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Biochemical Systematics and Ecology 38 (2010) 169–177
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Biochemical Systematics and Ecology
journal homepage: www.elsevier.com/locate/biochemsyseco
Prey detection of aquatic predators: Assessing the identity of chemical
cues eliciting prey behavioral plasticity
Bastien Ferland-Raymond a, *, Raymond E. March b,1, Chris D. Metcalfe c,1, Dennis L. Murray a,1
a
Department of Biology, Trent University, Peterborough, Ontario, Canada
Department of Chemistry, Trent University, Peterborough, Ontario, Canada
c
Environmental and Resource Studies Program, Trent University, Peterborough, Ontario, Canada
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 October 2009
Accepted 29 December 2009
Chemical cues transmitted through the environment are thought to underlie many prey
responses to predation risk, but despite the known ecological and evolutionary significance of such cues, their basic composition are poorly understood. Using anuran tadpoles
(prey) and dragonfly larvae (predators), we identified chemical cues associated with
predation risk via solid phase extraction and mass spectrometry of the extracts. We found
that dragonfly larvae predators consistently produced a negative ion, m/z 501.3, when they
fed on bullfrog (Rana catesbeiana) and mink frog (Rana septentrionalis) tadpoles, but this
ion was absent when dragonflies were fasted or fed invertebrate prey. When tadpole
behavioral responses to dragonfly chemical cues were examined, tadpoles reduced their
activity, particularly in response to dragonflies feeding on tadpoles. Furthermore, a negative correlation was noted between the level of tadpole activity and the concentration of
the m/z 501.3 compound in dragonfly feeding trials, indicating that this ion was possibly
responsible for tadpole anti-predator behavior.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Chemical cues
Kairomones
Predation risk
Tadpoles
Dragonfly larvae
1. Introduction
Animals use a variety of means to communicate with conspecifics and heterospecifics, including exchange of auditory,
visual, or chemical cues (Bradbury and Vehrencamp, 1998). The specific method used for communication between individuals
depends largely upon the type of information being transferred and the medium available for transmission of the cue.
Chemical cues serve as the primary vehicle for conveying information concerning impending predation risk in aquatic
predator-prey systems (Brönmark and Hansson, 2000; Eklöv, 2000). It is understood that to be effective, such cues should be:
i) easily identified by the recipient, ii) easily transmitted in water, and iii) comprise an accurate index of predation risk
(Bradbury and Vehrencamp, 1998). Yet, surprisingly the composition of chemical cues used in perceived predation risk, their
general etiology, and their role in the evolution of predator-prey systems, are not well known (Dicke and Grostal, 2001;
Kiesecker et al., 2002; Li and Jackson, 2005).
Aquatic cues of predation risk can constitute either alarm pheromones that emanate directly from killed or injured
individuals (Smith, 1992), or kairomones that are released by predators themselves (Dicke and Grostal, 2001). It follows that
the primary utility of such cues is to alter behavior or morphology of prey (e.g. Maerz et al., 2001; Van Buskirk and McCollum,
* Corresponding author.
E-mail addresses: [email protected] (B. Ferland-Raymond), [email protected] (R.E. March), [email protected] (C.D. Metcalfe),
[email protected] (D.L. Murray).
1
Main address: Trent University, 1600 West Bank Drive, Peterborough, Ontario K9J 7B8, Canada. Fax: þ1 705 748 1003.
