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Pumiliotoxin metabolism and molecular physiology in a poison frog 1
2
Aurora Alvarez-Buylla1, Cheyenne Y. Payne1, Charles Vidoudez2, Sunia A. Trauger2 and Lauren 3
A. O’Connell*1 4
5
1 Department of Biology, Stanford University, Stanford, CA 94305, USA 6
2 Harvard Center for Mass Spectrometry, Harvard University, Cambridge, MA 02138, USA 7
8
Running title: Physiology of pumiliotoxin uptake 9
Word count (including methods): 2470 10
Word count (Abstract): 180 11
Key words: alkaloid, cytochrome P450, allopumiliotoxin, RNA sequencing, Dendrobatidae, 12
decahydroquinoline 13
14
* To whom correspondence should be addressed: 15
Lauren A. O’Connell 16 Department of Biology 17 Stanford University 18 371 Jane Stanford Way 19 Stanford, CA 94305 20 loconnel@stanford.edu 21 22
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ABSTRACT 23
Poison frogs bioaccumulate alkaloids for chemical defense from their arthropod diet. These 24
small molecules are sequestered from their gastrointestinal tract and transported to the skin for 25
storage. Although many alkaloids are accumulated without modification, some poison frog 26
species can metabolize pumiliotoxin (PTX 251D) into the more potent allopumiliotoxin (aPTX 27
267A). Despite extensive research characterizing the chemical arsenal of poison frogs, the 28
physiological mechanisms involved in the sequestration and metabolism of individual alkaloids 29
is unknown. We performed a feeding experiment with the Dyeing poison frog (Dendrobates 30
tinctorius) to ask if this species can metabolize PTX 251D into aPTX 267A and what gene 31
expression changes are associated with PTX 251D exposure in the intestines, liver, and skin. 32
We found that D. tinctorius can metabolize PTX 251D into aPTX 267A, and that PTX 251D 33
exposure changed the expression of genes involved in immune system function and small 34
molecule metabolism and transport. These results show that individual alkaloids can modify 35
gene expression across poison frog tissues and suggest that different alkaloid classes in wild 36
diets may induce specific physiological changes for accumulation and metabolism.
37
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1. INTRODUCTION 38
Poison frogs (Family Dendrobatidae) are chemically defended against predators [1–3] 39
using alkaloids that are sequestered from dietary arthropods [4,5]. Many poison frog alkaloids 40
have been found in the ants and mites they consume, suggesting most alkaloids are 41
sequestered unchanged [6–9]. However, poison frogs can also metabolize specific alkaloids into 42
more potent forms [10], although the underlying physiological mechanisms of this are unknown. 43
Controlled alkaloid-feeding experiments have been crucial in understanding alkaloid metabolism 44
in poison frogs [10–12]. Although many alkaloids are sequestered unmodified, some 45
dendrobatids metabolize pumiliotoxin (PTX) 251D into the more potent allopumiliotoxin (aPTX) 46
267A [10]. After PTX 251D feeding, both PTX 251D and its metabolite, aPTX 267A, were 47
detected on the skin of Dendrobates auratus, but only PTX 251D was detected in Phyllobates 48
bicolor and Epipedobates tricolor [10]. These results suggest that an unidentified enzyme 49
performs the 7'-hydroxylation of PTX 251D into aPTX 267A in certain species. Whether other 50
poison frog species can metabolize PTX 251D into aPTX 267A remains unknown, along with 51
the metabolic mechanisms involved. 52
We conducted an alkaloid feeding study with the Dyeing poison frog (Dendrobates 53
tinctorius) to test whether this species can metabolize PTX 251D into aPTX 267A and to explore 54
gene expression changes associated with PTX 251D exposure. To test whether PTX 251D 55
elicited specific gene expression changes, we fed control frogs decahydroquinoline (DHQ) and 56
treatment frogs a mixture of PTX 251D and DHQ. We predicted metabolic enzymes involved in 57
the hydroxylation of PTX 251D into aPTX 267A may be upregulated in response to their 58
metabolic target. Furthermore, if specific alkaloid sequestration pathways exist for PTX 251D, 59
the proteins involved in that process may also be enriched upon exposure. 60
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2. MATERIALS AND METHODS 61
Alkaloid feeding 62
Lab-reared (non-toxic) Dendrobates tinctorius were housed in terraria with live plants, a 63
