DOI: 10.5061/dryad.vx0k6dk2g
title: Salty suitors: High larval sodium intake reduces adult survival and influences reproductive behavior in a lepidopteran herbivore

description: Sodium is a vital element for animals, essential for physiological
 function and individual-level development and behavior. When animals are
 sodium-limited, they often engage in behaviors such as cannibalism, or
 visiting salt licks to obtain sodium. Even so, how variation in sodium
 availability at larval stages influences mating behavior and reproductive
 output has received relatively little attention and those of Lepidoptera
 report conflicting results. We used the bordered patch butterfly, Chlosyne
 lacinia (Geyer, 1837; Nymphalidae), to investigate the role of larval
 sodium uptake in adult mating behavior, reproductive success, and
 lifespan. Males and females reared on host plants with higher sodium
 concentrations mostly did not pair differently than individuals reared on
 host plants with lower sodium concentrations and did not differ in their
 immediate reproductive success. However, we observed that females reared
 on High-Sodium host plants avoided mating with males reared on High-Sodium
 host plants, suggesting the possibility that larval sodium acquisition
 could influence adult choices. High-sodium individuals had shorter
 lifespans compared to Low-Sodium individuals, further adding to the
 evidence that sodium in high concentrations can be detrimental to C.
 lacinia. There are many opportunities throughout the mating and
 reproductive processes for individuals to exercise choice or to influence
 the decisions of potential mates, and to maximize their reproductive
 success. In addition to the behavior of conspecifics, environmental
 variation may play a role in how these individuals make these decisions
 throughout mating.
description: <em>Collection sites and colony
 maintenance</em> We collected <em>C.
 lacinia </em>caterpillars on two separate occasions, referred to as
 “Colony 1” and “Colony 2” for two behavioral experiments (see below
 regarding Choice and No Choice trials). Colony 1 was used for the Choice
 trials and Colony 2 was used for the No Choice trials. We collected
 <em>C. lacinia </em>caterpillars for Colony 1 from eight
 different locations in southern Texas in April 2023 (Fig. 1A; Table S1).
 Colony 2 caterpillars were collected from 12 locations in southern Texas
 in June 2023 (Fig. 1A, Table S1). Each location was labeled as a distinct
 population, and we maintained each population separately. We collected
 3<sup>rd</sup>-5<sup>th</sup> instar larvae (Fig.
 1B) to minimize the development time individuals would spend in the
 laboratory. We kept each population in a jar (16.5 cm height, 8.5
 diameter). We maintained caterpillars in their respective jars, feeding
 them <em>ad libitum</em> <em>Helianthus annuus
 </em>L. (Asteraceae) or <em>Ambrosia trifida </em>(L.,
 1753; Asteraceae) leaves sourced locally in Louisiana. We checked the jars
 daily, adding more food and removing frass as needed. We separated any
 pupating individual into its own smaller individual container (5.5 oz),
 with filter paper in the bottom and subsequently checked daily for
 eclosion. Upon eclosing, we sexed the individual. Although there is some
 size dimorphism, there is substantial overlap and accurate sexing requires
 viewing the external genitalia. <em>C. lacinia</em> can be
 sexed by their external genitalia; females have a genital pore, or ostium,
 visible near the ventral tip of the abdomen (Fig. 1C). Males have smaller
 abdomens, and each has a visible valve (Fig. 1D). Once sexed, we numbered
 individuals on the lower left wing with a fine-tipped permanent marker
 (Fig. 1E). We segregated these F0 males and females into same-sex
 butterfly cages (40 x 40 x 60 cm) to prevent unplanned mating between
 closely related individuals. We brought males and females from different
 populations together to mate F0 individuals to avoid inbred lines. After
 mating, each female was placed in a cage with an <em>H.
