DOI: 10.5061/dryad.9w0vt4bnx
title: Consistent traffic noise impacts few fitness-related traits in a field cricket

description: Anthropogenic habitat change is occurring rapidly, and organisms can
 respond through within-generation responses that improve the match between
 their phenotype and the novel conditions they encounter. But, plastic
 responses can be adaptive or maladaptive and are most likely to be
 adaptive only when contemporary conditions reasonably mimic something
 experienced historically to which a response has already evolved. Noise
 pollution is a ubiquitous anthropogenic stressor that accompanies
 expanding urbanization. We tested whether the amplitude of traffic noise
 influences a suite of fitness-related traits (e.g. survival, life history,
 reproductive investment, immunity) and whether that depends on the life
 stage at which the noise is experienced (juvenile or adult). Our
 treatments mimic the conditions experienced by animals living in urban
 roadside environments with variable vehicle types, but continuous movement
 of traffic. We used the Pacific field cricket, an acoustically
 communicating insect that was previously shown to experience some negative
 behavioral and life history responses to very loud, variable traffic
 noise, as a model system. 
description: <strong>General experimental
 design</strong> We used a fully factorial design
 with four possible noise levels experienced during development and/or at
 adulthood. Each cricket had their own unique
 <strong>"id."</strong> We assigned juvenile crickets
 to one of four acoustic environments: a no traffic noise treatment or one
 of three chronic traffic noise treatments (50dBA, 60dBA, or 70dBA) as seen
 in the column <strong>"juv_treatment."</strong> At
 adulthood, we randomly reassigned the crickets to a noise treatment, as
 seen in the <strong>"adult treatment"</strong>
 column. The <strong>"trial_type"</strong> column
 represents the combination of the juvenile and adult treatments for each
 individual cricket (juvenile:adult). We then measured a suite of
 fitness-related traits, which we categorized as related to basic
 life-history (survival, development time, and adult size), reproductive
 investment (mating success, sperm viability, number of eggs hatched after
 1 week of laying, and male and female investment in reproductive organs),
 and immunity (hemocyte counts and melanization of a foreign
 body). <strong>Rearing and traffic noise
 exposure</strong> To expose crickets to different
 noise levels, we reared the animals inside of replicate, acoustically
 isolated Percival incubators (model I36VLC). Because the incubators
 themselves generate white noise when on, we left the power off, but
 retained a 12:12 light:dark schedule in each using clamp lights outfitted
 with 60W LED bulbs attached to mechanical timers. The lighting also
 maintained incubator temperatures within those naturally experienced by
 the animals (21°C to 23°C), and we maintained 60-65% humidity by placing a
 1.89L bin of water in each incubator. We measured temperature and humidity
 every two weeks. Each acoustically isolated incubator
 housed a Bluetooth EcoXBT speaker that either played nothing (leaving
 background ambient noise of 37-38dBA only; hereafter “silent”) or an
 uncompressed .wav traffic noise track standardized to 50dBA, 60dBA, or
 70dBA (when measured at 1m away). We created the traffic tracks following
 Gurule-Small et al. 2018 and used the same traffic noise recordings as
 those studies. In brief, we first spliced together ten randomly chosen 30
 second clips of traffic noise originally recorded for Gurule-Small et al.