0305-1978/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bse.2009.12.035
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B. Ferland-Raymond et al. / Biochemical Systematics and Ecology 38 (2010) 169–177
2000a, 2000b), and thereby confer increased survival and fitness (Kishida and Nishimura, 2004). Although the occurrence of
chemical cues in aquatic predator-prey systems is well-documented (reviewed by Chivers and Mirza, 2001b; Kats and Dill,
1998), chemical structure and function of such cues have been rarely studied. Past attempts to identify predator-prey kairomones in aquatic environments were restricted largely to systems with Daphnia serving as prey versus a range of predator
species (Boriss et al., 1999; Tollrian and von Elert, 1994; von Elert and Loose, 1996; von Elert and Pohnert, 2000). In general,
these studies proposed that chemical cues typically are comprised of anions (<500 Daltons) containing thiol- and hydroxylgroups. In contrast, in a terrestrial predator-prey system predator-prey chemical cues appear to be comprised of sulphurbased compounds (Nolte et al., 1994). However, it is surprising that there are no published reports to date identifying specific
chemical cues that induce amphibian anti-predator responses, especially considering the extensive research documenting
such responses across a variety of frog species (e.g., Fauth, 1990; McCollum and Van Buskirk, 1996; Peacor, 2002; Skelly and
Werner, 1990; Wilson and Lefcort, 1993). Clearly, additional work is needed to fully reveal the basic structure, function, and
evolutionary significance of chemical cues in aquatic predator-prey systems.
We used solid phase extraction (SPE) combined with mass spectrometry to examine chemical cues released by dragonfly
larvae (Anax junius) fed mink frog (Rana septentrionalis) and bullfrog (Rana catesbeiana) tadpoles, and to test the possible
relationship between relevant compounds and tadpole phenotypic responses. Because Rana tadpoles can modify behavior
more intensively in response to chemical signals from predators feeding on conspecifics or congeners (Chivers and Mirza,
2001a; Ferland-Raymond and Murray, 2008; Kiesecker et al., 2002; Laurila et al., 1998; Relyea and Werner, 2000), we
considered that some cues eliciting anti-predator responses were released exclusively by predators eating tadpoles. Therefore, we expected a unique chemical signature among predators fed either ranid species, and that such cues would constitute
large and complex molecules that are distinct from other compounds released by predators. Exposure to such chemical cues
should elicit change in tadpole activity patterns.
2. Methods and materials
Mink frog and bullfrog egg broods (3 per species) were collected during June-July 2006 from permanent ponds near
Peterborough, Ontario (44 170 N, 78 190 W). Study animals were maintained in an outdoor rearing facility until their
development reached Gosner stage 25 (Gosner, 1960), at which time they were used in experiments. Dragonfly larvae (Anax
junius) predators and Libellulidae larvae (Libellula spp.), which served as a control diet, were caught in the same ponds using
dip nets. Dragonfly predators were housed individually whereas libellulids were housed collectively.
2.1. Experimental design: water chemistry experiment
Solid phase extraction (SPE, Gorecki and Pawliszyn, 1996) in concert with mass spectrometry (MS) was used to identify
chemical compounds released by predators. To obtain water samples for analysis, dragonfly larvae were starved for 5 days and
then transferred to 40 ml of artificial pond water (0.5 mM NaCl, 0.05 mM KCl, 0.4 mM CaCl2 and 0.2 mM NaHCO3 in HPLC
grade water), using a protocol similar to that described by Prosser (1973). To minimize potential transference of chemical
cues, dragonfly larvae were rinsed with distilled water and dried using a paper towel prior to being moved. Dragonfly larvae
were maintained for 24 h in the water sample to allow them to excrete a sufficient amount of cue compounds before
removing them and saving the water for further analysis. Dragonfly larvae were then fed w0.12 g of either; i) bullfrog (R.
catesbeiana) tadpoles, ii) mink frog (R. septentrionalis) tadpoles, or iii) invertebrates (Libellulidae), and then placed in a clean
40 ml water sample for another 24 h. Dragonfly larvae were not fed in experimental containers to ensure that any chemicals
detected originated directly from predators. This experiment was repeated 3 times for each of the predator diets, as well as 9
times for fasting dragonfly larvae. A control baseline was obtained by conducting the above procedure on a single blank water
sample.