 annuus</em> as a potential oviposition substrate. These planned
 matings resulted in unique lineages, each of which was given a letter ID
 (Table S1) so that we could ensure that siblings were not placed together
 for any of the mating trials. The egg clutches from these matings were the
 F1 offspring used in the experimental trials. For the
 Choice<em> </em>trials' F1 egg clutches, each clutch was
 kept in a mesh-covered glass jar with a piece of filter paper that we
 moistened daily. When a clutch hatched, we moved it into a larger
 caterpillar jar. We treated No<em> </em>Choice<em>
 </em>trial clutches the same, except that we left each egg clutch on
 the plant where the female originally oviposited, because we learned from
 the Choice trials that we had difficulties maintaining optimal moisture
 levels in the jars. Once the No Choice clutches hatched, they were also
 moved into a larger caterpillar jar. These F1 individuals were used in the
 sodium treatments so that we could control diets during their entire
 larval development. <em>Sodium
 treatments</em> We potted seeds of domesticated
 common sunflower<em> </em>(<em>H. annuus</em>,
 Dwarf sunspot, Urban Farmer)<em> </em>and grew them in
 hydroponics following the protocol from Santiago-Rosario et al. (2023)
 using a Hoagland solution containing either no added sodium (0% added
 NaCl, i.e., Low-Sodium treatment) or 1.0% added NaCl (High-Sodium
 treatment). The Hoagland solution contained
 Ca(NO<sub>3</sub>)<sub>2</sub>,
 KNO<sub>3</sub>,
 NH<sub>4</sub>H<sub>2</sub>PO<sub>4</sub>, MgSO<sub>4</sub>*7H<sub>2</sub>0, MnCl<sub>2</sub>*4H<sub>2</sub>0, ZnSO<sub>4</sub>*7H<sub>2</sub>0, CuSO<sub>4</sub>*5H<sub>2</sub>0, MoO<sub>3</sub>, FeSO<sub>4</sub>*7H<sub>2</sub>0, and H<sub>2</sub>BO<sub>3</sub>. We grew the plants with a light regime of 16L:8D, with a temperature of 23.3 ⁰C and relative humidity of 60 ± 10%. We checked the plants daily and kept the water level constant. After two weeks, the old solution was removed, and new Hoagland solution (and NaCl for the High-Sodium treatment) was added to maintain the correct sodium concentrations for the plants. We grew a separate set of plants without added sodium for oviposition by test-subject butterflies during their behavioral trials. A set of 100 host plants were sown in nutrient-rich soil (Clegg’s Nursery, Louisiana) in 2 L pots and kept in natural, well-watered conditions. Prior to the 2<sup>nd</sup> or 3<sup>rd</sup> instar, their small size makes it difficult to move individuals without injuring or killing them (Santiago-Rosario et al., 2023). Once an F1 caterpillar reached the 2<sup>nd</sup> or 3<sup>rd</sup> instar, we placed it into an individual treatment of either feeding on Low-Sodium host plants or High-Sodium plants (LS or HS, respectively) in a 32-oz extruded polystyrene foam cup with a transparent lid and a hole in the lid's middle, covered with white mesh. Each cup contained a single caterpillar and a single host plant. Caterpillars were randomly assigned to treatments, and we alternated the placement of the treatment containers under the grow lights; every week the containers were randomly moved to avoid differences in the light conditions that could interact with or otherwise compromise our treatment differences. If a caterpillar consumed entirely its host plant, we replaced it with a new host plant under the same sodium treatment and continued to do so until pupation. We did not measure total consumption. Once an individual pupated, we followed the pupal-rearing protocol mentioned above. All individuals were maintained until dead. For the Choice colony we raised 100 individuals until adulthood (♀ LS n = 29, ♂ LS = 27, ♀ HS n = 17, ♂ HS = 27), and for the No Choice colony we raised 65 individuals as adults (♀ LS n = 19, ♂ LS = 18, ♀ HS n = 14, ♂ HS = 14). <em>Choice mating trials</em> We held the Choice trials in June 2023. In the Choice<em> </em>trials, a single female had equal access to one male of each treatment (15 LS and 9 HS females, for a total of n = 24 female-choice trials). For a single trial, we placed a LS and a HS male in the experimental cage (30.5 cm<sup>3</sup>), the female in a separate cage, and left the cages outdoors in shaded areas overnight. The next day, we moved the cages into the sun for 30-40 minutes before starting the trials between 8:30 to 9:00 AM. The average daytime temperature was 29.2⁰C and the average nighttime temperature was 23.4⁰C (Climate Report, 2023a). Once we added a female to an experimental cage (containing one of each type of male), we started timing. We recorded the identity of the male that succeeded in copulating, the time before a pair started copulating (latency to mate), and the duration of copulation. After a completed copulation, we moved the female into a larger oviposition cage (40 x 40 x 60 cm) with one sunflower