water pool, and a shelter. Ten adult females were size-matched, randomly assigned to control 64
or experimental groups (N=5 per group), and then housed individually. To measure the specific 65
effects of PTX 251D compared to a background toxicity, the control group was fed 0.01% DHQ 66
(Sigma-Aldrich, St. Louis, USA) in a solution of 1% EtOH and the experimental group was fed a 67
solution of 0.01% DHQ and 0.01% PTX (PepTech, Burlington, MA, USA) in a solution of 1% 68
EtOH. Each frog was fed 15 µL each day for five days by pipetting the solution directly into the 69
mouth between 10am-12pm. On the afternoon of the fifth day, frogs were euthanized by cervical 70
transection and the dorsal skin, liver, intestines, and eggs were dissected into Trizol (Thermo 71
Fisher Scientific, Waltham, USA). All procedures were approved by the Institutional Animal Care 72
and Use Committee at Stanford University (protocol number #32870). 73
RNA extraction and library preparation 74
RNA extraction followed the protocol outlined in Caty et al. 2019 [13] and according to 75
the manufacturer’s instructions. After the first spin, the organic layer was saved for alkaloid 76
extraction (see below). Poly-adenylated RNA was isolated using the NEXTflex PolyA Bead kit 77
(Bioo Scientific, Austin, USA) following manufacturer’s instructions. RNA quality and lack of 78
ribosomal RNA was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa 79
Clara, USA). Each RNA sequencing library was prepared using the NEXTflex Rapid RNAseq kit 80
(Bioo Scientific). Libraries were quantified with quantitative PCR (NEBnext Library quantification 81
kit, New England Biolabs, Ipswich, USA) and an Agilent Bioanalyzer High Sensitivity DNA chip, 82
both according to manufacturer’s instructions. All libraries were pooled at equimolar amounts 83
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and were sequenced on four lanes of an Illumina HiSeq 4000 machine to obtain 150 bp paired-84
end reads. 85
86
Transcriptome assembly and differential expression analysis 87
All scripts are detailed in supplementary materials. We created a reference 88
transcriptome using Trinity [14], and cleaned the raw assembly by removing contigs with BLAST 89
hits belonging to microorganisms and invertebrates in the Swiss-Prot database [15]. 90
Overlapping contigs were clustered using cd-hit-est [16,17] and contigs that were less than 91
250bp long were removed from the assembly. We mapped the paired quality-trimmed 92
sequences to the reference transcriptome using kallisto [18]. Samples were compared across 93
treatment groups (DHQ vs DHQ+PTX) for the skin, liver, and intestines, as these tissues 94
contained higher levels of PTX. Differences in gene expression levels were calculated using 95
DESeq2 [19] [P<0.05 false discovery rate (Benjamini–Hochberg FDR), 4-fold change]. Contigs 96
with significant expression differences were BLAST-ed to the non-redundant (nr) database 97
using an E-value cutoff of 1e-5. Many contigs did not have a BLAST hit, or aligned to 98
hypothetical or non-vertebrate proteins. Therefore, BLAST annotations were visually inspected 99
and contigs of interest were chosen based on candidates from existing literature. Boxplots were 100
made with R package ggplot2 (R version 3.6.3) using TMM (trimmed mean of M-values) 101
normalized expression. 102
Alkaloid extraction and detection 103
To isolate alkaloids, 0.3 mL of 100% EtOH was added to 1mL of organic layer from the 104
Trizol RNA extraction, inverted 10 times, and stored at room temperature for 2-3 minutes to 105
precipitate genomic DNA, which was pelleted by centrifugation at 2000g for 5 minutes at 4°C. 106
Then, 300 µL of supernatant was transferred to a new microfuge tube. Proteins were 107
precipitated by adding 900 µL of acetone, mixing by inversion for 10-15 seconds, incubating at 108
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room temperature for 10 min, and centrifuging at max speed for 10 min at 4°C. Then, 1 mL of 109
supernatant was moved into a glass vial and stored at -20°C until dried down completely under 110
a gentle nitrogen gas flow. 111
Samples were resuspended in 200 µl of methanol:chloroform 1:1 and 1 µM Nicotine-d3 112
(used as an internal standard). A 10-point standard curve was prepared in the same solution 113
with DHQ and PTX. A QE+ mass spectrometer coupled to an Ultimate3000 LC (ThermoFisher) 114