OasisÒ hydrophilic–lipophilic balance (HLB) SPE cartridges (6 cm3/500 mg, Waters, Oakville, ON, Canada) were used to
extract chemicals from water samples. These cartridges are appropriate for characterizing unknown compounds since they
retain at high capacity both hydrophilic and lipophilic compounds. We followed the protocol for SPE extraction of neutral
compounds described by Miao and Metcalfe (2003). Briefly, cartridges were preconditioned using acetone, methanol, and
HPLC-grade water (pH ¼ 7.0). The water sample was then passed through the cartridge at w10.0 ml/min. Once the entire
sample had passed through, cartridges were dried for 1 min and then eluted three times with 3-ml methanol. Every 3-ml
aliquot was kept in the cartridge for w10 min. Eluates were saved, reduced in volume just to dryness using a UVS 400 vacuum
centrifuge (Savant Instruments), and then reconstituted in 0.5 ml of methanol/water (50:50). Once extracted, samples were
refrigerated at 4 C before analysis by mass spectrometry. We also performed SPE extractions of acidic chemical compounds,
but neutral extraction provided similar results but with consistently higher sensitivity; accordingly we restrict our results to
those from the latter extraction method.
Extracts were analyzed by mass spectrometry using a Micromass (Manchester, U.K.) Quattro instrument equipped with
a Z-spray electrospray (ES) ionization source. This instrument is capable of tandem mass spectrometry (i.e. MS/MS) at low
resolution, but in this case, the instrument was used in single quadrupole mode (i.e. Q-MS). The extracts were introduced into
the MS by infusion with a syringe pump (KD Scientific, Boston, MA, USA) at a flow rate of 10 mL/min, and the resultant ions
were scanned in negative ion mode. For all samples, the electrospray needle was held at 3.00 kV, the reflector at 0.2 V, and the
cone voltage at 45 V, with the temperature of the source and the desolvation gas set at 100 C and 150 C, respectively.
B. Ferland-Raymond et al. / Biochemical Systematics and Ecology 38 (2010) 169–177
171
Samples were scanned for compounds ranging from m/z 50–700 and the total ion count (TIC) was measured for interesting
ion species. This mass range was selected because it was likely to include relevant compounds (von Elert and Loose, 1996) and
preliminary work conducted at a higher mass range revealed a paucity of compounds of potential interest.
2.2. Water chemistry experiment: data analysis
The occurrence of candidate compounds as chemical cues was first assessed in the water samples using qualitative analysis
of spectrometric graphs generated by Q-MS. Each of the 18 mass spectra was analyzed separately; but for simplicity, herein we
present pooled spectra for each treatment. Compounds occurring in the starved dragonfly treatment were considered to be
metabolic by-products, whereas those occurring exclusively in the diet treatments were inferred to be dietary compounds.
2.3. Potential effect of cue on behavior: experimental design
The level of activity of mink frog and bullfrog tadpoles exposed to caged dragonfly larvae was measured to link presence of
any chemical cues in the water to anti-predator response of tadpoles. Bullfrog and mink frog tadpoles were tested separately
by exposing each species to caged dragonfly larvae feeding on i) conspecific tadpoles of either species, ii) congeneric tadpoles
from the other Ranid species, or iii) heterogeneric larvae (Libellulidae), and comparing their activity to control tadpoles that
were not exposed to predators. Tadpoles were maintained in 15 L containers for 3 weeks in an indoor laboratory at a constant
temperature (20 C) and lighting schedule (12:12). Tadpoles were fed boiled spinach (1⁄2 teaspoon) twice per week and
containers were cleaned prior to each feeding. Dragonfly larvae were fed similar amounts of prey as in the chemistry
experiment (described above) each time experimental tadpoles were fed; as stated above, dragonflies were fed outside
experimental containers to avoid transference of alarm cues associated with prey. The experiment was replicated 15 times.
Tadpole activity was evaluated twice daily at 9:00 and 16:00, 5 days per week for the entire duration of the experiment. The
same observer performed all behavioral tests by checking tadpoles while standing still and moving carefully between aquaria.
All individuals displaying movement (i.e., any tail movement, see Laurila et al., 1997) within a 30 s. period were counted and
the count was converted to a proportion of tadpole moving. This proportion represents a snapshot measure of activity
(Ferland-Raymond and Murray, 2008).