host plant<em> </em>(not in hydroponics). Mated females were given a 20% sugar solution, which was replenished every day and kept in these cages for oviposition until natural death (date recorded when possible). We checked for new egg clutches daily and when found they were numbered. We did not reuse trial males (48 males total). <em>No Choice mating trials</em> We held the No<em> </em>Choice trials in August 2023. In the No<em> </em>Choice trials, a single female had access to a single male (8 LS and 6 HS females and males, for a total of n = 14 female-no-choice trials). Half of the High-Sodium females were assigned High-Sodium males, and the other half were assigned Low-Sodium males, and the same for the Low-Sodium females. For a given trial, we kept the male and female in separate butterfly cages and left them outside overnight in shaded locations to acclimate. We moved the cages the next day into place in partially sunny locations 30-40 minutes before starting the trials (average day temperature of 32.3⁰C and average night temperature of 25.9⁰C, Climate Report, 2023b). We started timing a trial once the female was added to the single-male experimental cage. We counted male abdominal bends toward the female in the first ten minutes, the latency to mate, and the duration of copulation. After a completed copulation, females were moved to an oviposition cage (40 x 40 x 60 cm) with a sunflower<em> </em>plant and kept as in the Choice trials. Trial males (n = 14) and trial females were not reused. <em>Reproductive output </em> We used the number of clutches, number of larvae hatched, and number of larvae surviving to adulthood of the F2 generation as fitness proxies. Number of larvae hatched was determined by photographing a freshly hatched clutch and then enlarging the image to count the number of larvae. We maintained the F2 caterpillars in the same manner as the colonies, keeping them in jars and feeding them <em>H. annuus </em>or <em>A. trifida</em> leaves <em>ad libitum</em>. Each pupa was kept in a separate container until eclosion, when it was frozen. We counted the number of pupae that successfully eclosed to determine the total number of adult offspring for each mated experimental F1 pair.<em> </em> <em>Statistical analyses</em> All statistical analyses were performed in R Studio Version 4.2.0 (R Core Team, 2024), and we used the <em>ggplot2</em> package (Wickham, 2016) for visualization. We ran a Chi-square test to determine if the chances of mating or not mating differed significantly between the Choice and No Choice trials, and another Chi-square test to analyze whether female mating outcomes in the Choice trials departed from what we would expect by chance. Because there was only one pairing between a High-Sodium female and a High-Sodium male, we had no direct estimate of inter-pair variance for this type of pairing and we removed it from the dataset for the following analyses. We ran Analyses of Variances (ANOVAs) to determine whether latency to mate and copulation duration differed among parental sodium pairings. Both variables were converted to seconds and time before copulation was log-transformed to achieve a more normal data distribution. The model notation for both ANOVAs is as follows, where β<sub>j</sub> is the Beta coefficient and ε<sub>i</sub> is the error: (latency to mate)<sub>i</sub> = β<sub>j</sub>(parental sodium treatment)<sub>i</sub> + ε<sub>i</sub> (copulation duration)<sub>i</sub> = β<sub>j</sub>(parental sodium treatment)<sub>i</sub> + ε<sub>i</sub> We ran ANOVAs to determine the effects of parental sodium treatments on the following reproductive variables: number of clutches, number of adult offspring, and the average pupation time. We log-transformed the number of adult offspring to obtain more normally distributed data, but the data for number of clutches, number of hatched offspring, and average pupation time were approximately normal. The statistical notation for the three models is as follows: (number of clutches)<sub>i</sub> = β<sub>j</sub>(parental sodium treatment)<sub>i</sub> + ε<sub>i</sub> (number of adult offspring)<sub>i</sub> = β<sub>j</sub>(parental sodium treatment)<sub>i</sub> + ε<sub>i</sub> (average pupation time)<sub>i</sub> = β<sub>j</sub>(parental sodium treatment)<sub>i</sub> + ε<sub>i</sub> For the subset of Choice F1 individuals with full eclosion and death data (LS = 44, HS = 37), we created survivorship curves in R using the packages “survival” and “survminer” (Therneau, 2024; Kassambara et al., 2021). We ran a generalized linear model (GLM) to determine whether sex or treatment independently influenced survival, as well as the interaction of sex and treatment on survival. We used the <em>Anova</em> function from the package “car” to create an analysis-of-variance table to analyze the resulting GLM (Fox & Weisberg, 2019).
