%) exhibited
abundant microbial growth and 8 (62%) exhibited some microbial growth. In other experi-
ments we replaced endogenous pitcher fluid with PSA cocktail prior to sampling and

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 Acid phosphatase in pitchers

Table 1. Growth of microbes from endogenous pitcher fluid from one representative pitcher as measured in average number of colony-forming
units per plate (average CFU/plate)1.

Incubation Temperature (C) Medium2 Day 1 Day 2 Day 3 Day 4

35 CMA 0 2 20.5 23.5
LB 1.5 11 29 36.5
NA 30 69 TNTC3 TNTC
PDA 0 12 22.4 28
TSA 0 22.8 43 54
25 CMA 0 0 TNTC TNTC
LB 30.5 69 TNTC TNTC
NA 39 80.5 TNTC TNTC
PDA 9.2 41.6 TNTC TNTC
TSA 17.2 47.6 TNTC TNTC
15 CMA 0 0 0.5 0.5
LB 0 5 27 57.5
NA 0 10.5 31 59.5
PDA 0 0 13.2 28
TSA 0 18.4 39 60
1
Duplicate 50 μL samples of endogenous pitcher fluid from growth chamber plants were plated on microbial media, incubated at indicated temperatures
and scored for colony formation at daily intervals after inoculation. Data represent average values of the duplicate samples for one representative pitcher.
2
CMA: Corn Meal Agar, LB: Luria-Bertani Agar, NA: Nutrient Agar, PDA: Potato Dextrose Agar, TSA: Trypticase Soy Agar.
3
TNTC: too numerous to count

https://doi.org/10.1371/journal.pone.0181252.t001

culturing. Of the fluid samples removed from 31 different pitchers 4 d after receiving PSA
cocktail, 19 samples (61%) exhibited abundant microbial growth, 6 (19%) samples exhibited
reduced microbial growth, and 6 (19%) samples exhibited no microbial growth. Neither cock-
tail (CCA or PSA) abolished the growth of microorganisms from inside the pitcher traps, but
the PSA cocktail slowed microbial growth compared to the CCA cocktail.
Finally, in experiments with 28 pitchers we rinsed the inside of the pitcher trap with 70%
ethanol prior to the addition of PSA cocktail and blocked the pitcher opening with a sterile
cotton plug prior to sampling and culturing. In these experiments, 7 (25%) fluid samples
showed abundant microbial growth, 8 (29%) showed reduced levels of growth, and the
remaining 13 (46%) showed no microbial growth. The combination of ethanol rinse followed
by PSA cocktail produced the greatest reduction in microbial growth.
When bacteria derived from 12 pitchers were plated in duplicate samples on NA with and
without CCA cocktail, 11 samples exhibited microbial growth on NA+antibiotics. One pitcher
sample and the E. coli control did not grow on the NA+antibiotics. In a mixture 2x the stan-
dard concentration of PSA in nutrient agar, 15 of 17 isolates grew. Fifteen isolates grew on
nutrient agar infused with 50 μg/mL tetracycline. Fourteen isolates grew on nutrient agar
infused with either 150 μg/mL chloramphenicol or 50 μg/mL gentamicin. All isolates grew on
the control NA without antibiotics.

Effects of prey, pitcher age, and microbes on acid phosphatase activity

Pitchers fed one sterilized ant on Day 0 generally had higher levels of acid phosphatase activity
on Day 5 (AcP(5)) than unfed pitchers, and fed pitchers exhibited a skewed frequency distribu-
tion of acid phosphatase activity (Fig 1 and S1 Supplementary Information). Log transforma-
tion of the data allowed for a one-tailed t-test assuming unequal variances (P = 5.6 x 10−10)

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 Acid phosphatase in pitchers