2.4. Potential effect of cue on behavior: data analysis
A general linear model based on bootstrapping was used to test the association between the main candidate ion identified
and tadpole activity level. Activity level (obtained from the behavioral experiment) was calculated as the difference between
the activity measured in the focal container (exposed to dragonfly larvae) minus the corresponding control container from the
same block. Five of 15 behavioral replicates were randomly selected per treatment, and these data were paired with
a chemistry sample from the same diet. The level of activity of the tadpoles was compared to the TIC level (obtained from the
chemistry experiment) by linear regression obtained from the chemistry experiment while keeping the results for the two
prey species separate. The exercise was repeated 1000 times to minimize the influence of individual specific pairings, and
bootstrapping was used to evaluate the significance level of the trend. This approach was required, rather than a paired design
with water being sampled directly from tanks, because preliminary analysis revealed that tank water was contaminated with
fecal and food residues and thereby confounded our chemical analysis. Accordingly, the link between water chemistry and
tadpole behavior remains correlative, although we maintain that our approach provided results that are representative of
chemical signaling in our system. Statistical tests were conducted in R (R-2.6.2, R project).
2.5. Identification of candidate chemical cues: experimental design
The structure of the ions isolated and partially characterized by Q-MS using the Quattro instrument were further elucidated by evaluating their accurate-mass using ES orthogonal acceleration quadrupole time-of-flight mass spectrometry using
a Micromass (Manchester, UK) Q-ToF 2 tandem mass spectrometer with a Z-spray ES source. Signal detection was performed
with a reflector, microchannel plate (MCP) detector and time-to-digital converter. A multi-point mass calibration, giving
a polynomial function, was carried out using a NaI/CsI standard solution from m/z 50–1000. Similar instrumental settings
were used between the Q-ToF 2 and the Q-MS, but in the former high resolution instrument the cone voltage was varied
between 30 and 50 V and the source temperature was kept at 80 C. To fragment the ions in MS/MS experiments, UHP
argon was used as the collision gas and the collision energy was varied from 4–65 eV. MassLynx v. 3.5 software was used for
data retrieval and processing in both the Q-MS and the Q-ToF 2 experiments.
2.6. Identification of candidate chemical cues: data analysis
We used three techniques independently or in pairs to further characterize the elemental composition of the putative
chemical cues of interest (see Herbert and Johnstone, 2003). First, we calculated accurate masses for relevant compounds
using elemental composition software (MassLynx v. 3.5) and determined possible elemental compositions for the ions falling
within a range of 2 mDa of the m/z 501.3 species observed. Second, we subjected ions to collision with argon gas to induce
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fragmentation, with product ions providing information on the composition of the specific molecule. Finally, the isotopic
signature was compared to those of the compounds obtained from the accurate mass experiment. We calculated the
Euclidean distance (ED) between the intensity of every isotopic version of our ion and the closest models obtained from
elemental composition software; the lower the ED, the better the model fit. To develop a threshold allowing us to reduce our
list of candidate chemical compounds, we calculated a theoretical distribution of the ED and retained only models within the
5% lower tail of the student-t distribution.
3. Results
3.1. Water chemistry experiment
The blank full scan Q-MS in negative-ion mode revealed a complex suite of ions within the m/z 50–350 range (Fig. 1), so we
focused our analysis on the more distinct m/z > 350 portion of the mass spectrum. Fasting dragonfly larvae produced a mass
spectrum (Fig. 2a) similar to that of the blank, implying that dragonfly larvae themselves produced no distinct metabolic ions
at m/z > 350. The same phenomenon was revealed when extracts were analyzed from the treatment where dragonfly larvae
were feeding on invertebrates (Fig. 2b). However, when dragonfly larvae were fed tadpoles, several new ions were observed
above m/z 350 (Fig. 2c, d), implying that these ions were related to tadpole diet. Dominating all of these peaks (including all
replicates for each species) was a negative ion generated at m/z 501.3. Although trace levels of m/z 501.3 were observed in the
blank (Fig. 1), the ion count of m/z 501.3 in the blank was 0.2% of that seen in samples from dragonfly larvae eating mink frog
tadpoles and, therefore, it is presumed to have arisen through contamination. It is important to note that ion species m/z 501.3
from Fig. 2d is the same as m/z 501.4 from Fig. 2c and therefore, we refer to this ion species as m/z 501.3. Interestingly, another
ion (m/z 515) seems to be predominant in dragonfly larvae feeding on bullfrog tadpoles; this ion was also present also in
dragonflies fed mink frog tadpoles but in the latter case was largely masked by the more abundant ion m/z 501.3 (Fig. 2d).
