DOI: 10.5061/dryad.hhmgqnkm9
title: Anticipatory plasticity: frog embryos respond to environmental cues by producing an adaptive phenotype at hatching

description: Developmental plasticity can occur at any life stage, but a context in
 which it might be crucial is when individuals that produce specific
 phenotypes early in development gain a competitive advantage at a later
 life stage. Here we asked if pre-hatching (embryonic) exposure to a
 nutrient-rich resource can impact hatchling morphology in tadpoles of
 Mexican spadefoot toads, Spea multiplicata. Induction of a distinctive
 carnivore morph can occur when a tadpole eats live fairy shrimp. We
 investigated whether cues from fairy shrimp, detected as embryos,
 determine hatchling morphology in a manner allowing individuals to take
 advantage of this nutritious resource. We found that hatchlings with
 embryonic exposure to shrimp were larger and had larger jaw
 muscles––traits that increase their ability to compete for shrimp. Thus,
 embryos can assess and respond to environmental cues by producing
 preemptive resource-use phenotypes. Such anticipatory plasticity may be an
 important but understudied form of developmental plasticity.
description: In July 2020, we added fairy shrimp
 eggs, <em>Streptocephalus</em> sp., to 5-gallon aquaria of
 distilled water placed by a
 window. <em>Streptocephalus</em> sp. occur in the natural
 ponds where our toads were collected. Their nauplii began hatching within
 24 hours. We started feeding the shrimp nauplii Brine Shrimp Food
 (Northeastern Brine Shrimp LLC) when one day old. We harvested shrimp from
 aquaria and transferred them to a tub of dechlorinated tap water. We
 repeated this step to rinse away residual food particles and concentrate
 the shrimp's density. In nature, fairy shrimp
 hatch within 48 hours of pond filling. Depending on the time of day a pond
 fills, <em>S. multiplicata</em> oviposit within 2-20 hours of
 pond filling (they breed only on the night after a pond fills).
 Because <em>S. multiplicata</em> eggs take another 48 hours to
 hatch, <em>S. multiplicata</em> embryos would typically be
 exposed to live shrimp for at least 2-20 hours. To mimic these conditions,
 we bred the frogs approximately 20 hours after the shrimp eggs were placed
 in water. We bred three pairs of <em>S. multiplicata</em> that
 were collected near Portal, Arizona, USA, and that had been part of a
 laboratory colony at UNC-Chapel Hill for up to four years. We induced
 breeding by injecting adults with 0.07 mL luteinizing hormone-releasing
 hormone at a concentration of 0.01 µg/µL and leaving the pairs in separate
 nursery tanks overnight. While the frogs were breeding, we filled 60
 replicate plastic boxes (11 x 11 x 11 cm) with 900 mL of dechlorinated tap
 water. We assigned boxes to the shrimp exposure treatment or control,
 adding 10 mL of concentrated shrimp nauplii in dechlorinated water to the
 treatment boxes. Control boxes received 10 mL of dechlorinated
 water. The next morning, we collected the frog eggs
 that had been laid overnight, placing a group of approximately 20 sibling
 eggs into each experiment box. We randomly distributed ten boxes per
 treatment per family across three shelves of a metal rack (N=30 replicates
 per treatment). Eggs were kept at 23 ℃ on a light-dark cycle of 14hL:
 10hD. We removed dead shrimp from treatment boxes daily and replenished
 the boxes with new concentrated shrimp (treatment) or water (control)
 daily until the tadpoles hatched. All tadpoles hatched approximately two
 days later. To assess whether pre-hatching shrimp
 exposure impacted tadpole morphology at hatching, we left 15 tadpoles per
 box to develop for another project and randomly sampled the remaining
 0-day-old tadpoles from each box (N = 125 hatchlings per treatment). We
 euthanized the sampled hatchlings via immersion in a 0.1% aqueous tricaine
 methanesulfonate (MS-222) solution according to IACUC protocol ID
 17-252.0-A and preserved them in 95% ethanol. We measured the preserved
 tadpoles using previously published methods. Specifically, using hand-held
 digital calipers, we measured each hatchling’s body size (snout-vent
 length; SVL) and orbitohyoideus (jaw) muscle width (OH; carnivores have
 larger OH). We also measured the length of its gut (carnivores have
 shorter guts) by removing and uncoiling the intestines against a ruler.
 Finally, we determined its Gosner developmental stage by visual
 inspection. Measurements were done blind with respect to treatment. We
 standardized OH width and gut length for body size by regressing ln(OH)
 and ln(gut length) on ln(SVL). Because we were
 concerned that we did not have enough fairy shrimp in the first trial, we
 ran another trial in Fall 2022. This second trial differed from the first
 in the following three ways. First, two families of eggs were used from
 adults collected from the field two months before the experiment began
 (also near Portal, AZ). Second, our shrimp treatments contained both fairy
 and brine shrimp (<em>Artemia</em> sp.). Brine shrimp are more
 easily raised at the high densities required to trigger carnivore
 development; these brine shrimp were hatched from eggs in 25 ppt
 formulated seawater and fed a solution of baker’s yeast. Third, we sampled
 up to four hatchlings from approximately 20 replicate boxes per treatment
 (control N = 66 hatchlings from 23 boxes, treatment N = 49 hatchlings from
 17 boxes). Across the two trials, we had 47 replicate treatment boxes and
 53 replicate control boxes across five families, representing 366
 hatchling tadpoles measured. To determine if
 pre-hatching exposure to shrimp impacted the morphology of hatchlings, we
 used linear mixed-effects models in the R package ‘lme4’. Because there
 was not a blocking effect of trial date, we pooled data across trials. Our
 response variables were SVL, Gosner stage, SVL-corrected jaw width, and
 SVL-corrected gut length. Our fixed effect was treatment, and we specified
 experimental box nested within family as a random intercept. We used
 an F-test of fixed-effects terms with Kenward-Roger’s method for
 denominator degrees of freedom for inference ('lmerTest'
 package). We performed all analyses in R version 4.1.2.