Fig 1. Frequency distribution illustrating the number of pitchers with a given level of acid phosphatase activity
(μg/mL) inside the pitcher on Day 5 of the experiment (AcP(5)). Fed pitchers (black bars) exhibited a more skewed
distribution with higher levels of acid phosphatase activity than unfed pitchers (gray bars).
https://doi.org/10.1371/journal.pone.0181252.g001

with antilog means: fed pitcher mean = 4.69 μg/mL (95% confidence interval = 3.38–6.39 μg/
mL; n = 56) vs. unfed pitcher mean = 0.98 μg/mL (95% confidence interval = 0.66–1.36 μg/mL;
n = 46). A Mann-Whitney U test also indicated that fed pitchers had significantly different dis-
tribution of data than unfed pitchers (P < 0.001).
Final acid phosphatase activity inside the pitchers, measured on Day 5 of the experiment
(AcP(5)), was significantly higher than initial acid phosphatase activity, measured on Day 0 of
the experiment (AcP(0)), for both fed and unfed pitchers. A paired t-test for the unfed pitchers,
using log-transformed data, indicated that AcP(5) of 0.98 μg/mL is significantly greater than
AcP(0) of 0.12 μg/mL (one-tailed P = 4.83 x 10−10). Similarly, in fed pitchers AcP(5) of 4.69 μg/
mL is significantly greater than AcP(0) of 0.17 μg/mL (one-tailed P = 1.02 x 10−18).
Regression analysis indicated that initial acid phosphatase activity (Log AcP(0)) was very
weakly related to pitcher age (R2 = 0.0763). Final acid phosphatase activity (Log AcP(5)) gener-
ally increased with pitcher age (Fig 2). Pitcher age accounted for almost 39% of the variation in
acid phosphatase activity on Day 5 in fed pitchers but less than 17% of the variation in acid
phosphatase activity in unfed pitchers (using log-transformed data). Pitchers older than 6 d
more readily responded to feeding (Fig 2).
As Fig 2 illustrates, pitcher ages fall into four categories: 0–6 d, 14–20 d, 26–30 d, and 40+
d. Log of final acid phosphatase activity (Log AcP(5)) data were sorted into these four age
groups. In fed pitchers, a one-way ANOVA of Log AcP(5) demonstrated significant differ-
ences across four age groups (P < 0.001) (Fig 3). Acid phosphatase activity increased with
increasing age of the pitcher (Fig 3). In unfed pitchers, a one-way ANOVA of Log AcP(5) dem-
onstrated significant differences across four age groups (P = 0.008); however, Levine’s test

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 Acid phosphatase in pitchers

Fig 2. Regression analysis of log acid phosphatase activity across pitcher age (d) for fed and unfed pitchers.
Both fed and unfed pitchers exhibited an increase in acid phosphatase activity with increasing pitcher age, with a
greater increase in the fed pitchers. Pitcher age accounted for almost 39% of variation in enzyme activity in fed pitchers
and just under 17% of the variation in enzyme activity in unfed pitchers.
https://doi.org/10.1371/journal.pone.0181252.g002

indicated a lack of homogeneity of variances (P = 0.042). The youngest pitchers had relatively
low levels of acid phosphatase activity, but there is not an overall trend with age (Fig 3). A
2-way ANOVA (Feeding Status + Age + Interaction) was precluded in that error variances
were unequal based on Levine’s Test (P = 0.010).
Are older pitchers more responsive to feeding than younger pitchers? For this analysis, we
used the differences between final and initial measurements of acid phosphatase activity (AcP
(5)-AcP(0)). Because of the small samples sizes of the oldest pitchers, they were combined into
a group 26+ d old. The data, both raw and transformed, failed to meet the assumption of
homogeneous variances required by a 2-way ANOVA. Therefore, AcP(5)-AcP(0) values were
analyzed using the nonparametric test of Friedman’s two-way analysis of variance by ranks
comparing across three age groups and across two groups of feeding status, and the null
hypothesis was rejected (P = 0.003) Older pitchers responded more readily to feeding than
younger pitchers (Fig 4).
In spite of efforts to control microbial populations, 32% of pitchers produced culturable
microbes on Day 0, and 56% of pitchers produced culturable microbes at Day 5. Sixty-four
percent of fed pitchers produced culturable microbes, whereas 46% of unfed pitchers produced
culturable microbes at Day 5. Microbe levels at Day 0 (Microbes(0) measured in CFUs) were
very weakly related to pitcher age (R2 = 0.0605). Also, microbe levels at Day 5 (Microbes(5)
measured in CFUs) were very weakly related to pitcher age (R2 = 0.0782). In regression analy-
ses of samples grouped on the basis of Day 5 microbe levels (Microbes(5): Zero CFUs; 1–99
CFUs; 100+ CFUs), log acid phosphatase activity increased with pitcher age for all groups (Fig
5). Pitcher age explains more variation in AcP activity for samples with lower microbe levels.
R2 values for Zero CFUs, 1–99 CFUs, and 100+ CFUs are 48%, 30%, and 28%, respectively.