 2018 to create a five-minute track. To investigate how noise levels impact
 plastic responses to noise, we next leveled the sound from each recording
 clip so that the average amplitude was consistent across the track. A
 sound engineer (JHG) adjusted the gain of each clip in Logic Pro X
 (version 10.4.8, Apple Inc., Los Altos, CA, USA) to level the sound so
 that each segment and the full five-minute track was at the appropriate
 amplitude (RMS level) for each treatment (50dBA, 60dBA, or 70dBA). Noise
 treatments were broadcast to crickets living inside of the incubators 24
 hours a day (including during the daily peaks of communication by song),
 mimicking noise that would be experienced near a highway with high traffic
 fluidity. We measured the amplitude of our treatments using a PCE-430
 sound level meter and class 1 microphone at 1m away from the speaker
 inside of the Percival incubators. Every two weeks we confirmed the
 amplitude of the traffic noise tracks. From October
 2020 to August 2021, we isolated Pacific field crickets just prior to
 their 2nd instar (~14 days after hatching, as early as was possible
 without causing mortality) from a lab-reared population that was
 originally collected in Hilo, Hawaii in 2017. We randomly assigned them to
 one of the four traffic noise treatments
 (<strong>"juv_treatment"</strong>) and housed them
 in groups of 15 in 1.89L boxes inside of the incubators for the duration
 of their developmental exposure. Each 1.89L box contained wet cotton for
 water, egg carton for shelter, and ad libitum Flukers Cricket Chow for
 food. We did this eight times over the course of eight months, yielding
 eight cohorts of crickets. For each cohort
 (<strong>"cohort"</strong> column), we isolated 4-7
 boxes (<strong>"box"</strong> column) of crickets
 per treatment, depending on the number of animals available in the lab
 colony, for a total of 3060 crickets assigned to treatments. The
 <strong>"cohort_box"</strong> column represents the
 combination of the cricket's cohort letter and box number.
 We provided clean housing and fresh food and water twice per
 week. To ensure that animals remained unmated prior to mating trials (see
 below), we isolated them individually in 0.47L deli cups when they
 approached eclosion (during the final instar before adulthood) with water,
 shelter and food (ad libitum Kaytee rabbit chow). We checked the deli cups
 twice weekly for eclosion. At eclosion we randomly reassigned individuals
 to one of the four noise treatments
 (<strong>"adult_treatment"</strong>). Throughout the
 experiment, we changed the stacking order of the boxes within incubators
 once a week (to account for the possibility that crickets would experience
 different loudness of noise depending on their location within the
 incubator) and rotated treatments among incubators every two weeks to
 avoid incubator effects. <strong>Assay
 Overview</strong> To measure a comprehensive set
 of fitness-related traits following developmental and/or adult exposure to
 different noise levels, we used the following general workflow: (1) at
 eclosion we determined development time, survival to eclosion, and
 measured pronotum width; (2) two weeks later we assessed mating success in
 controlled mating trials, allowed females to lay eggs for one week, and
 measured male sperm viability; (3) following the sperm viability assay or
 week of egg laying, we moved the crickets into the immune assays; (4)
 finally, when all other assays were complete, we dissected out and weighed
 the reproductive organs of all males and females and determined survival
 from eclosion to dissection.  <strong>Life
 History Trait Assays</strong> When we assigned
 newly eclosed adults to their adult noise treatments, we measured the
 width of their pronotum (<strong>"pronotum"</strong>
 column) to the nearest mm using digital calipers (n = 454), recorded their
 sex (<strong>"sex"</strong> column), and recorded
 the date on which they eclosed. To obtain a metric of development time
 (<strong>"days_to_eclosion"</strong> column), we
 subtracted the date at which they were assigned to treatments
 (<strong>"hatch_date"</strong> column) from the date
 of eclosion (<strong>"ecolsion_upper"</strong>
 column). Thus, development time here is from treatment assignment (0-14
 days after hatching) to adulthood (assay: development time, n = 518).