description: # Salty suitors: High larval sodium intake reduces adult survival but does
 not affect reproductive behaviors in a lepidopteran herbivore
 [https://doi.org/10.5061/dryad.vx0k6dk2g](https://doi.org/10.5061/dryad.vx0k6dk2g) ## Description of the data and file structure Chlosyne lacinia 2023-2024 Project ReadMe This GitHub project corresponds to my second dissertation chapter regarding the effect of larval sodium access on adult mating behavior in C. lacinia. In this project folder, There are raw data files, cleaned up data files, a metadata document, and R scripts used to run my analyses. ### **Files and descriptions:** * 2023_choice_trials_2025_V_26.csv * 2023_choice_trials_subset_2025_V_26.csv: a subset of the “raw_data” file with only the Choice trial data * 2023_choice_trials_subset_HH_removed_2025_V_26.csv: the “2023_choice_trials_subset” with one entry removed, the single High-Sodium x High-Sodium pairing. This was used for analysis * 2024_4_2_modeling.Rmd: R script for the ANOVAs, using “2023_choice_trials_subset_HH_removed” and “choice_f2_fitness_data_HH_removed” * all_mate_no_mate_CL.csv: the mating vs. not mating outcomes for both the Choice and No Choice trials * c_lac_mapping.Rmd: The code to create a map of the sites in Texas (US) where we collected *C. lacinia* individuals in the summer of 2023. * chi_squared_code.Rmd: The R code used to run the Chi-Square test using chi_treatment_choice_numeric.csv * chi_treatment_choice_numeric_V_26.csv: the cleaned up choice data that has been formatted to run a Chi-Square test. This was to determine if female mating choices deviated from what we would expect by chance (random mating). * choice_f2_fitness_data_2025_V_26.csv: a subset of the raw data file with only the Choice trial fitness data * choice_f2_fitness_data_HH_removed_2025_V_2026.csv: the “choice_f2_fitness_data_2025_V_26.csv” with one entry removed, the single High-Sodium x High-Sodium pairing * colony_1_coordinates.csv: the latitude and longitude of the C. lacinia populations from Texas used in the Choice trials * colony_2_coordinates.csv: the latitude and longitude of the C. lacinia populations from Texas used in the No Choice trials * metadata.pdf: A document with all the necessary metadata for the experiments and data files * raw_data_choice_trials_2025_V_26.csv: the trial data from the Choice experiments; used Colony 1 * raw_data_colony1_f1_larvae.csv: the information on individuals for the F1 generation for Colony 1 * raw_data_colony2_exp_larvae_2025_V_26.csv: the information on experimental individuals for the F1 generation for Colony 2 * raw_data_colony2_f0_2025_V_26.csv: the information on individuals for the F0 generation of Colony 2 * raw_data_colony2_f1_2025_V_26.csv: the information on individuals for the F1 generation of Colony 2 * raw_data_f0_crosses_2025_V_26.csv: the parental information for the F0 crosses that resulted in F1 individuals for Colony 2 * raw_data_f0_individuals_2025_V_26.csv: the information on individuals for the F0 generation of Colony 1 * raw_data_no_choice_trials_2025_V_26.csv: the trial data from the No Choice experiment; used Colony 2 individuals ### **Data files and variables:** #### 2023\_choice\_trials\_2025\_V\_26.csv: * trial_number: the unique identifier (numeric) of the Choice trial * date: the date (DD-MM-YYYY) of the trial; month is in Roman Numerals * female: the unique identifier of the female in the trial * female_sodium: the larval sodium regime, Low or High, of the trial female * mating_male: the unique identifier of the mating male in the trial * mating_male_treatment: the larval sodium regime, Low or High, of the mating male * combination_treatment: the mating female and mating male sodium treatments; female is listed first * non-mating_male: the unique identifier of the male that did not mate during the trial * non-mating_male_treatment: the larval sodium regime, Low or High, of the male that did not mate * start_time: the start time, minutes and seconds, the trial began * mating_begins: the time, minutes