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 Acid phosphatase in pitchers

Fig 3. Means (± 1 SE) of the log of acid phosphatase activity measured on Day 5 (Log AcP(5)) for fed and
unfed pitchers in four age groups. Fed pitchers exhibited a trend of increasing enzyme activity with pitcher age.
Homogeneous subsets identified by Tukey HSD are labeled with the same letter. Sample sizes for Fed are: Age 0–6,
n = 13, Age 14–20, n = 21, Age 26–30, n = 19, Age 40+, n = 3; and for Unfed are: Age 0–6, n = 14, Age 14–20, n = 14,
Age 26–30, n = 14, Age 40+, n = 4.
https://doi.org/10.1371/journal.pone.0181252.g003

Are microbe levels related to enzyme activity within the pitcher? Regression analysis indi-
cated that initial acid phosphatase activity (Log AcP(0)) was not related to Microbes(0) (R2 =
0.0147). Final acid phosphatase activity (Log AcP(5)) was very weakly related to Microbes(5)
in unfed pitchers (R2 = 0.0697) and in fed pitchers (R2 = 0.1265).
For statistical analyses samples were grouped into two categories: High Microbes(5) in
which CFUs on Day 5 were equal to or greater than 100; and Low Microbes(5) in which CFUs
on Day 5 were less than 100 (including zero CFUs). High Microbes(5) samples had signifi-
cantly higher levels of AcP(5) than Low Microbes(5) samples (one-tailed t-test assuming
unequal variances, on log-transformed data, P = 0.003; Mann Whitney U test P = 0.007). Anti-
log mean of acid phosphatase activity in High Microbes(5) samples = 4.46 μg/mL (95% confi-
dence interval = 2.66–7.16 μg/mL, n = 31) and in Low Microbes(5) samples = 1.92 μg/mL
(95% confidence interval = 1.39–2.58 μg/mL, n = 71).
Do fed pitchers have higher microbe levels? There were 20 samples that were Fed and had
High Microbes(5), 11 samples were Unfed, High Microbes(5), 36 were Fed, Low Microbes(5),
and 35 were Unfed, Low Microbes(5). A G-test of independence using Williams’s correction
for small sample size results in Gadj = 1.652 (P > 0.05), indicating that microbe levels and
feeding status were independent. Fed pitchers were not more likely to have higher microbe
levels.
When samples were removed from the pitcher on Day 0 and incubated for 5 d outside the
pitcher, did they exhibit the same amount of enzyme activity as samples incubated inside the
pitchers? To address this question, we did two separate analyses. In the first analysis, samples

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 Acid phosphatase in pitchers

Fig 4. Initial acid phosphatase activity (AcP(0)) (μg/mL) subtracted from final acid phosphatase activity (AcP
(5)) μg/mL) in fed (dark gray bars) and unfed (light gray bars) pitchers for three age groups of pitchers.
Differences (mean ± 1 SE) increased with pitcher age, to a greater degree in fed pitchers than in unfed pitchers.
(Samples sizes are: Age 0–6, Unfed, n = 14, Fed, n = 13; Age 14–20, Unfed, n = 14, Fed, n = 21; Age 26+, Unfed,
n = 18, Fed, n = 22).
https://doi.org/10.1371/journal.pone.0181252.g004

