DOI: 10.5061/dryad.2v6wwpzw2
title: Data from: Phenotypically plastic responses to environmental variation are more complex than life history theory predicts

description: For insects that exhibit wing polyphenic development, abiotic and biotic
 signals dictate the adult wing morphology of the insect in an adaptive
 manner such that in stressful environments the formation of a
 flight-capable morph is favored and in low stress environments a
 flightless morph is favored. While there is a relatively large amount
 known about the environmental cues that dictate morph formation in wing
 polyphenic hemipterans like planthoppers and aphids, whether those cues
 dictate the same morphs in non-hemipteran (i.e. cricket) wing polyphenic
 species has not been explicitly investigated. To experimentally test the
 generality of environmental cue determination of wing polyphenism across
 taxa with diverse life histories, in this study we tested the importance
 of food quantity, parasitic infection, and tactile cues on wing morph
 determination in the wing polyphenic sand field cricket, Gryllus firmus.
 Our results also show that certain stress cues, such as severe diet
 quantity limitation and parasitic infection, actually led to an increase
 in the production of flightless morph. Based on these findings, our
 results suggest that physiological and genetic constraints are important
 to an organism’s ability to respond to environmental variation in an
 adaptive manner beyond simple life history trade-offs.
description: <strong><em>Cricket populations and
 rearing</em></strong> We used<em> G.
 firmus</em> crickets from two laboratory populations originally
 collected from Gainesville, Florida. These colonies have been artificially
 selected for &gt;40 generations by Dr. Anthony Zera to produce lines
 with increased production of either the flight-capable long-wing (LW)
 morph or the flight-incapable short-wing (SW) morph. Although these lines
 were selected for increased long-wing and short-wing morph formation, they
 still exhibit phenotypic plasticity for morph (i.e. the colony selected
 for long-wing can still produce short-winged adults). Additionally, we
 derived an unselected population from <em>G. firmus</em>
 collected from Tamarac, Florida in 2019. Because of the different origins
 of the individuals, we refer to these three separate lines using the
 following abbreviations: Selected Long-Wing (S–LW), Selected Short-Wing
 (S–SW), and Unselected (US). However, it is important to note that we were
 most interested in the question of the degree of influence that genetic
 and environmental factors have on morph determination at the individual
 level, rather than explicitly setting out to test differences between
 populations. Our goal was to identify individual level physiological
 responsiveness to the cues.  Populations were housed separately in large
 plastic bins in a climate-controlled room (26 – 28 ºC at 50 – 70%
 humidity) and provided <em>ad libitum</em> water and food. We
 used a standard lab diet of wheat germ, wheat bran, powdered milk and
 nutritional yeast (Zera and Larsen 2001).
 <strong><em>Morphological
 ontogeny</em></strong> To document
 morphological changes during development and to identify if there were any
 observable juvenile traits that indicate when the adult morph decision was
 made, crickets from the two selected populations (S–LW and S–SW) were
 separated within 24 hours of hatching. We used the morph-selected
 populations to ensure that roughly an equal number of long-wing and
 short-wing adults would be available to compare trait growth across the
 final juvenile instars to test for any differences between morphs. Each
 hatchling was assigned a random ID and housed individually in an 8 oz deli
 cup (Fabri-Kal® Pro-Kal™) containing an egg carton shelter and
 <em>ad libitum</em> food and water. We checked for nymphal
 molting events every 24 hours. To better visualize molting, we placed a
 small dot of non-toxic acrylic paint (Apple Barrel® 20591E Bright Magenta,
 21476E Lime Tree) on the side of the abdomen; upon molting this paint was
 shed with the exoskeleton. After a nymph molted, we recorded the date and
 photographed the nymph from above using a Leica M125 automontage. This
 procedure was repeated for each instar until maturity was reached (8 – 11
 nymphal instars). We waited 48 hours after adult eclosion to ensure full
 sclerotization of the exoskeleton before we euthanized individuals via
 freezing. Adults were photographed dorsally by a Nikon® D7500 mounted
 automontage both before and after forewing dissection. Upon completion of
 rearing, we randomly selected 25 long-wing and short-wing individuals of
 each sex to collect measurements of the terminal instar series and adult
 stages using ImageJ (Version 1.52s). For each of these 200 crickets,
 photographs from the adult, terminal juvenile instar (T), and the previous
 two juvenile instars (T-1 and T-2) were measured for head width, thorax
 width, abdomen length, total body length, forewing bud lengths, hindwing
 bud length, and ovipositor length in accordance with Supplemental Figure
 1. <strong><em>Food quantity
 treatments</em></strong> To establish the
 quantity of food to provide crickets assigned to the high- and low-
 quantity food treatments, we measured food consumption of individuals in
 their T-1 juvenile instar. Thirty T-1 crickets of both sexes were removed
 from both the S–LW and S–SW lines within 24 hours of eclosing and placed
 in individual deli cups with 500 mg of food (enough to simulate
 <em>ad libitum</em> conditions), a cotton-plugged water vial,
 and a shelter. After 7 days, we removed the shelter, water, and frass to
 weigh the remaining uneaten food. This process was repeated weekly until
 adult eclosion. Average consumption per week was calculated as 203.5 mg (±
 17.9 standard error). To identify the impact of food
 quantity on morph determination, crickets were housed individually in deli
 cups within 24 hours of eclosion into the T-1 instar from each of the
 S–LW, S–SW, and US populations and assigned to the high-quantity or
 low-quantity food treatment. Crickets in the high-quantity treatment group
 were provided food <em>ad libitum</em>, whereas crickets
 assigned to the low-quantity group were provided only 58 mg of food every
 fourth day, which is approximately 50% of the average <em>ad
 libitum</em> consumption. All deli cups were provisioned with a
 shelter and <em>ad libitum</em> water. One day after adult
 eclosion, each cricket was weighed, euthanized by freezing, and wing morph
 recorded. <strong><em>Density
 treatments</em></strong> To identify the
 mechanism underlying the impact of local conspecific density on morph
 determination, crickets were housed individually in deli cups within 24
 hours of eclosion into the T-1 instar from each of the S–LW, S–SW, and US
 populations and assigned to the simulated high- or low-density treatment.
