DOI: 10.5061/dryad.573n5tbm2
title: Living with males leads to female physical injury in the leaf-footed cactus bug

description: Males in many species possess sexually selected weapons that they use to
 fight for mating opportunities. It is well established that male-male
 competition can lead to physical injuries for males. However, very few
 studies have looked at the physical consequences for conspecific females.
 We hypothesized that living with males in a species with male-male
 competition would result in female injury. Because larger female
 invertebrates typically have greater reproductive output, they have higher
 resource value for males and can elicit aggression and fighting. Thus, we
 further hypothesized that larger females in this context would receive
 more injuries. For this study, we focused on the leaf-footed cactus
 bug, Narnia femorata (Hemiptera: Coreidae), a species of
 insect in which males fight using their spiny and enlarged hindlegs. In
 just two hours of observation, we documented males competing with other
 males in 61% of 103 trials. In 43% of these 63 competitions, females were
 physically contacted and sometimes attacked with a kick or squeeze. We
 left insects in social groups for 74 hours and found that females living
 with multiple males had a higher likelihood of obtaining injuries (26.2%
 of 103 trials) compared to those living only with females (9.7% of 103
 trials). In addition, larger females were more likely to be injured
 compared to smaller females. Our study highlights the harm that females
 can experience in species with male-male competition.
description: <strong>Insect rearing</strong> We
 used <em>Narnia femorata</em> (Stål, 1892) (Hemiptera:
 Coreidae) from a lab colony established in 2022 and supplemented in 2023
 with wild-caught individuals from Live Oak, Florida (30.2642 N°, 83.1768°
 W). All the insects were kept in a rearing room with temperatures ranging
 from 26–28 °C. We reared nymphs in plastic deli cups capped with mesh
 lids. We kept the density at 5-20 siblings per cup since nymphs are
 usually found in groups in nature (Allen and Miller 2020). We also
 provided each cup with a prickly pear cactus pad (<em>Opuntia
 mesacantha </em><em>ssp</em><em>.
 </em><em>l</em><em>ata</em>) planted in soil
 with an adjacent cactus fruit. We monitored the quality of cacti and
 cactus fruit throughout the project and moved bugs to a new cup if cactus
 quality declined. When the nymphs emerged into their
 penultimate instar, we separated each one into an individual deli cup with
 a planted cactus pad and a fruit. They were kept in deli cups by
 themselves to minimize social interactions and to ensure the females
 remained unmated. Once an individual emerged as an adult, we assigned it a
 unique code and marked it using a non-toxic paint pen. All adults were
 marked at least one day before the experiments started to minimize any
 effects of this disruption during the experimental period.
 <strong>Treatments</strong> There
 were two treatments, each with 103 replicates: a 1) female-only treatment
 and a 2) mixed-sex treatment. Each group of the female-only treatment
 consisted of one focal female and three background females; each group of
 the mixed-sex treatment consisted of one focal female and three males. For
 the mixed-sex treatment, we mimicked an operational sex ratio where more
 males are seeking matings than are females, a situation likely common in
 nature (Kokko et al. 2014). The distribution of these insects in the wild
 suggests the formation of hidden leks (<em>sensu </em>Fletcher
 & Miller 2006), and it is also not uncommon in the leaf-footed bug
 superfamily to find multiple males on a host plant as they vie for a
 coveted territory (Miyatake 1995). Not considering reproductive state or
 social groupings, the primary sex ratio of <em>N.
 femorata</em> in the wild is typically 1:1 (Cirino and Miller 2017).
 Females mate multiply, and work on other hemipteran insects suggests that
 last-male sperm precedence is common (Parker 1970; Gwynne 1984).