were grouped into categories based on feeding status and the quantity of microbes found
inside the pitcher on Day 5 (Microbes(5), measured in CFUs). This resulted in four groups:
Unfed, Low Microbes(5); Unfed, High Microbes(5); Fed, Low Microbes(5); Fed, High
Microbes(5). Within each of the four groups, a paired t-test comparing the enzyme activity
inside the pitcher (Inside AcP(5)) with paired samples incubated outside the pitcher (Paired
Outside AcP(5)) resulted in a significant difference (Fig 6). Inside AcP(5) samples were higher
in acid phosphatase activity than Paired Outside AcP(5) samples (Fig 6).
In the second analysis, samples were grouped into categories based on feeding status
and the quantity of microbes found on Day 5 in the samples incubated outside the pitcher
(Microbes(o-5)). There were four groups analogous to the ones in the previous analysis. Paired
t-tests comparing the enzyme activity outside the pitcher (Outside AcP(5)) with paired sam-
ples incubated inside the pitcher (Paired Inside AcP(5)) indicated significant differences in
two of the four groups. In the Fed, Low Microbes(o-5) group, the Paired Inside AcP(5)
mean = 4.19 μg/mL (95% confidence interval = 2.93–5.86 μg/mL) was significantly greater
than the Outside AcP(5) mean = 0.74 μg/mL (95% confidence interval = 0.56–0.95 μg/mL)
(n = 49, P = 2.035 x 10−10). In the Unfed, Low Microbes(o-5) group, the Paired Inside AcP(5)
mean = 0.92 μg/mL (95% confidence interval = 0.60–1.32 μg/mL) was significantly greater
than the Outside AcP(5) mean = 0.16 μg/mL (95% confidence interval = 0.06–0.27 μg/mL)
(n = 41, P = 1.077 x 10−5). Very few samples incubated outside the pitcher fell into the High
Microbes (o-5) groups (Fed, High Microbes(o-5), n = 5; Unfed, High Microbes(o-5), n = 4).
Mean enzyme activity was higher in the samples from inside the pitchers, but confidence

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 Acid phosphatase in pitchers

Fig 5. Regression analysis of log acid phosphatase activity on Day 5 across pitcher age (d) for fed pitchers
separated on the basis of microbe levels on Day 5 (Microbes(5)), taken from samples of pitcher fluid.
Enzyme activity increased with pitcher age in all three microbe levels. The amount of variation explained by pitcher
age (R2) decreased with increasing microbe levels.
https://doi.org/10.1371/journal.pone.0181252.g005

intervals were extremely large. Lack of significant differences between inside and outside sam-
ples in these two groups is likely due to the small sample sizes.

Discussion
The initial goal of the preliminary experiments was to determine how best to eliminate
microbes from the inside of the pitchers so that we could measure the acid phosphatase pro-
duced by the pitcher itself. Our approach differed from previous studies in that every sample
of pitcher fluid was not only assayed for enzyme activity but also plated onto agar for microbial
counts. Of the five agars tested, nutrient agar (NA) resulted in the most numerous CFUs.
Pitcher-derived bacteria grew best at 25 C, consistent with incubation temperatures used in
previous experiments (e.g., [20]). This culturing technique does not definitively indicate the
absence of microbes [19,46] however, it certainly can indicate the presence of microbes.
Two combinations of antibiotics were tried, and in each series of experiments the pitchers
were inoculated either at the start of the experiment or repeatedly every other day. Using a
combination of antibiotics including carbenicillin, cefotaxime, and ampicillin (CCA cocktail)
at the start of the experiment, microbes were present by Day 2. With the same combination of
antibiotics given repeatedly every other day, microbes were present by Day 4. In another
experiment we used a combination of buffered penicillin, streptomycin and amphotericin B
(PSA cocktail) at the start of the experiment. Again, microbes were present within days; and
renewing the antibiotics every other day did not change the results. This is in marked contrast
to findings in Nepenthes, in which microbial growth is inhibited by pitcher fluid [47]. How-
ever, the microbes used in the Nepenthes study were not microbes naturally associated with
Nepenthes; and this study may not reflect the relationship between Nepenthes and its associated
microbes.