was used for analysis. Five µl of each sample were injected on a single Gemini C18 column 115
(100x2mm, Phenomenex). The mobile phases were A: water and B: acetonitrile, both with 0.1% 116
formic acid. The gradient was 0% B for 1min, then increased to 100% B in 14 min, followed by 5 117
min at 100% B and 3.5 min at 0% B. Data were quantified using accurate mass, and specific 118
transitions for DHQ and PTX used the standard curve for absolute quantification. aPTX was 119
identified by accurate mass and MS/MS fragmentation similarity to PTX. 120
Data analysis of liquid chromatography/tandem mass spectrometry (LC-MS/MS) data 121
R version 3.6.3 was used for all statistical analyses. There were instances in the LC-122
MS/MS data where the molecules of interest (DHQ, PTX 251D, or aPTX 267A) were not found, 123
and these were converted to zeros prior to statistical analyses. A generalized linear mixed 124
model was used (glmmTMB package in R) [20] to test for differences in alkaloid abundance 125
across tissues and treatment type, with the frog as a random effect and a negative binomial 126
error distribution. PTX 251D and DHQ were analyzed separately. The aPTX 267A abundance 127
was approximated using the area-under-the-curve divided by the internal nicotine standard, as 128
there is no standard for aPTX 267A, and therefore exact pmol values could not be calculated. A 129
Wilcoxon rank-sum test (wilcox.test) was used to compare the aPTX values in the skin between 130
treatment groups and the Kruskal-Wallis test (kruskal.test) was used to compare the aPTX 131
values across tissues. 132
133
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3. RESULTS 134
The Dyeing poison frog metabolizes PTX to aPTX 135
We conducted a feeding experiment to determine if the Dyeing poison frog can 136
metabolize PTX 251D into aPTX 267A (Figure 1A). Alkaloids were most abundant in the skin 137
and liver, followed by the intestines. Alkaloid abundance in the eggs was very low. DHQ 138
abundance did not differ by treatment group (GLMM treatment, p = 0.377), confirming that both 139
groups were fed equal amounts. DHQ abundance differed across tissue types (GLMM tissue, X2
140
(3) = 203.642, p < 2e-16), with the highest levels occurring in the liver and skin (Figure 1B). PTX 141
251D abundance differed by tissue and treatment (GLMM tissue:treatment, X2 (3) = 57.265, p < 142
2e-12), with the highest levels in the liver and skin in the DHQ+PTX feeding group (Figure 1C). 143
We detected aPTX 267A in the skin of all individuals in the DHQ+PTX feeding group at higher 144
levels than the DHQ-fed group (Wilcoxon test, W = 0, p = 0.012, Figure 1D). The amount of 145
aPTX 267A differed across tissues (Kruskal-Wallis, X2 (3) = 13.727, p = 0.003), with the 146
abundance in the skin greater than eggs (post-hoc Dunn test, p = 0.001) and intestines (post-147
hoc Dunn test, p = 0.035, Figure 1E). These data show D. tinctorius can metabolize PTX 251D 148
into aPTX 267A and that some alkaloid metabolism may occur in the liver and intestines. 149
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150
Figure 1: Alkaloid sequestration in different tissue types. (A)
Frogs were orally
151 administered either DHQ or DHQ+PTX once a day for five days. (B)
DHQ abundance differed
152 by tissue but not treatment group, and was highest in the liver and skin (GLMM tissue, X
2
(3) =
153 203.642, p < 2e-16). (C)
PTX levels differed by tissue and treatment, and were higher in the
154 liver and skin of the DHQ+PTX fed group (GLMM tissue:treatment, X
2
(3) = 57.265, p < 2e-
12).
155 (D) The hydroxylated metabolite aPTX was found in the DHQ+PT
X fed frogs (Wilcoxon test, W
156 = 0, p-value = 0.012, n = 5). (E) aPTX abundance differed across tissues within the DHQ-
PTX
157 group (Kruskal-Wallis, X
2
(3) = 13.727, p = 0.003), and was found primarily in the skin, with
158 some in the liver. 159 160
PTX alters gene expression across tissues 161
We next quantified gene expression changes associated with PTX sequestration and
162
metabolism. Although hundreds of genes were differentially expressed in each tissue, most did
163
not have annotations, or aligned with unknown, hypothetical, or non-
vertebrate proteins.
164
Cytochrome P450 (CYP3A29), an enzyme family well-
known for their involvement in small
165
molecule hydroxylation, was upregulated in the intestines (log2FC = 5.72, p = 0.0045; Figure
166
lly
ed
=
he
2).
W
X
ith
nd
id
s.