and seconds, the pair began copulating * mating_ends: the time, minutes and seconds, the pair stopped copulating * time_before_copulation: the time between the start of the trial and the start of mating in minutes and seconds * copulation_duration: the time between the start and end of copulation in minutes and seconds * duration_rounded: the duration of copulation rounded to the nearest half minute * abdomen_bends_in_first_ten_minutes: the number of times a male flanked a female and bent his abdomen at a 90+ degree angle in the attempt to couple his genitalia with the female’s genitalia * oviposition_1: the date (DD-MM-YYYY) of the oviposition of clutch 1 * c1_size_(mm) : the size, in mm, of clutch 1; measured at its widest and longest points * c1_hatch: the data (DD-MM-YYYY) of the hatching of clutch 1 * c1_hatching_number: the number of larvae hatching from clutch 1 * oviposition_2: the date (DD-MM-YYYY) of the oviposition of clutch 2 * c2_size_(mm): the size, in mm, of clutch 2; measured at its widest and longest points * c2_hatch: the date (DD-MM-YYYY) of the hatching of clutch 2 * C2_hatch_num: the number of larvae hatching from clutch 2 * oviposition_3: the date (DD-MM-YYYY) of the oviposition of clutch 3 * c3_size_(mm) : the size, in mm, of clutch 3; measured at its widest and longest points * c3_hatch: the date (DD-MM-YYYY) of the hatching of clutch 3 * c3_hatch_num: the number of larvae hatching from clutch 3 * oviposition_4: the date (DD-MM-YYYY) of the oviposition of clutch 4 * c4_size_(mm): the size, in mm, of clutch 4; measured at its widest and longest points * c4_hatch: the date (DD-MM-YYYY) of the hatching of clutch 4 * c4_hatch_num: the number of larvae hatching from clutch 4 * oviposition_5: the date (DD-MM-YYYY) of the oviposition of clutch 5 * c5_size_(mm): the size, in mm, of clutch 5; measured at its widest and longest points * c5_hatch: the date (DD-MM-YYYY) of the hatching of clutch 5 * c5_hatch_num: the number of larvae hatching from clutch 5 Relevant acryonyms: NMO: no mating occurred DNE: did not eclose NA: missing/unavailable for that individual Additionally, not all females had multiple clutches. For example, if a female had 2 clutches, the cell for any clutch after will say “no additional oviposition.”   #### 2023\_choice\_trials\_subset\_2025\_V\_26.csv: * trial_number: the unique identifier (numeric) of the Choice trial * date: the date (DD-MM-YYYY) of the trial; month is in Roman Numerals * family: the lineage cross of the mating individuals; female lineage listed first * female: the unique identifier of the female in the trial * female_sodium: the larval sodium regime, Low or High, of the trial female * mating_male: the unique identifier of the mating male in the trial * mating_male_treatment: the larval sodium regime, Low or High, of the mating male * combination_treatment: the mating female and mating male sodium treatments; female is listed first * non-mating_male: the unique identifier of the male that did not mate during the trial * non-mating_male_treatment: the larval sodium regime, Low or High, of the male that did not mate * start_time: the start time, minutes and seconds, the trial began * mating_begins: the time, minutes and seconds, the pair began copulating * mating_ends: the time, minutes and seconds, the pair stopped copulating * time_before_copulation: the time between the start of the trial and the start of mating in minutes and seconds * time_before_seconds: the time between the start of the trial and the start of mating in seconds * copulation_duration: the time between the start and end of copulation in minutes and seconds * duration_seconds: the time between the start and end of copulation in seconds * duration_rounded: the duration of copulation rounded to the nearest half minute   #### 2023\_choice\_trials\_subset\_HH\_removed\_2025\_V\_26.csv: * trial_number: the unique identifier (numeric) of the Choice trial * date: the date (DD-MM-YYYY) of the trial; month is in Roman Numerals * family: the lineage cross of the mating