Because m/z 501.3 was by far the predominant ion generated in mass spectra at the m/z > 350 range, this compound was
considered a candidate cue in tadpole anti-predator signaling. Accordingly, further compound structural analysis and
correlation between cue concentration and prey behavior was focused on this particular ion.
3.2. Assessment of effect of cue on behavior
Regression of ion m/z 501.3 TIC versus tadpole activity level showed a correlation between the level of tadpole behavior and
intensity of the ion m/z 501.3. Both bullfrog (slope ¼ 0.011, P < 0.001) and mink frog (slope ¼ 0.007, P < 0.001) tadpoles
showed reduced activity in treatments where there was a higher TIC level of ion m/z 501.3 (Fig. 3).
3.3. Identification of the structure of m/z 501.3
Further analysis with the Q-ToF 2 revealed that m/z 501.3 had an accurate mass of m/z 501.2841, which leads to 40 different
possible elemental compositions within 2 mDa (Appendix 1). The mass/charge ratio was determined to four decimal places
using (NaI)2I (m/z 426.6930), deprotonated quercetin-3-O-galactoside (m/z 463.0877), and (NaI)3I (m/z 576.5872)
255.2
100
279.3
MAX TIC: 3.80e5
311.2
325.2
% 120.9
136.9
199.1
227.1
139.0
339.2
353.1
0
100
150
200
250
300
350
381.2
400
451.0 501.1
450
500
550
600
650
700
750
m/z
800
Fig. 1. Full-scan mass spectrum of blank water lacking dragonfly larvae, generated by Q-MS in negative ion mode. The ordinate corresponds to the proportion of
the ion count from the different ions normalized to the ion count of the base peak. The base peak corresponds to a total ion count of 3.80 105. The abscissa is the
mass/charge ratio (m/z) of the ions found in the sample.
B. Ferland-Raymond et al. / Biochemical Systematics and Ecology 38 (2010) 169–177
173
MAX TIC: 1.99e7
a
100
%
118.8
311.2
0
b
100
%
107.9
0
c
255.2 311.2
100
%
501.4
255.2 311.2
515.4
0
d
501.3
100
%
255.2 281.3
253.2
347.3
0
100
200
300
400
500
600
700
80
m/z
Fig. 2. Full-scan mass spectrum generated by Q-MS in negative ion mode of fasting dragonfly larva (a), dragonfly eating Libellulidae larva (b), dragonfly larva
eating bullfrog tadpoles (c), or dragonfly eating mink frog tadpoles (d). The ordinate corresponds to the proportional representation of each ion relative to ion m/z
501.3 in the treatment involving dragonflies eating mink frog tadpoles (total ion counts of 1.99 107), while the abscissa is the mass/charge ratio (m/z).
alternately as lock masses. Under these conditions, it is expected that the mass/charge ratio is accurate to within 1 mDa
(March et al., 2004). Ion species m/z 501.3 also showed a complex isotopic signature, with peaks at m/z 502, m/z 503, m/z 504
and m/z 505 and intensities of 30.4%, 11.5%, 3.2% and 1.6% respectively. This isotopic signature was compared with models
obtained using accurate mass analysis (within 10 mDa.) by calculating the ED of candidates (n ¼ 187) and selecting those
within the 5% lower tail threshold of the distribution obtained. The threshold indicated that compounds with an ED distance
value <6.81 should be retained, thereby reducing our previous list of candidate compounds from 40 to 19.
