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Supplementary Material: Animal collection and stimulus preparation.
Animal Collection and Holding:
For Experiment 1, we collected adult, non-gravid guppies (Poecilia reticulata)
from two sites of known predation risk, the Upper Aripo River (low predation) and the
Lower Aripo River (high predation). The Lower Aripo is characterized as a high
predation site [1,2], containing several species that actively prey on juvenile and adult
guppies. Common predators include pike cichlid (Crenicichla species), blue acara
(Aequidens pulcher), brown coscarub (Cichlasoma taenia), wolf fish (Hoplias
malabaricus) and twospot sardine (Astyanax bimaculatus). The Upper Aripo River is
characterized as a low predation site [1,2] as it contains Hart’s rivulus (Rivulus hartii) and
a freshwater prawn (Macrobrachium crenulatum), which opportunistically prey on small,
juvenile guppies [3,4]. Guppies were collected with a seine net and transported to the
laboratory at the University of the West Indies, St. Augustine, Trinidad & Tobago.
Guppies were held in separate 100L tanks for at least 48 hrs prior to testing. Tank water
was filtered and maintained at ~ 25°C under a 12:12 Light:Dark cycle. Prior to testing,
guppies were fed a diet of commercial flake food (Tetramin) and brine shrimp nauplii
twice daily. Adult pike cichlids (Crenicichla frenata) were collected using hand-seine
nets from the Lower Aripo River, downstream of our observation sites as predator odour
donors for Experiment 2. Prior to stimulus collection (see below), pike cichlids were
held, in pairs, in 100 L glass aquaria (~25°C, 12:12 L:D cycle) and fed 1-2 guppies per
day. Nile tilapia (Oreochromis niloticus) were obtained from a local hatchery
(Experiments 1 and 2) and held in 900 L continuously filtered holding tanks (~ 25°C,
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12:12 L:D cycle). Prior to stimulus collection, tilapia were fed ad libitum with
commercial pellets.
For Experiment 2, we conducted in situ behavioural observations of guppies in
three streams, the Upper and Lower Aripo Rivers and the Tacarigua River. The
Tacarigua is characterized as an intermediate predation risk stream. Though it contains a
similar predator guild as the Lower Aripo River, the density of predators is lower [1,2].
The juvenile convict cichlids used for Experiment 3 were laboratory-reared
descendants of laboratory stock crossbred with wild-caught cichlids from Costa Rica.
Prior to testing, cichlids were held 60 L glass aquaria containing filtered dechlorinated
tap water (26°C, pH ~7.2, 12:12 L:D cycle) and a gravel substrate. Cichlids were fed
twice daily with commercial flake food and brine shrimp nauplii. Rainbow trout
(Onchoryncus mykiss) used as predator odour donors (see below) originated from a
commercial hatchery (Pisciculture des arepent verts, Ste. Edwidge-de-Clifton, Quebec).
Prior to use, trout were housed in recirculating 390 L tanks (~18°C, pH ~ 7.0, 12:12 L:D
cycle) and fed, ad libitum, daily with commercial trout chow.
Woodfrog egg masses were collected in the middle of the breeding season at our
field site, from a local pond in Central Alberta . They were placed in a plastic pool filled
with conditioned well, which was left floating on the pond, so the developing embryos
and tadpoles would experience identical temperature and weather conditions as the wild
ones. The conditioned well water was obtained by filling a 1900-L tub with well water
and seeding it with plankton and aquatic plants from the pond. This water is hereafter
referred to simply as well water. The embryos and tadpoles were left to develop until
Gosner stage 25 [5].
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Stimulus Collection:
For Experiments 1 and 2, we generated two chemosensory stimuli. Initially, we
collected injured conspecific cues, also known as alarm cues, from non-gravid female
guppies from both the Upper and Lower Aripo River populations. Fish alarm cues are
located in the epidermis, and can only be released in the water column via mechanical
damage to the skin, which usually occurs during a predator capture. These cues are
known to elicit a strong, innate antipredator response in nearby conspecifics. Cue donors
were euthanized via cervical dislocation. Due to the small body size, we used whole body
extracts rather than skin extracts [6,7]. We immediately removed the head and tail and
placed the remaining tissue into 200 mL of dechlorinated tap water. Tissue samples were
homogenized and filtered through polyester filter floss and diluted to the desired final
volume with the addition of aged tap water. To control for potential confound associated
with population bias in the response to conspecific alarm cues [8], we blended equal
quantities of alarm cue from Upper and Lower Aripo guppies and used the combined
solution as our stimulus. We collected tissue from 30 Upper Aripo (mean ± SD standard
length = 24.43 ± 2.55 mm) and 41 Lower Aripo (21.09 ± 2.83 mm) guppies. We collected
a total of 54.72 and 55.57 cm2 of tissue surface area (Upper and Lower Aripo guppies,
respectively) and adjusted the final volume to 1100 mL. The final concentration (~0.1
cm2/mL) has previously been shown to reliably elicit increased antipredator responses in
Trinidadian guppies [6,7]. The alarm cue was frozen in 20 mL aliquots at -20°C until
needed. Sufficient alarm cue was generated for Experiments 1 and 2.