 Survival to eclosion is the percent of crickets that lived to eclosion of
 the original 3060 crickets assigned to treatments (assay: juvenile
 survival, n = 3060), whereas the survival to dissection describes the
 percent of survivors from eclosion to the end of the experiment (assay:
 adult survival, n = 518).  <strong>Reproductive
 Investment Assays</strong> <em>Mating
 Trials</em> To determine whether experience with
 noise impacts the likelihood of individuals mating, we conducted
 standardized no-choice mating trials. We allowed each individual to mate
 only once. Whenever possible, we paired males and females from the same
 adult treatment for mating, but because the sexes eclosed at different
 rates this was not always possible. In these cases, we paired the focal
 cricket with a mate pulled randomly from the large, freely breeding
 laboratory colony (51% of trials; whether the mate was pulled from the
 breeding colony or the experimental group is included as a covariate in
 the model). The column <strong>"pop_pair"</strong>
 indicates whether this stimulus cricket came from the same experimental
 treatment or the laboratory colony,
 <strong>"pair_virgin"</strong> indicates whether
 this stimulus cricket was a virgin, and
 <strong>"pronotum_pair"</strong> indicates the
 pronotum size of the stimulus male. Each mating trial consisted of placing
 the male and female together inside of a deli cup (9cm diameter) at
 ambient room temperature under dim light for up to two hours each day for
 up to four days (or until a single successful mating took place). We
 checked deli cups every 15 minutes for successful mating as evidenced by
 spermatophore transfer. After the crickets mated, or four days of
 unsuccessful mating trials, we again isolated the males and females in
 separate deli cups. We recorded the number of days that they failed in the
 <strong>"num_fails"</strong> column, whether they
 mated in the <strong>"mated"</strong> column, and
 the date of successful mating in the
 <strong>"date_mating"</strong> column. Using the
 date of the mating and the cricket's ecolsion date, we calculated the
 <strong>"age_mating."</strong> Not all individuals
 successfully mated in the time allotted, so we were able to assess mating
 success (mated or not mated in four opportunities) (assay: mated, n =
 445). Number of Hatchlings Next,
 allowed all females who mated successfully to lay eggs for one week. After
 one week, we recorded the date as
 <strong>"end_egg_lay."</strong> We then placed each
 female’s cotton pad in a labeled Tupperware container, providing food and
 shelter when hatchlings appeared. Not all females that mated had offspring
 hatch, allowing us to assess hatching success (zero live hatchlings or
 more than zero live hatchlings) (assay: hatching success, n = 178). Five
 weeks after placing the cotton pad in the Tupperware, we counted the
 number of offspring (<strong>"num_f2"</strong>
 column, n = 138). We recorded the date we counted the number of offspring
 in the <strong>"date_f2_counted"</strong> column and
 calculated the number of days these offspring had to hatch in the
 <strong>"days_F2_hatched"</strong> column.
 Sperm Viability Following successful mating
 trials, we isolated males for sperm viability analysis the following day.
 If males did not mate in the four days (n = 18/243, 7.4%) or if we were
 not able to conduct the assay the exact day after mating, we standardized
 time since spermatophore production by manually removing their
 spermatophore (externally with no injury to the cricket) 24 hours before
 their sperm viability assay. We used a THERMOFisher LIVE/DEAD sperm
 viability kit to stain live and dead sperm following the protocol outlined
 in Garcia-Gonzalez et al. 2005. (assay: sperm viability, n = 167).
 Briefly, we removed a spermatophore non-invasively from the male cricket,
 placed it on a glass slide in Beadle saline, and cut it open with
 dissection scissors to evacuate the sperm. We pipetted 5µL of the sperm
 and Beadle saline mixture to a clean part of the glass slide and gently
 mixed it with a sterilized pin. Then, we stained the sperm with SYBR-14
 and propidium iodide (each addition followed by a 10-minute incubation
 period in the dark) and photographed the sperm with a Leica M165FC scope
 outfitted with an EC3 camera on a computer running LAS X imaging software.