all
re
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2A). In the liver, vitellogenin 2 (VTG2) was downregulated in the PTX feeding group (log2FC =
-
167
7.73, p = 0.0421, Figure 2B). MHC Class I
was upregulated in both the liver (log2FC = 3.49, p
168
= 0.0005) and intestines (log2FC = 5.41, p = 0.0001) in the presence of PTX 251D
(Figure
169
2A,B). In the skin, Sy
ntaxin 1A (STX1A) was upregulated (log2FC = 2.58, p = 0.0385) and a
170
Solute Carrier Family 2 protein (SLC2) was downregulated (log2FC = 6.73, p = 0.0496) in
171
response to PTX 251D (Figure 2C). 172
173
Figure 2: Differentially expressed genes in different tissues*. (A)
Differentially expressed
174 genes in the intestines include CYP3A29, Cytochrome P450 Family 3 Protein 29 and MHCI
,
175 MHC Class I alpha. (B) Differentially expressed genes in the liver included
VTG2, vitellogenin 2
176 and MHCI , MHC Class I alpha again. (C)
Differentially expressed genes in the skin included
177
STX1A, syntaxin 1A and SLC2, solute carrier family 2. (FC indicates log2 fold change values, *
178 indicates adjusted p-value < 0.05, *** indicates adjusted p-value < 0.005) *Note: y-
axis of
179 individual plots have different scales 180 181
4. DISCUSSION 182
Performing a controlled feeding study with DHQ and PTX 251D
allowed us to determine
183
that D. tinctorius can metabolize PTX 251D into aPTX 267A. Although previ
ous studies have
184
documented wild D. tinctorius with aPTX on their skin [2,6]
, this is the first experimental
185
evidence that this species metabolizes PTX 251D into aPTX 267A
. The accumulation of both
186
DHQ and PTX 251D
in the liver and intestines, along with the skin, indicates that these tissues
187
play an important role in the sequestration of alkaloids. Indeed, the liver and intestines are
188
-
, p
re
a
in
ed
,
2
ed
, *
of
ne
ve
tal
th
es
re
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important sites of alkaloid metabolism in mammals due to high levels of Cytochrome P450s [21–189
23]. Together, these results show that D. tinctorius is able to metabolize PTX 251D into aPTX 190
267A and that the tissue distribution of alkaloids includes the skin, liver, and intestines. In the 191
future, a better understanding of alkaloid pharmacokinetics could be achieved through finer 192
time-course feeding experiments. 193
PTX 251D feeding changed gene expression in the intestines, liver, and skin, suggesting 194
a single alkaloid can change poison frog physiology. Specifically, the upregulation of CYP3A29 195
in response to PTX in the intestines implicates this enzyme in the metabolism of PTX 251D into 196
aPTX 267A, or of PTX into a metabolic byproduct to be later discarded. Although we originally 197
expected to identify metabolism enzymes in the liver, it is possible the liver instead acts as a 198
detoxification site. In the dendrobatid Oophaga sylvatica, feeding DHQ compared to a non-199
alkaloid vehicle control led to a downregulation of CYP3A29 in the intestines, suggesting that 200
expression is regulated differently by specific alkaloids [24]. The upregulation of MHC class I 201
proteins in the intestines and liver in response to PTX 251D supports previous findings that frog 202
immune systems respond to alkaloids [13,25]. We also found VTG2 (vitellogenin-2) was 203
downregulated in response to PTX 251D. Although vitellogenins are typically thought to be egg-204
yolk proteins, they also play regulatory roles and protect cells from reactive oxygen species that 205
may arise from alkaloid metabolism [26–28]. Finally, SLC2 (solute carrier family 2) which 206
encodes for the GLUT family of glucose transporters, was downregulated in the skin with PTX 207
feeding. Alkaloids have been found to be potent inhibitors of GLUTs in mammalian cell lines, 208
and the downregulation of GLUTs in this case may be due to the presence of concentrated PTX 209
251D in the frog skin [29]. Together, these data support an argument for physiological “fine-210
tuning” of gene expression in response to certain alkaloids. 211
We provide evidence that D. tinctorius can metabolize PTX 251D into aPTX 267A and 212
that PTX 251D exposure changes gene expression across tissues, demonstrating that specific 213
alkaloids can change poison frog physiology [24,25]. Following up on candidate genes with 214
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biochemical studies is needed in order to fully characterize the genetics of alkaloid 215
sequestration and metabolism. In the wild, where chemically defended dendrobatids carry many 216
different alkaloids, subtle alkaloid differences may induce distinct gene expression changes. 217
More broadly, modulating gene expression in response to specific alkaloids may set the stage 218
for local adaptation to environmental resources. 219
5. ACKNOWLEDGEMENTS 220
We thank Stephanie Caty and Nora Moskowitz for their comments. We acknowledge that the 221
land on which this research was conducted is the ancestral and unceded land of the Muwekma 222
Ohlone tribe. 223
224
6. FUNDING 225
This work was supported by a National Science Foundation (NSF) [IOS-1822025] grant 226
to LAO. AAB is supported by a NSF Graduate Research Fellowship (DGE-1656518) and an 227
HHMI Gilliam Fellowship. LAO is a New York Stem Cell – Robertson Investigator. 228
229
6. DATA ACCESSIBILITY 230
All LC-MS/MS data from the alkaloid analysis is available from the Dryad Digital Repository 231
(pending). All Illumina fastq files are available on the Sequence Read Archive (pending). All 232
data and code is available in the supplementary material. 233
7. AUTHOR CONTRIBUTIONS 234
AAB and LAO designed the experiment. AAB and CYP carried out the experimental procedures. 235
CV and SAT quantified alkaloids. AAB analyzed the data. AAB and LAO wrote the manuscript 236
with contributions from all authors. 237
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