We noted qualitatively that inducing molecular fragmentation upon m/z 501.3 using tandem mass spectrometry yielded
generally low recovery, implying that the compound was relatively stable. Following fragmentation, a major peak at m/z 97 was
observed. Fragmentation of m/z 502 (one 13C) and 503 (two 13C or one 34S) revealed 2 additional peaks to m/z 97; m/z 98 and m/z
99 (Fig. 4). Because the m/z 99 peak is higher than m/z 98, we infer that m/z 501.2841 contains at least one sulfur element. Also,
because the ratio between m/z 98 and m/z 97 is 4.4%, our reconstruction suggests that the ion contains approximately 20 carbon
atoms and a single sulfur atom. Indeed, we discount the possibility that ion m/z 501.2841 is comprised of >1 sulfur because such
a model would be restricted to 10 carbons but no such candidate model was under serious consideration (Table 1).
Finally, we reconstructed the m/z 97 product ion and inferred that the only valid candidate model could be C5H5S-. Because
this ion contains 3.5 double bond equivalents (DBE, number of rings and double bonds), we can eliminate all compounds
having <3 DBE in our restricted list of possible models. Thus, by process of elimination of models with more or less than one
sulfur with <3 DBE and <6.81 ED, only 5 molecules remained in contention (Table 1). From these, a single candidate
(C20H39N9O4S-) has 20 carbons, making it the most likely ion representing m/z 501.3.
B. Ferland-Raymond et al. / Biochemical Systematics and Ecology 38 (2010) 169–177
-0.04
-0.02
Bullfrog
Mink frog
-0.06
Activity level
0.00
174
0
1
2
3
TIC of ion m/z 501.3
4
Fig. 3. Activity level observed in bullfrog and mink frog tadpoles relative to the estimated total ion count (TIC 104) of ion species m/z 501.3.
4. Discussion
A single ion (m/z 501.3) stands out when we compared the chemical signature of dragonfly larvae feeding on different
prey species. Ion m/z 501.3 was produced by dragonflies feeding on diets of either frog species, but was essentially absent
from the invertebrate diet or fasting predators. These results are consistent with companion work conducted on dragonfly
larvae feeding on R. sylvatica tadpoles (B. Ferland-Raymond, unpublished), indicating that this compound may be
widespread through the genus Rana. Indeed, because ranid tadpoles have been shown to respond more strongly to
predators fed conspecifics or closely-related heterospecific tadpoles (Chivers and Mirza, 2001a; Ferland-Raymond and
Murray, 2008; Kiesecker et al., 2002; Laurila et al., 1997, 1998; Relyea and Werner, 2000), we infer that there likely is
a chemical cue that is specific to the genus and that m/z 501.3 is a viable candidate. Indeed, our data show a negative
relationship between ion TIC concentration and tadpole activity level, although we recognize that this relationship
remains in need of further confirmation. Additional work should involve assessment of the linkage between ion species
m/z 501.3 and tadpole activity, via chemical analysis of water samples taken directly from tadpole behavior tanks. To
effectively conduct such work, we assume that waste material can be adequately filtered from the sample while retaining
chemical compounds of interest.
Although molecular reconstruction did not confirm the identity of m/z 501.3, it is likely a large and complex carbon
chain containing a single sulfur atom. We propose C20H39N9O4S- as a logical candidate, that is [M–H] where
M ¼ C20H40N9O4S. Our extensive search of the literature has not revealed previous identification of this compound,
however, we can confirm that it shares similar properties with cues reported in other predator-prey systems: The
compound is comparable in size with the chemical cues released in a Daphnia-Chaborus system (Parejko and Dodson,
Fig. 4. Product ion mass spectrum of the isotopic cluster m/z 501, 502, and 503 in negative ion mode. The ordinate corresponds to the proportion of the ion count
from the different product ions adjusted to the ion count of undissociated m/z 502. The abscissa is the mass/charge ratio (m/z).