 The GFP3 (blue) and DSR (green) fluorescent filters allowed us to take
 pictures of live (green) and dead (red) sperm, respectively, from the same
 view window on each sample. We recorded the date on which we performed
 this assay (<strong>"date_sv"</strong>) and used the
 cricket's eclosion date to calculate the
 <strong>"age_sv."</strong> We
 then crowdsourced the live and dead sperm counting using the world’s
 largest community science platform Zooniverse ([61]; https://www.zooniverse.org/projects/marywestwood/the-cricket-wing). We divided each sperm image into 36 smaller images and uploaded the resulting 12,304 images to our Zooniverse project. We developed a detailed protocol and training tutorial that taught volunteers to identify sperm cells and click on each cell in an image that either showed live (green) or dead (red) sperm. Volunteers were not aware of the crickets’ noise treatment, and we had 784 volunteers count sperm cells. Clicks were automatically counted and deposited in a spreadsheet accessible to the researchers. Each image was counted 6 times. We also scanned for errors in all data caused by such issues as volunteers submitting highly inaccurate or empty counts, and removed these from the dataset. Next, we calculated the mean number of sperm in each image and identified outlier images (those below the 1st quartile or above the 3rd quartile); we did not find that certain individual volunteers regularly submitted counts that were identified as outliers. Thus, the “wisdom of the crowd” offset any one-off outlier counts. After removing erroneous counts, each image was counted an average of 5.2 +/- 0.98 times. Finally, to calculate sperm viability, we summed the counts for each male cricket across the 36 images and divided the number of live sperm by the number of dead sperm. Numbers of live sperm are found in the <strong>"live"</strong> column, number of dead sperm in the <strong>"dead.y"</strong> column, and sperm viabilty in the <strong>"sperm_viability"</strong> column. Reproductive Organs We dissected male and female reproductive organs after the completion of all other assays. We recorded the date we dissected them (<strong>"date_weigh_w"</strong>) and used this date and the eclosion date to calculate <strong>"age_dissection."</strong> We froze crickets at -20°C for 15 minutes to euthanize them, then dissected them under a dissecting scope (Wild Heerbrugg M3Z) to extract and weigh reproductive organs. We recorded the initials of the dissector in the <strong>"dissector_initials"</strong> column. For males we removed the testes, spermatophore mold, and accessory glands (assays: testes, n = 158, spermatophore mold, n = 149, accessory gland, n = 157). For females we removed the ovaries (assay: ovaries, n = 196). We weighed each organ type separately on a VWR-64B scale immediately after dissection. Females were 33.3 +/- 9.4 days post eclosion and males were 34.8 +/- 13.8 days post eclosion at the time of dissection (age is included as a covariate in the reproductive organ models; see below). Female ovary weight is recorded in the <strong>"ovary_w"</strong> column, male testes in the <strong>"testes_w"</strong> column, spermatophore mold in the <strong>"smold_w"</strong> column, and accessory glands in the <strong>"ag_w"</strong> column. All weights were measured in grams. Immunity Assays We assessed cricket immunity using hemocyte counts and melanization. We conducted the hemocyte counts after sperm viability (males) or one week of egg laying (females) (average age: 25 +/- 3.8 days post eclosion (females) and 23.9 +/- 5.4 days post eclosion (males)). We counted the numbers of two different types of hemocytes, plasmatocytes (<strong>"total_count_plasmatocytes"</strong> column) and granulocytes (<strong>""</strong> column), following Triggs et al. 2012. We placed the crickets at 5°C for two minutes to anesthetize them, and then poked the cricket’s pronotum using a sterilized pin and pipetted 2µL of the hemolymph that emerged into 4µL of an anticoagulant buffer Smilanich et al. 2018. We then pipetted 5µL of the mixture onto a Weber Scientific Hemocytometer and placed it under a Keyence VXH Digital Microscope with a Keyence VH-Z100UR/W/T lens. We imaged the 5x5 grid of the hemocytometer at 400X and employed the Zooniverse platform again to count the number of each hemocyte type (on average 10 counts per image; assay: granulocytes, n= 213; plasmatocytes, n = 212). We recorded this date as the <strong>"date_hem"</strong> and used the ecolsion date to calculate the <strong>"age_immunity."</strong> We recorded the total number of hemocytes in the <strong>"total_hems"</strong> column. Immediately following the hemocyte assay, we inserted a 3mm long piece of nylon monofilament fishing line with a knot tied at the end into the previously created hole in the cricket’s pronotum. We left the filament in the crickets for 24 hours then removed and imaged the filaments under a Keyence VHX Digital Microscope (Keyence Corporation, Itasca, IL USA) scope with a Keyence VH-Z20R/W/T lens at 50x magnification, recording this date as "date_fil_out". We used the GNU Image Manipulation Program (v 2.10) to measure the amount of melanization on the filaments (assay: melanization, n = 359). The level of melanization was calculated in the <strong>"mean_white"</strong> column. Survival Measurements To calculate the proportion of individuals that survived to eclosion and dissection, we created a separate data sheet titled “20231027_F1_SurvivalDataset.” All column names with the same name as those in “20231027_F1_CompleteDataset” contain the same type of data. However, each box of crickets started with 15 individuals, but many died before reaching adulthood and therefore never received an ID. The <strong>“individual”</strong> column provides a temporary individual ID for each of these crickets. The <strong>“cohort_start_date”</strong> column contains the date the crickets were placed in the treatments, and the <strong>“dead_date”</strong> contains the day the individual cricket died. These two dates were used to calculate <strong>“days_until_death.”