 Nymphs assigned to the low-density treatment were housed in individual
 deli cups placed in a larger rearing container (Whitmor ClearVue® shoe
 box, 29.8 x 18.7 x 9.5cm) containing two egg carton shelters, <em>ad
 libitum</em> food, and two cotton-plugged water vials, but no
 crickets. Individuals assigned to the high-density treatment were housed
 in individual deli cups which possessed small airholes, placed in a larger
 rearing container containing two shelters, food, and two cotton-plugged
 water vials, five adult female short-wing crickets, and five adult male
 short-wing crickets. The crickets in the larger container simulating
 density were replenished on a weekly basis. We provided all focal crickets
 with <em>ad libitum</em> food, water, and shelter and
 monitored them daily for eclosion. One day after adult eclosion, each
 cricket was weighed, euthanized by freezing, and its wing morph
 recorded. <strong><em>Parasitic
 infection treatments</em></strong>
 Horsehair worm cysts (<em>Paragordius varius</em>)
 were obtained from a stream near Lincoln, Nebraska via collection of their
 intermediate aquatic snail host (<em>Physa sp.</em>). Snails
 were euthanized via freezing, then removed from their shells. Snail tissue
 was confirmed to be encysted prior to use in our
 experiment.<strong><em> </em></strong>To identify
 the impact of parasitic infection on morph determination, crickets were
 housed individually in deli cups within 24 hours of eclosion into the T-1
 instar from each of the S–LW, S–SW, and US populations and assigned to the
 uninfected or infected treatment.<strong><em>
 </em></strong>Crickets were fasted for two days then fed
 either 5 mg of their standard diet or encysted snail tissue for the
 uninfected and infected treatments respectively. Crickets were monitored
 under red-light conditions for 15 minutes to verify food/snail consumption
 and then housed individually for the remainder of the study and monitored
 daily for eclosion. Upon molting, adult wing morph was recorded, crickets
 were euthanized via freezing and crickets in the infected treatment were
 dissected to confirm parasitic infection. Crickets that we did not detect
 a parasite in were excluded from the experiment as we could not
 distinguish between crickets that were never infected and those who
 successfully evaded parasite development via immune action. We did not
 collect weight data for this experiment given the inherent conflation
 between host and parasite weight.
description: # Phenotypically plastic responses to environmental variation are more
 complex than life history theory predicts
 [https://doi.org/10.5061/dryad.2v6wwpzw2](https://doi.org/10.5061/dryad.2v6wwpzw2) We have submitted the ontogenetic morphometric data under “Corrected DFA data" as well as the raw data on the mass and morph outcome for three different environmental cue experiments under the file names “Food Quant”, “Density”, and “Infect” which correspond to the cues of food quantity, simulated density, and parasite infection respectively. ## Description of the data and file structure Ontogenetic morphometric data (“Corrected DFA data”): Group: wing morph outcome x sex (SW F, LW F, SW M, LW M) wherein SW = Short-winged population, LW = Long-winged population, M= males and F= females Sex: M = male, F= female Morph: wing length at adult eclosion, LW= long-winged, SW = short-winged Instar: this refers to the terminal instar series as described in the text wherein T= terminal instar (directly before adult eclosion), T1= T-1 instar (directly before terminal instar), T2= T-2 instar (directly preceding T-1 instar, and adult = adult life stage Head width: Width of head capsule as measured in mm by Image J in accordance with the supplemental diagram of measurement locations. Thorax width: Width of thorax as measured in mm by Image J in accordance with the supplemental diagram of measurement locations. Abdomen Length: Length of abdomen as measured in mm by Image J in accordance with the supplemental diagram of measurement locations. Total Body Length: Length of body as measured in mm by Image J in accordance with the supplemental diagram of measurement locations. Hind Wing Length: Length of hind wing as measured in mm by Image J in accordance with the supplemental diagram of measurement locations. Fore Wing Length: Length of fore wing as measured in mm by Image J in accordance with the supplemental diagram of measurement locations. Ovipositor Length: Length of ovipositor as measured in mm by Image J in accordance with the supplemental diagram of measurement locations Environmental Cues Data (“Food Quant”, “Density”, and “Infect”) ID: A unique identification code that was assigned to each cricket to enable tracking through the experiments, also denotes population of origin wherein SW = Short-winged population, LW = Long-winged population, M= males and F= females Treatment: Dense = simulated density treatment, Loner = control treatment (reared alone), Restrict = restricted diet treatment, Buffet = *ad libitum* diet treatment, Infect = infected, Uninfected = uninfected Sex: M = male, F= female Morph = wing morph outcome, LW = long-winged morph, SW = short-winged morph