B. Ferland-Raymond et al. / Biochemical Systematics and Ecology 38 (2010) 169–177
175
Table 1
Candidate elemental compositions for ion m/z 501.2841, a possible chemical cue in dragonfly-tadpole signaling. We provide ion mass/charge ratio (Th), the
difference in mass (experimental – calculated) (mDa), the number of double bond equivalents (DBE), the elemental composition chemical formula, and the
Euclidian distance between the isotopic signature of our compound and the model proposed. The models are presented in descending order of likelihood.
Mass
Difference
DBE
Formula
Euclidean distance
501.2846*
501.2834
501.2830
501.2827
501.2859
0.5
0.7
1.1
1.4
1.8
6
4.5
9
13.5
5.5
C20
C25
C29
C33
C22
5.69
3.54
3.41
6.63
4.55
H39 N9 O4 S
H47 N2 O2 P2 S
H44N O2 P S
H41 O2 S
H41 N6 O5 S
1990). The size of the molecule is relevant when considering cue efficacy, as large molecules are more complex and likely
to possess an increased number of specific binding sites leading to stronger species-specificity and reliability in predation
risk assessment (Bradbury and Vehrencamp, 1998). It is also noteworthy that the presence of sulphur in ion m/z 501.3 is
similar to its occurrence in chemical cues from a terrestrial vertebrate predator-prey system (Nolte et al., 1994). It seems
logical that sulfur would be an important chemical element in signaling in predator-prey systems, given its prevalent role
in a variety of olfactory responses (Johnson, 1998).
Of primary ecological and evolutionary importance is whether a chemical cue is an alarm pheromone (originating from
consumed prey) or a kairomone (originating from predators), and the results of this work provide some insight into this
area of inquiry. Because dragonfly larvae were fed outside the experimental container, alarm pheromones directly
associated with dying prey were presumably removed (see Chivers and Smith, 1998), making dragonflies themselves the
proximate source of ion m/z 501.3. However, it cannot be fully discounted that alarm pheromones emanating from
tadpoles were involved because of the clear role of predator diet in chemical cue production and its apparent relationship
to attendant tadpole behavioral responses. Thus, the chemical cue may have been produced by the tadpoles and released
as a digestive by-product following consumption by predators (Brown et al., 1995; Lefcort, 1996). Indeed, it is known for
other systems that prey extracts mixed with predator digestive extracts can induce a stronger response than either
treatment alone (Jacobsen and Stabell, 2004; Stabell et al., 2003). It was also shown by Fraker et al. (2009) that the use of
a detergent (Triton X-100) was required to solubilize cell membranes and to release the chemical cue causing behavioral
inhibition. This detergent is maybe playing the role of digestive enzyme in the guts of predators, making the chemical
cues responsible for anti-predator behavior a latent alarm pheromones (Stabell, 2005). At the present stage in our
investigation we cannot fully support this possibility, and additional experimental work specifically on the genesis of
compound m/z 501.3 in aquatic predator-prey systems is needed to fully understand its ecological and evolutionary
significance.
Our experimental design potentially precludes the discovery of certain compounds (e.g. rare compounds under m/z
350 or compounds passing through the SPE process) potentially responsible for perceived predation risk in tadpoles.
However, considering the wide variety of prey responses exhibited in response to predation risk, (Kiesecker et al., 2002;
Relyea, 2001; Richardson, 2001; Teplitsky et al., 2003; Van Buskirk, 2002), there are likely a range of compounds
involved in eliciting changes in prey phenotype (Van Buskirk and Arioli, 2002). Thus, the challenge for future research
will be to differentiate among the multiple chemical cues found in the environment, as well as those involved in various
types of predator-prey signaling, to provide a mechanistic understanding of the determinants of phenotypic plasticity
among prey. Future work should focus specifically on testing experimentally the influence of chemical cues such as ion
m/z 501.3 on prey anti-predator responses. Such studies should involve either direct application of extracted compounds
in prey environments lacking predators, or neutralizing compound activity in environments where predators are
present. Ultimately, these efforts will lead to a more robust understanding of the role of chemical compounds in antipredator responses, and thereby help unravel the mechanisms underlying chemical signaling in aquatic predator-prey
systems.