 We assigned insects randomly to a treatment group. Some unmated
 females were used as background females in two different replicates of the
 female-only treatment. Each male was used in the mixed-sex treatment up to
 five times. We visually inspected each focal insect before assigning it to
 a treatment and did not include those with obvious physical
 damage. <strong>Experimental
 Design</strong> There were two parts of the
 experiment: a two-hour observation period followed by a 72-hour
 non-observation period. During the observation period, we conducted
 behavioral trials at approximately 29 °C, noting their fighting and
 copulating behaviors. Before each trial, we allowed selected insects to
 acclimate to the environment in mesh-covered plastic cups alone for around
 30-60 minutes before putting them into their assigned group. All insects
 were gently moved into their assigned deli cup with a cactus and a ripened
 fruit. We also placed pine needles into the container, which females used
 as their main egg-laying substrate. Each observation
 period lasted for 2 hours, and we watched up to 9 groups at a time. It was
 feasible to track behaviors for multiple groups at the same time because
 these insects interact only intermittently and sometimes appear to ignore
 each other completely (Nolen et al. 2017). We defined male-male
 competition (or male contest) as one or more behaviors that include the
 following: leg displaying, charging, mounting, kicking, or squeezing. We
 defined these behaviors as in the ethogram (Table S1) modified from Nolen
 et al. (2017). We conducted observation on these behaviors for both
 mixed-sex and female-only treatments. The squeeze attack, where males
 squeeze each other using their spiny hindlegs, and often end-to-end, is
 part of male-male competition, and is the behavior most likely to cause
 injury because of the sharp spines and the force generated as the tibia
 and femur of one or both legs are squeezed together. In fact, squeezing
 can be felt as a pinch and can even draw blood when some leaf-footed bugs
 are handled by humans (Miller et al. 2024). Thus, we paid special
 attention to hindleg squeezes in one of our statistical analyses.
 Copulation was noted when a male mounted a female, initiated genital
 contact, and females opened their genital plates to allow copulation.
 Copulation was only considered successful when a male subsequently
 dismounted and rotated outward to form an obtuse angle with the genitalia
 still connected. We documented the number of copulating pairs that were
 contacted by rivals and whether a kick or squeeze occurred.
 After the two-hour observation period, we moved all the groups
 back to the 26-28 °C rearing room for another 72 hours. We provided this
 extended period to allow insects to interact further and females in the
 mixed-sex treatment to lay eggs so that we could quantify their
 reproductive output. Females in the female-only treatment were always
 unmated. After this non-observation period, we froze the focal female in
 each group for both treatments for later measurement and bodily injury
 scoring. <strong>Injury
 Scoring</strong> We scored female injury blind to
 treatment using a Leica M165C stereo microscope. We used ImageJ v.1.54d
 (Schneider et al. 2012) to measure pronotum width as a proxy for body size
 (Allen and Miller 2017). We counted and categorized observed physical
 injuries as follows: (1) antennal injury, including broken antennae and
 missing setae; (2) leg injury, including broken spines, melanization,
 exoskeletal punctures, and missing legs; and (3) wing injury, including
 wing tears and wing fragmentation. We noted evidence of melanization
 because it suggests wound healing in insects (Gillespie et al.
 1997). <strong>Offspring
 production</strong> We assessed one potential
 negative consequence of injury – a decrease in the production of
 offspring. We only collected these reproductive output measurements for
 the mixed-sex treatment groups because females remained unmated in the
 female-only groups. We assessed the number of live offspring produced from
 eggs laid during the 74-hour experimental period. We counted the number of
 hatchlings based on the number of opened pseudo-opercula on Day 14 after
 the experiment. Hatchling counts were collected by two people, blind to
 each other’s counts, and we used the average of these two
 numbers. <strong>Statistical
 Analyses</strong> All statistical analyses were
 conducted using IBM SPSS v.28. We first tested the consequence of the
 mixed-sex versus the female-only treatment on female injury after the
 74-hour experimental period. We used a Generalized Linear Model (GLM)
 assuming a binominal distribution with female injury (presence or absence)
 as a binary response variable. The explanatory variable was social group
 treatment (mixed-sex or female-only), which was considered as the main
 effect. We used female body size as a continuous covariate. In this and in
 all models, we first included two-way interaction. We <em>a
 </em><em>priori </em>decided to use the stepwise
 elimination procedure (Hardy and Field 1998), where non-significant
 interactions are removed. We conservatively decided <em>a priori
 </em>that only interactions with <em>p </em>&gt;
 0.10 would be removed. Next, we examined whether an
 observed aggressive squeeze attack on a female was predictive of female
 injury at the end of the 74 hours. We constructed a binomial GLM with
 female injury (presence or absence) as the binary response variable. Our
 model tested if an observed squeeze attack on a female predicted female
 injury at the end of the study. This GLM included female body size as a
 continuous covariate. Finally, we examined the effects
 of female body size and injury on offspring production. The hatchling
 count data was over-dispersed, thus we ran a GLM fit to the negative
 binomial distribution. Our model included the number of hatchlings as the
 response variable and both female injury (presence or absence) and female
 body size as explanatory variables.
