Salty suitors: High larval sodium intake reduces adult survival and influences reproductive behavior in a lepidopteran herbivore

Vincent, Maggie 1 ; Salgado, Ana1 ; Santiago-Rosario, Luis 2 ; Harms, Kyle1

Research facility: Louisiana State University

Published Jul 02, 2025 on Dryad. https://doi.org/10.5061/dryad.vx0k6dk2g

Abstract

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.

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 >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 >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

Collection sites and colony maintenance

We collected C. lacinia 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 C. lacinia 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^rd-5^th 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 ad libitum Helianthus annuus L. (Asteraceae) or Ambrosia trifida (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. C. lacinia 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 H. annuus 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 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 Choice 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.

Sodium treatments

We potted seeds of domesticated common sunflower (H. annuus, Dwarf sunspot, Urban Farmer) 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(NO₃)₂, KNO₃, NH₄H₂PO₄, MgSO₄*7H₂0, MnCl₂*4H₂0, ZnSO₄*7H₂0, CuSO₄*5H₂0, MoO₃, FeSO₄*7H₂0, and H₂BO₃. 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^nd or 3^rd 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^nd or 3^rd 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).

Choice mating trials

We held the Choice trials in June 2023. In the Choice 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 cm³), 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 (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).

No Choice mating trials

We held the No Choice trials in August 2023. In the No 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 plant and kept as in the Choice trials. Trial males (n = 14) and trial females were not reused.

Reproductive output

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 H. annuus or A. trifida leaves ad libitum. 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.

Statistical analyses

All statistical analyses were performed in R Studio Version 4.2.0 (R Core Team, 2024), and we used the ggplot2 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_j is the Beta coefficient and ε_i is the error:

(latency to mate)_i = β_j(parental sodium treatment)_i + ε_i

(copulation duration)_i = β_j(parental sodium treatment)_i + ε_i

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)_i = β_j(parental sodium treatment)_i + ε_i

(number of adult offspring)_i = β_j(parental sodium treatment)_i + ε_i

(average pupation time)_i = β_j(parental sodium treatment)_i + ε_i

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 Anova function from the package “car” to create an analysis-of-variance table to analyze the resulting GLM (Fox & Weisberg, 2019).