</strong> Date Cleaning Following data collection, we cleaned our dataset by fixing any errors, such as typos, and making sure there was only one row for each individual ID. Statistical Analyses We performed all statistical analyses in R (v2022.12.0+353). First, we visualized histograms and q-q plots and determined the best distribution fit for each variable (see accompanying code and data); all variables fit the assumptions of a normal distribution. We then ran linear mixed-effect models to test whether the continuous fitness traits we measured depended on juvenile and/or adult noise treatment and generalized linear mixed-effect models for the four binomial traits (juvenile survival, mated or not, successful hatching or not, and adult survival). The basic model structure included juvenile treatment (silent, 50dBA, 60dBA, or 70dBA), adult treatment (silent, 50dBA, 60dBA, or 70dBA), and their interaction as main effects; the interaction allowed us to determine whether plasticity is stage dependent. For pronotum, development time, and juvenile survival, juvenile noise treatment (silent, 50dBA, 60dBA, or 70dBA) was the only main effect because the crickets had not experienced the adult noise treatment at the time of the assays. All models included the cohort blocking variable as a random effect. We Bonferroni corrected all p-values to account for the number of models run.  We included several appropriate covariates, and these differed across models. For pronotum, development time, and adult survival, we included sex as a covariate. The adult survival model also included mating success. The binomial model addressing whether crickets mated or not also included sex as a covariate as well as whether the cricket they were paired with was from the experimental treatment or from the breeding colony. Both the binomial model assessing mated females’ hatching success and the linear model investigating the number of hatchlings included pronotum size and age at the end of the egg-laying period as covariates. We included two covariates in the model assessing sperm viability, pronotum size, and age at the time of the assay. The model structures for all reproductive organs included pronotum size and age at dissection as covariates. In the reproductive organ models, we also included whether the cricket successfully mated or not as a covariate, as mating may generate differences in reproductive organ mass. For the ovary model, we also included the number of hatchlings as a covariate. Finally, for both hemocyte models (number of granulocytes, number of plasmatocytes) and the melanization model, the covariates were pronotum size, mating success, and age at the time of the assay. 
description: # Consistent traffic noise impacts few fitness-related traits in a field
 cricket Journal: BMC Ecology and Evolution Authors: Gabrielle T Welsh,
 Sophia C Anner, Mary L Westwood, Victoria Rockwell, Hannah O'Toole,
 Megan Holiday, Robin M Tinghitella Contact Gabrielle Welsh
 () for questions regarding analyses. ## Data
 description The assay data are located in the file title
 "20231027_F1_CompleteDataset.csv," and the survival data are
 located in the file "20231027_F1_SurvivalDataset.csv". The data
 analysis is located in the file titled
 "20231027_WelshAnner_Analysis.rmd" Here is the summary of column
 names and if there are NA values: ### 20231027\_F1\_CompleteDataset.csv *
 id: individual numerical ID for each cricket in the experiment * sex: sex
 of experimental treatments. F=female and M=male. * pronotum: width of the
 pronotum of each cricket (mm). There are NA values if the pronotum
 measurement wasn't recorded or there was a typo * adult_treatment:
 one of 4 acoustic treatments (silent, 50 dBA, 60 dBA, and 70 dBA) that
 crickets received as adults * juv_treatment: one of 4 acoustic treatments
 (silent, 50 dBA, 60 dBA, and 70 dBA) that crickets received as juveniles *
 trial_type: combination of juv_treatment:adult_treatment for each
 individual cricket * cohort: group of crickets placed into treatments at
 the same time. Crickets with the same letter were in the same cohort *
 box: numerical values for one of seven boxes into which 0-2 week old
 crickets were placed at the start of the experiment. There are NA values
 if the box number wasn't recorded * cohort_box: combination variable
 of the cohort letter and box number. There are NA values if the box number
 wasn't recorded * eclosion_upper: the date on which an individual
 cricket eclosed * pop_pair: population from which the stimulus cricket for
 mating trials came from. "MAF" indicates the stimulus cricket
 was another experimental cricket from the same treatment while any other
 population name indicates the stimulus cricket was from a general
 laboratory population. There are NA values if this wasn't recorded. *
 pair_virgin: 0 indicates the stimulus mating cricket was not a virgin
 while 1 indicates that it was. There are NA values if this wasn't
 recorded * pronotum_pair: width of pronotum of stimulus mating pair
 cricket (mm). There are NA values if this wasn't recorded. *
 num_fails: number of days a cricket did not mate before mating
 successfully. There are NA values if this wasn't recorded * mated: 0
 indicates the individual did not mate while 1 indicates that it did mate.