description: # Living with males leads to female physical injury in the leaf-footed
 cactus bug Dataset DOI: [10.5061/dryad.573n5tbm2](10.5061/dryad.573n5tbm2)
 ## Description of the data and file structure This dataset contains the
 data for our experimental study investigating the effect of living with
 multiple males on female physical injury in a species with male-male
 competition. We used *Narnia femorata* (Hemiptera: Coreidae), the
 leaf-footed cactus bug, as our model system. The data cover 103 groups for
 two treatments: 1) female-only treatment, and 2) mixed-sex treatment.
 Through the experiment, we documented male competing behavior in 61% of
 the mixed-sex treatment. In 43% of these 63 competitions, females were
 physically contacted and sometimes attacked with a kick or squeeze. For
 the 74 hours that we left the insects in their social groups, we found
 that females living with multiple males were more likely to obtain
 injuries (26.2% of 103 trials) compared to those living only with females
 (9.7% of 103 trials). In addition, larger females were more likely to be
 injured compared to smaller females. In the first sheet within the
 dataset, we included information about treatment, individual insect ID and
 body size, incidences of different types of interactions, and female
 reproductive outputs. We also documented different types of injuries found
 on each female, which is recorded in the second sheet within the dataset.
 We used the third sheet to explain our variables and abbreviations used in
 the entire dataset. ### Files and variables #### File:
 Manuscript\_Data.xlsx **Description:** This file contains all the data we
 used for statistical analyses. The "Data" sheet includes metrics
 for insect social interactions, insect body size, offspring production for
 each treatment. The "Injury Type" sheet contains female physical
 injury details including number of injuries and injury types. The
 "Read Me" sheet contains explanation of variables and
 abbreviations used in the entire dataset. ##### Variables | Spreadsheet |
 Legend | Description | | :---------- | :---------------- |
 :-------------------------------------------------------------------------------------------------------------------------------------------------------------- | | Data | Treatment | M=Mixes-sex treatment, F=Female-only treatment | | Data | ID | Insect sample ID | | Data | PronotumWidth(mm) | Female pronotum width used as proxy for body size | | Data | #Squeeze | Number of direct squeeze a female received from male(s) | | Data | #Injury(total) | Total number of injury incurred on a female | | Data | MMC? | Whether male-male competition occurred in a group; Yes=male-male competition was observed, NA=male-male competition was not observed; MMC=male-male competition | | Data | #MPH(A) | Number of attack a mating pair received; MPH=mating pair harassment | | Data | #MPH(C) | Number of contact without combat a mating pair received | | Data | #mates | Number of mates a female had during the 2-hour observation | | Data | EggCount | Number of egg observed | | Data | HatchlingCount | Number of hatchling observed | | Injury Type | Treatment | M=Mixes-sex treatment, F=Female-only treatment | | Injury Type | ID | Insect sample ID | | Injury Type | #Injury | Number of injury incurred on a female | | Injury Type | InjuryType | MS=missing Setae | | Injury Type | InjuryType | BA=broken antennae; BS=broken spine; MEL=melanization; EP=exoskeletal puncture; ML=missing leg; WT=wing tear; WF=wing fragmentation | * In the "Data" sheet, we denoted "NA" (not applicable) to all Female-only treatment for the following columns: "MMC?", "#MPH(A)", "#MPH(C)", "#mates", "EggCount", and "HatchlingCount." This is because there were no males in this treatment, and therefore, there were no male-female interaction and all females remained unmated throughout the experiment. ## Code/software We used IBM SPSS v.28 to conduct all statistical analyses.