individuals; female lineage listed first * female: the unique identifier of the female in the trial * female_sodium: the larval sodium regime, Low or High, of the trial female * mating_male: the unique identifier of the mating male in the trial * mating_male_treatment: the larval sodium regime, Low or High, of the mating male * combination_treatment: the mating female and mating male sodium treatments; female is listed first * non-mating_male: the unique identifier of the male that did not mate during the trial * non-mating_male_treatment: the larval sodium regime, Low or High, of the male that did not mate * start_time: the start time, minutes and seconds, the trial began * mating_begins: the time, minutes and seconds, the pair began copulating * mating_ends: the time, minutes and seconds, the pair stopped copulating * time_before_copulation: the time between the start of the trial and the start of mating in minutes and seconds * time_before_seconds: the time between the start of the trial and the start of mating in seconds * copulation_duration: the time between the start and end of copulation in minutes and seconds * duration_seconds: the time between the start and end of copulation in seconds * duration_rounded: the duration of copulation rounded to the nearest half minute   #### all\_mate\_no\_mate\_CL.csv: * outcome: the number of trials in which mating occurred or mating did not occur; numeric * choice: the results (mating or not mating) from the Choice trials * no_choice: the results (mating or not mating) from the No Choice trials   #### chi\_treatment\_choice\_numeric\_V\_26.csv: * treatment: the potential mating outcomes for female, male_high (High-Sodium male), male_low (Low-Sodium male), or no_mating (mating did not occur) * female_high: the number of each mating outcome for High-Sodium females * female_low: the number of each mating outcome for Low-Sodium females   #### choice\_f2\_fitness\_data\_2025\_V\_26.csv: * female: the unique identifier of the female in the trial * female_sodium: the larval sodium regime, Low or High, of the trial female * male_sodium: the larval sodium regime, Low or High, of the mating male * cross_type: the mating female and mating male sodium treatments; female is listed first * clutch_number: the number of clutches the female oviposited * hatch_number: the total number of larvae hatched from all the female’s clutches * adult_number: the total number of adult offspring from all the female’s clutches * average_pupation_time: average time from the start to the end of pupation for all pupae of the female * prop_surviving_to_adulthood: the total number of adult offspring divided by the number of hatching offspring   #### choice\_f2\_fitness\_data\_HH\_removed\_2025\_V\_2026.csv: * female: the unique identifier of the female in the trial * female_sodium: the larval sodium regime, Low or High, of the trial female * male_sodium: the larval sodium regime, Low or High, of the mating male * cross_type: the mating female and mating male sodium treatments; female is listed first * clutch_number: the number of clutches the female oviposited * hatch_number: the total number of larvae hatched from all the female’s clutches * adult_number: the total number of adult offspring from all the female’s clutches * average_pupation_time: average time from the start to the end of pupation for all pupae of the female * prop_surviving_to_adulthood: the total number of adult offspring divided by the number of hatching offspring   #### colony\_1\_coordinates.csv: * population_number: the unique identifier for each collection site; each collection site was &gt;20m apart * latitude: degrees latitude north * longitude: degrees longitude west   #### colony\_2\_coordinates.csv: * population_number: the unique identifier for each collection site; each collection site was &gt;20m apart * latitude: degrees latitude north * longitude: degrees longitude west   #### raw\_data\_choice\_trials\_2025\_V\_26.csv: * trial number: the unique identifier (numeric) of the Choice trial * date: the date (DD-MM-YYYY) of the trial; month is in Roman Numerals * female: the unique identifier of the female in the trial * sodium: the larval sodium regime, Low or High, of the trial female * mating male: the unique identifier (numeric) of the male that mated during the trial * mating male treatment: the larval sodium regime, Low or High, of the male that mated during the trial * non-mating male: the unique identifier (numeric) of the male that did not mate during the trial * non-mating male treatment: the larval sodium regime, Low or High, of the male that did not mate during the trial * start time: the time (in minutes and seconds) that the males and female were placed in the trial chamber * mating begins: the time (in minutes and seconds) that genital contact was made * mating ends: the time (in minutes and seconds) that genital contact stopped * time_before_copulation: the time between the start of the trial and the start of mating in minutes and seconds * copulation duration: the time between the start and end of genital contact in minutes and seconds * abdomen bends in first ten minutes: the number of times a male flanked a female and bent his abdomen at a 90+ degree angle in the attempt to couple his genitalia with the female’s genitalia * oviposition_1: the date (DD-MM-YYYY) of the oviposition of clutch 1 * c1_size_(mm) : the size, in mm, of clutch 1; measured at its widest and longest points * c1_hatch: the data (DD-MM-YYYY) of the hatching of clutch 1 * c1_hatching_number: the number of larvae hatching from clutch 1 * oviposition_2: the date (DD-MM-YYYY) of the oviposition of clutch 2 * c2_size_(mm): the size, in mm, of clutch 2; measured at its widest and longest points * c2_hatch: the date (DD-MM-YYYY) of the hatching of clutch 2 * C2_hatch_num: the number of larvae hatching from clutch 2 * oviposition_3: the date (DD-MM-YYYY) of the oviposition of clutch 3 * c3_size_(mm) : the size, in mm, of clutch 3; measured at its widest and longest points * c3_hatch: the date (DD-MM-YYYY) of the hatching of clutch 3 * c3_hatch_num: the number of larvae hatching from clutch 3 * oviposition_4: the date (DD-MM-YYYY) of the oviposition of clutch 4 * c4_size_(mm): the size, in mm, of clutch 4; measured at its widest and longest points * c4_hatch: the date (DD-MM-YYYY) of the hatching of clutch 4 * c4_hatch_num: the number of larvae hatching from clutch 4 * oviposition_5: the date (DD-MM-YYYY) of the oviposition of clutch 5 * c5_size_(mm): the size, in mm, of clutch 5; measured at its widest and longest points * c5_hatch: the date (DD-MM-YYYY) of the hatching of clutch 5 * c5_hatch_num: the number of larvae hatching from clutch 5   #### raw\_data\_colony1\_f1\_larvae.csv: * population ID: the unique identifier for the source population of the individual; used to reduce inbreeding * treatment type: High- or Low-sodium * treatment number: the unique identifier for the individual * treatment started: the date (DD-MM-YYYY) the larva was placed into its treatment jar * mortality: the date (DD-MM-YYYY) the individual died, if known; NA if not known * pupation: the date (DD-MM-YYYY) the larva entered pupation * eclosed: the date (DD-MM-YYYY) the pupae eclosed * sex: male or female, known after ecolsion * to_be_mated: the date (DD-MM-YYYY) an individual was planned to be mated; this was only used partially for planning and most matings were determined the day before and not entered here * mated (y/n): whether or not an individual was mated (Yes or No)   #### raw\_data\_colony2\_exp\_larvae\_2025\_V\_26.csv: * mother ID: the unique identifier of the mother of the larva * cross: the population cross of the parents; used to reduce inbreeding * population: the unique identifier for the source population of the individual; used to reduce inbreeding * treatment: Low- or High-Sodium * number: the unique identifier of the individual * pupation_date: the date (DD-MM-YYYY) the larva entered pupation * eclose_date: the date (DD-MM-YYYY) the pupae eclosed * mortality: the date (DD-MM-YYYY) the individual