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 Acid phosphatase in pitchers

Fig 6. Mean acid phosphatase activity of fluid inside the pitcher, measured on Day 5 (Inside Pitcher AcP(5)),
was significantly higher than paired controls. Samples taken from each pitcher on Day 0 were incubated outside
the pitcher, and acid phosphatase activity was measured on Day 5 (Paired Outside AcP(5)). Paired Outside AcP(5) and
Inside Pitcher AcP(5) are sorted by feeding status and microbe levels inside the pitcher on Day 5 (Microbes (5)). The
asterisks represent a statistically significant difference between Paired Outside AcP(5) and Inside Pitcher AcP(5) using
paired t-tests, * = P < 0.01, ** = P < 0.001. Paired t-tests were performed on log-transformed data, and antilogs of
means are represented with 95% confidence intervals. (Sample sizes are: Unfed, Low Microbes (5), n = 35; Unfed, High
Microbes (5), n = 11; Fed, Low Microbes (5), n = 36; Fed, High Microbes (5), n = 20).
https://doi.org/10.1371/journal.pone.0181252.g006

A final preliminary experiment demonstrated multiple microbial strains that were resistant
to various combinations of antibiotics. Our results are consistent with Tran [48], who per-
formed antibiotic sensitivity tests on strains of mycobacteria isolated from pitcher plant fluid.
Tran’s [48] results were mixed, with certain strains resistant to certain antibiotics and suscepti-
ble to others. In our study antibiotics did not effectively control microbial populations in the
pitchers for more than a day or two. Unopened pitchers were entirely covered with sterile
Whirl-pacs; although they were not in contact with the pitcher and not air-tight, a Whirl-pac
would prevent air movement across the pitcher. We conclude that the most likely source of the
microbes was the potting medium or the adjacent pitchers on the same plant. Sarracenia pur-
purea pitchers produce sugary nectar on the outside of the pitcher [49], and bacterial commu-
nities in pitchers are often limited by carbon availability [22]. We speculate that microbes
arrive on the inside of the pitcher by way of the outside surface of the pitcher. Further explora-
tion of the function of external nectar glands is warranted.
Prey, pitcher age, and presence of microbes all influenced the acid phosphatase activity in
fluid from S. purpurea pitchers. The addition of prey increased acid phosphatase activity over
that of unfed pitchers by almost 5x. Both fed and unfed pitchers increased acid phosphatase
activity over the 5 d incubation period. Fed pitchers increased enzyme activity over 27x in 5 d,
while unfed pitchers increased enzyme activity approximately 8x over the same time period.
These results are consistent with the literature suggesting that enzyme production is induced
by prey [26].

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 Acid phosphatase in pitchers