 There are NA values if this wasn't recorded * date_mating: date on
 which a cricket successfully mated. There are NA values if this
 wasn't recorded * age_mating: number of days between eclosion date
 and date of successful mating. There are NA values if this wasn't
 recorded * age_sv: number of days between eclosion date and date_sv. There
 are NA values if this wasn't recorded * date_sv: date on which sperm
 viability assay was performed * age_immunity: number of days between
 eclosion date and date_hem. There are NA values if this wasn't
 recorded * date_hem: date on which hemocyte assay was performed. There are
 NA values if this wasn't recorded * date_fil_out: date on which the
 filament was removed from the cricket. There are NA values if this
 wasn't recorded * dissector_initials: initials of the person who
 dissected the reproductive organs. All dissectors had unique initials.
 There are NA values if this wasn't recorded * age_dissection: number
 of days between eclosion date and date_weigh_w. There are NA values if
 this wasn't recorded * date_weigh_w: date on which cricket was
 dissected. There are NA values if this wasn't recorded * ovary_w:
 weight of ovaries (g). There are NA values for all males * testes_w:
 weight of testes (g). There are NA values for all females * smold_w:
 weight of spermatophore mold (g). There are NA values for all females *
 ag_w: weight of accessory glands (g). There are NA values for all females
 * end_egg_lay: date on which female cotton pads were collected after one
 week of egg laying. There are NA values for all males * date_f2_counted:
 date on which number of F2 offspring per female were counted. There are NA
 values for all males and for females if they didn't lay eggs. *
 num_f2: number of hatchlings that hatched i the 5 weeks after egg laying.
 There are NA values for all males and for females if they didn't lay
 eggs * hatch_date: date on which 0-2week old crickets were placed into
 treatments. There are NA values if this wasn't recorded *
 days_to_eclosion: number of days between hatch date and eclosion date.
 There are NA values if this wasn't recorded * days_F2_hatched: number
 of days (almost all were 35) during which F2 offspring were allowed to
 hatch. There are NA values for all males and for females if they
 didn't lay eggs * live: number of live sperm. There are NA values for
 all females * dead.y: number of dead sperm. There are NA values for all
 females * sperm_viability: ratio of live to dead sperm. There are NA
 values for all females * mean_white: mean proportion of the filament that
 was originally black from a mounted immune response. A larger mean_white
 value indicates a stronger immune response. There are NA values if this
 wasn't recorded * total_count_granulocytes: number of granulocytes
 counted in hemolymph sample of each individual. There are NA values if
 this wasn't recorded * total_count_plasmatocytes: number of
 plasmatocytes counted in hemolymph sample of each individual. There are NA
 values if this wasn't recorded * total_hems: total number of
 hemocytes (plasmatocytes and granulocytes) counted in hemolymph sample of
 each individual. There are NA values if this wasn't recorded ###
 20231027\_F1\_SurvivalDataset.csv * individual: individual id for all
 crickets originally placed in treatments, even those that died before
 reaching eclosion and receiving a formal id * cohort_start date: same as
 hatch_date above * dead_date: date the crickets died. There are NA values
 if cricket did not die until dissection * days_until_death: number of days
 from cohort start date to death date. There are NA values if cricket did
 not die until dissection * eclosion_date: same as eclosion_upper above *
 id: individual numerical ID for each cricket. There are NA values for
 crickets that died before reaching adulthood/eclosion