Acknowledgment
We are grateful to T. Hossie for his help gathering and maintaining the animals for the experiment. The success of this
research was only possible through help from M. Fiqueroa and L. Hongxia who provided much needed assistance in the
laboratory. Funding for this project came from the National Sciences and Engineering Research Council of Canada (NSERC).
Appendix 1
List of 40 possible elemental compositions within 2 mDa for the ion m/z 501.2841. We provide ion mass/charge ratio (Th), the
difference in mass (experimental–calculated) (mDa), the number of double bond equivalents (DBE), the elemental composition
chemical formula, and the Euclidian distance between the isotopic signature of our compound and the model proposed. The five
elemental compositions provided in Table 1 are included in bold.
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B. Ferland-Raymond et al. / Biochemical Systematics and Ecology 38 (2010) 169–177
Mass
Difference
DBE
Formula
Euclidean distance
501.2841
501.2840
501.2842
501.2840
501.2839
501.2839
501.2843
501.2837
501.2837
501.2845
501.2837
501.2846
501.2846
501.2846
501.2834
501.2834
501.2848
501.2849
501.2832
501.2832
501.2851
501.2830
501.2852
501.2852
501.2829
501.2829
501.2854
501.2854
501.2828
501.2855
501.2855
501.2827
501.2825
501.2824
501.2858
501.2859
501.2823
501.2859
501.2821
501.2861
0
0.1
0.1
0.1
0.2
0.2
0.2
0.4
0.4
0.4
0.4
L0.5
0.5
0.5
0.7
0.7
0.7
0.8
0.9
0.9
1
1.1
1.1
1.1
1.2
1.2
1.3
1.3
1.3
1.4
1.4
1.4
1.6
1.7
1.7
L1.8
1.8
1.8
2
2
0
0.5
5.5
13.5
10
15.5
9
5
0.5
1
0
6
0.5
4.5
9.5
4.5
9
1.5
1
1.5
4.5
9
9.5
15
6
0.5
0
0.5
0.5
5
10.5
13.5
10.5
0
6
5.5
0.5
0.5
4.5
8.5
C20 H47 N5 O3 S3
C18 H47 N8 P2 S2
C23 H42 N4 O6 P
C33 H43 P2
C27 H39 N3 O6
C26 H33 N10 O
C29 H46N P3
C22 H44 N7 P S2
C23 H50 O5 P S2
C19 H45 N5 O6 P2
C21 H50 N3 O2 P3 S
C20 H39 N9 O4 S
C21 H45 N2 O9 S
C25 H49 N2 P4
C26 H41 N6 S2
C25 H47 N2 O2 P2 S
C28 H43 N3 O S2
C16 H42 N10 O4 P S
C19 H43 N5 O8 S
C17 H43 N8 O5 P2
C24 H46 N4 O P S2
C29 H44N O2 P S
C29 H41 O7
C28 H35 N7 O2
C21 H40 N7 O5 P
C22 H46 O10 P
C20 H49 N5 O P2 S2
C22 H49 N2 O4 S3
C18 H45 N8 O2 S3
C25 H44 N O7 P
C24 H38 N8 O2 P
C33 H41 O2 S
C25 H37 N6 O5
C21 H48 N3 O4 P S2
C20 H41 N9 O2 P2
C22 H41 N6 O5 S
C19 H48 N6 O P3 S
C21 H47 N2 O7 P2
C25 H45 N2 O4 S2
C30 H45 O2 S2
6.96
6.28
8.29
8.18
6.56
7.19
7.44
2.70
3.87
11.20
6.95
5.69
6.27
8.68
3.20
3.54
4.32
9.46
7.59
12.40
2.40
3.41
6.00
7.09
9.19
9.37
5.14
6.78
7.44
7.55
7.79
6.63
7.06
4.82
10.14
4.55
8.04
10.05
2.80
5.45
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