Fed pitchers exhibited a trend of increasing acid phosphatase activity with increasing
pitcher age. Young pitchers (Ages 0–6 d) were far less capable of responding to prey than older
pitchers. The second age group (Ages 14–20 d) increased enzyme production (final minus ini-
tial AcP) approximately 7x the increase of the first age group (Ages 0–6 d). The third age
group (Ages 26+) increased enzyme production approximately 11x the increase of the first age
group. Unfed pitchers exhibited less variation in acid phosphatase activity with pitcher age.
Again, the youngest unfed pitchers had the lowest levels of enzyme activity. These results sug-
gest that using newly opened pitchers for enzyme assays, in an effort to avoid microbial symbi-
onts, may bias the measurement of enzyme activity toward the lowest values.
In general, pitchers with high levels of microbes exhibited over 2x higher levels of acid
phosphatase activity than pitchers with low levels of microbes. Microbe levels at Day 0 and
Day 5 were not a function of pitcher age, and fed pitchers were not more likely to have high
microbes. When samples were grouped into four categories (Unfed, Low Microbes; Unfed,
High Microbes; Fed, Low Microbes; Fed, High Microbes), all measurements of acid phospha-
tase activity inside the pitcher were significantly higher than the controls incubated outside the
pitcher. In other words, the milieu inside the pitcher was necessary for enzyme production.
The Fed, Low Microbe group exhibited approximately 4x the enzyme activity of the Unfed,
Low Microbe group. Similarly, the Fed, High Microbe group showed a little over 4x the
enzyme activity of the Unfed, High Microbe groups. The High Microbe groups and Low
Microbe groups responded similarly to feeding. Microbes are generally capable of up-regulat-
ing enzyme production [35], and microbes can likely account for the increased level of enzyme
activity within the pitcher fluid. Unless pitchers and microbes respond in the same way, these
data suggest that microbes are responsible for the increase in acid phosphatase activity with
the addition of prey.
Can we disentangle the relative importance of plant-derived enzymes from microbe-
derived enzymes? The difficulty in eliminating microbes indicates that they are closely associ-
ated with the plant. They may be embedded in the waxy surface such that an ethanol rinse
does not affect them. That microbes are responsible for some of the enzyme activity is apparent
when the Low Microbe and High Microbe groups are compared. The fact that both Low
Microbe and High Microbe groups responded similarly to feeding also suggests that the
enzyme production being induced is that of microbes. However, fed plants were not more
likely to have high levels of microbes, so feeding did not increase the population of microbes
during the span of the experiment. This result contrasts with other experiments done in a nat-
ural setting, in which addition of prey resulted in higher levels of bacterial cells over a span of
15 d, a much longer time span than we used [21].
Our data on microbes do not explain all the patterns. In particular, the increase in enzyme
activity with pitcher age appears to be unrelated to the level of microbes in the fluid of the
pitchers. Microbe levels on Day 0 and Day 5 are not closely related to pitcher age. Also, in fed
pitchers the trend of increasing enzyme activity with increasing pitcher age holds across all
microbe levels. Older pitchers appear to be more competent to respond to prey. Young pitch-
ers with high levels of microbes in pitcher fluid did not respond to feeding as strongly as older
pitchers with lower levels of microbes.
However, our method does not rule out the presence of microbes that are undetectable by
culturing [50] or that are embedded in the inner surface of the pitcher. Krieger and Kourtev
[46] identified three distinct sub-habitats within pitchers of S. purpurea: the pitcher fluid, the
bottom sediment, and the pitcher wall. They found high diversity of microbes within the
pitcher using molecular techniques. In addition, the microbial populations differed among the
three sub-habitats. Our pitchers had no bottom sediment since they were grown in a growth
chamber. If microbes are sufficiently embedded in the waxy surface of the pitcher such that

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 Acid phosphatase in pitchers

they unaffected by an ethanol rinse, our methods would not detect them. The pitcher wall and/
or pitcher fluid may be repopulated after antibiotic treatments by microbes that are not
culturable.
We hypothesize that older pitchers might have a more deeply embedded and more diverse
community of microbes. Hence, the increase in acid phosphatase activity with pitcher age
might be a function of the microbial community embedded in the pitcher wall, rather than a
function of the microbes present in the pitcher fluid or of the pitcher itself. This would involve
the process of succession in the bacterial community. Gray [51] described succession in the
bacterial community within S. purpurea pitchers, and found a decrease in abundance and rela-
tively constant species richness of culturable bacteria over several months. Using molecular
techniques, Peeples and Kourtev [52] found diverse bacterial communities 48 h after the open-
ing of pitchers. Over a two month period, bacterial communities in ten pitchers became more
different. Further studies of enzyme activity on the pitcher wall, such as those by Stauffer [25]
and Płachno et al. [12], could differentiate between enzyme production by the pitcher itself vs.
by embedded microbes in pitchers of different ages.
Our data support the conclusion that microbes are important components of the digestion
of prey by S. purpurea pitchers. The relative importance of the pitcher itself, beyond the main-
tenance of a habitat for symbionts, remains elusive.

Supporting information
S1 Supplementary Information. Complete data set of the main experiment.
(XLSX)

Acknowledgments
We thank A. M. Harding and R. L. Rountree for assistance with the preliminary experiments.
J. Duchamp and R. Winstead provided statistical advice. H. Travis, E. Yerger, and two anony-
mous reviewers offered valuable suggestions on the manuscript.

Author Contributions
Conceptualization: Carl S. Luciano, Sandra J. Newell.
Data curation: Carl S. Luciano, Sandra J. Newell.
Formal analysis: Sandra J. Newell.
Investigation: Carl S. Luciano, Sandra J. Newell.
Methodology: Carl S. Luciano, Sandra J. Newell.
Project administration: Sandra J. Newell.
Resources: Carl S. Luciano.
Writing – original draft: Sandra J. Newell.
Writing – review & editing: Carl S. Luciano, Sandra J. Newell.

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