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Secondly, we collected Nile tilapia odour as a novel predator cue. Nile tilapia do
not occur in our test streams and thus represent a ‘novel odour’ to guppies. We placed 10
tilapia (approximately 10 – 12 cm standard length) into a 60-L glass aquarium, filled with
45 L of dechlorinated tap water (~25° C). The tank was unfiltered and tilapia were not fed
while in the donor tank. Tilapia were held for 48 hrs and then returned to the holding
tanks. Water from the tilapia donor tank was filtered and frozen at -20° in 20 mL aliquots
until needed. Sufficient tilapia odour was generated for Experiments 1 and 2 (see below).
For Experiment 2, we generated two additional cues: pike cichlid odour and
lemon odour. Pike cichlid odour was collected from two donors (14.5 and 12.8 cm SL).
Pike cichlids were food deprived for three days prior to odour collection and then were
placed, individually, into 20-L glass aquaria (filled with 10 L of dechlorinated tap water)
and left for 48 hours. During this time, the cichlids were not fed and the tanks were left
unfiltered. Water from pike cichlid donor tanks was filtered and frozen in 20 mL aliquots
at -20°C. We also generated a lemon oil cue as a novel, but non-fish and ecologically
irrelevant stimulus. We diluted 120 drops of McCormicks Lemon Oil in 300 mL of
dechlorinated tap water and froze the resulting solution in 20 mL aliquots at -20°C.
Previous studies demonstrate that this concentration of lemon oil is detectable by prey
fish [9].
For the cichlid trials (Experiment 3) we generated convict cichlid skin extract
from 40 donor cichlids (mean ± SD SL= 35.30 ± 3.80 mm). Donors were killed via
cervical dislocation and skin fillets were removed from either side and immediately
placed into 300 mL of chilled distilled water. Skin fillets were then homogenized, filtered
through polyester filter floss and diluted to the final volume with distilled water. We
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collected a total of 168.95 cm2 of skin, diluted in 1690 mL of distilled water, to obtain
our cue solution (final concentration ~ 0.1 cm2 ml-1). Similar alarm cue concentrations
have been shown to elicit an antipredator response in juvenile convict cichlids [10,11].
The alarm cue was frozen in 20 mL aliquots at -20°C until required. We collected the
odour of a novel predator (juvenile rainbow trout). Trout odour donors (n = 6; mean ± SD
= 9.58 ± 0.44 cm FL) were placed into a 60-L glass aquarium (~ 18°C) filled with 30 L of
dechlorinated tap water for 48 hours. The tank water was aerated but not filtered. Trout
were not fed during the stimulus collection period. We removed approximately 2 L of
water from the tank and filtered it through filter floss to remove any particulate matter.
Trout odour was frozen at -20°C in 20 mL aliquots until needed.
For the tadpole trials (Experiment 3) injured tadpole cues were obtained by
sacrificing tadpoles, and getting a cue solution by using a mortar and pestle and filtering
and diluting the solution so that each pail would receive the equivalent of 1.5 crushed
tadpoles. We treated 9 pails with injured tadpole cues and 9 pails with water. Following
the treatment, 2-3 tadpoles from each bucket were tested for their response to water and
others to their response to salamander odour. Tiger salamander odour was obtained by
individually soaking 6 salamanders in 2 L of well water for 24 h and combining the odour
of 2 randomly chosen salamanders (ranging between 12.6 – 14.8 cm total length).
References:
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comparison. Am. Nat. 167, 867-878.
2 Botham, M.S., Hayward, R.K., Morrell, L.J., Croft, D.P., Ward, J.R., Ramnarine, I. &
Krause, J. 200). Risk-sensitive antipredator behavior in the Trinidadian guppy,
Poecilia reticulata. Ecology 89, 3174-3185.
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3 Endler, J.A., & Houde, A.E. 1995. Geographic variation in female preferences for
male traits in Poecilia reticulata. Evolution 49, 456-468.
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Supplementary Figures.
Figure S1: Mean (± SE) change in time spent moving (A) and foraging attempts (B) for
juvenile convict cichlids pre-exposed to low risk cues (distilled water) or high risk cues
(alarm cues) and tested for response to distilled water controls (open bars) versus rainbow
trout odour (solid bars). N = 20 per treatment combination.
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Figure S2: Mean (± SE) percent change in the number of line crosses by tadpoles preexposed to low risk cues (well water) or high risk cues (injured conspecific cues) and
tested for their response to well water controls (open bars) versus tiger salamander odour.