died, if known; NA if not known * sex: male or female, known after ecolsion * ready_to_mate: what date (DD-MM-YYYY) an individual would be sexually mature; males are ready to mate immediately following eclosion and females are ready 2-3 days after ecosion * notes: any additional information that would be helpful; DNE = did not eclose; says “none” if there were no additional notes   #### raw\_data\_colony2\_f0\_2025\_V\_26.csv: * ID: the unique identifier of the individual * populationNumber: the unique identifier for the source population of the individual; used to reduce inbreeding * Sex: male or female, known after eclosion * PupationDate: the date (DD-MM-YYYY) the larva entered pupation * EclosionDate: the date (DD-MM-YYYY) the pupae eclosed * MatingDate: the date (DD-MM-YYYY) the individual mated; “no” if mating did not occur * Mortality: the date (DD-MM-YYYY) the individual died, if known; NA if not known   #### raw\_data\_colony2\_f1\_2025\_V\_26.csv: * ID: the unique identifier of the individual * Population: the unique identifier for the source population of the individual; used to reduce inbreeding * pupation_date: the date (DD-MM-YYYY) the larva entered pupation * eclose_date: the date (DD-MM-YYYY) the pupae eclosed * sex: male or female, known after eclosion   #### raw\_data\_f0\_crosses\_2025\_V\_26.csv: * female_ID: unique identifier for the mating female * female_population: source population of the mating female * male_ID: unique identifier for the mating male * male_population: source population of the mating male * cross: resulting cross from the mating male and female * date: date (DD-MM-YYYY) the mating occurred   #### raw\_data\_f0\_individuals\_2025\_V\_26.csv: * ID: unique identifier of the individual * Population_number: source population of the individual, used to reduce inbreeding * Sex: male or female, known after eclosion * date_eclosed: the date (DD-MM-YYYY) of eclosion * Generation: the generation, F0 * Mate: whether or not an individual mated, date (DD-MM-YYYY) if known * Mortality: the date (DD-MM-YYYY) the individual died, if known; NA if not known * Notes: any additional notes that researchers thought might be helpful; “none” if there was nothing of note   #### raw\_data\_no\_choice\_trials\_2025\_V\_26.csv: * date: the date (DD-MM-YYYY) of the mating trial * female: the unique identifier of the trial female * male: the unique identifier of the trial male * cross: the population cross of the female and male; female listed first * start_time: the time (minutes and seconds) the male and female were placed in the trial chamber * copulation_start_time: the time (minutes and seconds) when genital coupling began * copulation_end_time: the time (minutes and seconds) when genital coupling ended * copulation_length: the time (minutes and seconds) between the start and end of genital coupling * abd_bends_first_10_minutes: the number of times a male flanked a female and bent his abdomen at a 90+ degree angle in the attempt to couple his genitalia with the female’s genitalia * copulation_(y/n): whether copulation occurred (y) or not (n) * oviposition_1_date: the date (DD-MM-YYYY) the clutch was laid * O1_size: the length and width (mm) of the clutch * O1_hatch_date: the date (DD-MM-YYYY) the clutch hatched * O1_hatch_number: the number of larvae hatched from the clutch ## Code/software All analyses were performed in R Studio Version 4.2.0 (R Core Team, 2024), and we used the *ggplot2* package (Wickham, 2016) for visualization. For our survival curves we used the packages “survival” and “survminer” (Therneau, 2024; Kassambara et al., 2021). To analyze our general linear models, we used the *Anova* function from the package “car” to create an analysis-of-variance table to analyze the resulting GLM (Fox & Weisberg, 2019). All code is uploaded in our files. ## Access information Other publicly accessible locations of the data: * The data is also available on Github at the following URL: [https://github.com/mvcarneal/clacinia](https://github.com/mvcarneal/clacinia)