Mountain Lake research 2015
1. Question: Does the corticosterone (CORT) response of male and female red-spotted
newts change with sex ratio?
Rationale: Because males interfere in the courtship of other males (Verrell 1983), we
expect males that are experiencing male biased sex ratios to have higher stress levels
than males that are experiencing female biased sex ratios. Furthermore, females
exposed to male biased sex ratios experience greater physiological stress (as suggested
by lymphocyte abundance and weight loss) than females that are not exposed to male
biased sex ratios during the mating season (Grayson et al. 2012).
Methods: We will obtain water-borne baseline CORT of adult male and female newts
(N=20 of each sex) collected from the field .Then we will place them in mesh boxes in
the field at different sex ratios. We will place 1 subject male or female with (1) 2 males
and 1 female, or (2) 1 male and 2 females. Each newt will be tested 2 times at the
different sex ratios in random order with treatment one first half the time. Newts will
be maintained in the mesh boxes for up to 2 weeks in the field. We will then obtain
water-borne post CORT values. We will obtain water-borne CORT values by placing
each newt in a 250 ml-glass beaker with 80 ml of spring water for 1 hr following Reedy
et al. (2014). We will then compare the change in CORT values (CORT response)
between the two treatments for both males and females.
2. Question: Do male and female red-spotted newts show a corticosterone response to
amplexus?
Rationale: Reedy et al. (2014) found that both male and female red-spotted newts have
higher CORT levels when found in amplexus than when found alone. We need to test the
assumption that those animals did not just have higher CORT levels then solo newts.
Methods: We will collect amplexed newts in the field (n = 30 pairs) and get their
baseline CORT levels following Reedy et al. (2014). We will then place them in mesh
boxes in the field for one week and record their behavior daily. Newts will be exposed
to one of two treatments: (1) placed with a new partner or (2) placed into a cage alone.
After one week, we will obtain a final CORT sample for each individual. We will then
examine if their behavior and treatment over the week is related to their CORT values.
3. Question: Do larval red-spotted newts show anti predator behavior and associated
hormonal (corticosterone) stress response to chemical cues of (1) larvae, (2) adults, (3)
Rana catesbiena tadpoles, (4) water control?
Rationale: Predation is a predominant force acting on prey populations. Prey must be
able to recognize and avoid predators to persist. In some aquatic systems, cannibalism
can be common especially when there are large size asymmetries in co-occuring
individuals. In red spotted newts, Notophthalmus viridescens, there are large size
differences between adults and larvae and adults cannibalize larvae (Gabor 1996).
Chemical cues are important in predator detection in amphibians and larval redspotted newts recognize and show antipredator response (freezing) to chemical cues of
adult red-spotted newts (Mathis 2003). Larval red-spotted newts did not freeze in
response to the chemical cues of non-predatory tadpoles (Hyla versicolor) or other
larval newts (Mathis & Vincent 2000; Mathis 2003).
Predation may also affect stress hormones (CORT), however, few studies have
examined the effect of predation on stress response in amphibians. The CORT response
to predation may differ between systems. Fraker et al. (2009) found a decrease in
CORT in Rana sylvatica tadpoles following exposure to high risk predator diet cues.
Whereas, Davis (2012) found that San Marcos salamanders, Eurycea nana, froze in
response to the chemical cues of a native fish predator and showed a significant
increase in CORT response. We want to assess whether larval red-spotted newts show a
greater CORT response to predators concomitant with a decrease in activity
(antipredator response) and if this response differs in response to non-predators.
Methods: We will place larval newts in a small shoebox sized test chamber with
dechlorinated water >12 h before the experiment. We will attach a polypropylene tube
to the center of one of the long sides of the chamber with the end of the tube sitting 1cm
above the surface of the water. Before the behavioral experiment we will obtain a prestimulus water-borne hormone sample by placing each newt in a 250 ml-glass beaker
with 80 ml of treated water for 1 hr. Following this, we will introduce the larva into the
center of the test chamber. After a 20 min acclimation period we will record the time
that the larva spend active for 8 min. Activity will be defined as any movements except
gill flicks. Following the pre-stimulus activity period, we will inject 40 ml of a stimulus
solution at a rate of 1ml/s, from one of the four treatments. We will test larval redspotted newts (n = 15 per treatment) anti predator behavior and associated hormonal
(cortisol) stress response to chemical cues of :(1) non predatory conspecific redspotted newt larvae, (2) a predatory adult newt, (3) a non predator heterospecific, Rana
catesbiana tadpoles, (4) water control. Following stimulus introduction we will record
post-stimulus activity for another 8 min. Immediately following the behavioral trial we
will obtain a post-stimulus water-borne hormone sample by placing each newt in a 250
ml-glass beaker with 80 ml of treated water for 1 hr. All experiments will be performed
at the same time of day to control for circadian rhythm in CORT production. All waterborne hormone samples will be frozen until processed.
Stimulus Acquisition
A minimum of four of each type of stimulus animal will be used. Prior to the
collection of chemical cues, stimuli will be fed a neutral diet of frozen brine shrimp
(Artemia) for at least five days to minimize the effects of prior diet. The volume of
the stimulus animal will be determined by displacement. To maintain similar
chemical concentrations between treatments, approximately 240 ml of water per 1
ml of stimulus animal will be used in the collecting chamber. Before acquisition of
the stimulus water, animals will be removed from the tanks and the water will be
stirred. Equal proportions of water from all individuals will be mixed to control for
individual effects before being frozen. The stimulus water will be frozen for at least
24 hours after its removal from the stimulus tank before its introduction into the
testing chamber. This method has been used successfully in previous studies
(Mathis et al., 1993; Epp & Gabor, 2008). The stimulus samples will be coded after
freezing and only thawed immediately prior to testing.
Supplies needed
CORT Plates
SPES
Nitrogen
Methanol
Mesh boxes