DOI: 10.5061/dryad.2ngf1vhvw
title: Inbreeding depression in a sexually selected weapon and the homologue in females

description: Theory predicts that traits with heightened condition-dependence, such as
 sexually selected traits, should be affected by inbreeding to a greater
 degree than other traits. The presence of environmental stress may
 compound the negative consequences of inbreeding depression. In this study
 we examined inbreeding depression across multiple traits and whether it
 increased with a known form of environmental stress. We conducted our
 experiment using both sexes of the sexually dimorphic leaf-footed cactus
 bug, Narnia femorata (Hemiptera: Coreidae). Adult male cactus bugs have
 enlarged hind legs used as weapons in male-male contests; these traits,
 and their homologue in females, have been previously found to exhibit high
 condition-dependence. In this study, we employed small developmental group
 size as an environmental stress challenge. Nymph N. femorata aggregate
 throughout their juvenile stages, and previous work has shown the negative
 effects of small group size on survivorship and body size. We found
 evidence of inbreeding depression for survival and seven out of the eight
 morphological traits measured, in both sexes. Inbreeding depression was
 higher for the size of the male weapon and the female homolog.
 Additionally, small developmental group size negatively affected survival
 to adulthood. However, small group size did not magnify the effects of
 inbreeding on morphological traits. These findings support the hypothesis
 that traits with heightened condition-dependence exhibit higher levels of
 inbreeding depression.
description: <strong>Parental generation
 (<em>F<sub>p</sub></em>)</strong>: Fifth
 instar nymphs were collected from the Ordway-Swisher Biological Station
 (Melrose, FL) in September 2014. Individual nymphs were kept in plastic
 deli containers, with topsoil, and a cactus pad
 (<em>Opuntia</em> <em>mesacantha</em>) with ripe
 fruit attached to it. This rearing protocol works for all life stages in
 this species (Allen et al., 2018). After nymphs molted into adults and
 reached sexual maturity (two weeks after molting), we randomly paired
 males and females. <strong>Parental pairs
 (<em>F<sub>p</sub></em>):</strong>
 Male-female pairs were allowed to mate freely, and ripe fruit was provided
 (one per cup). After eggs were laid, we removed them and placed them in a
 new container with cactus and no fruit (1<sup>st</sup> instar
 nymphs do not feed). We selected the first 15 eggs from each of 32 females
 over a two-week period. <strong>Founding
 generation nymphs
 (<em>F<sub>0</sub></em>):</strong> Once the
 eggs hatched and nymphs reached the 2<sup>nd</sup> instar,
 each female’s nymphs (15) were split equally into three cups. We supplied
 two ripe fruit per cup. After nymphs molted into adults and reached sexual
 maturity (two weeks after molting) we paired them as described
 below. <strong>Founding generation adults
 (<em>F<sub>0</sub></em>):</strong> To create
 inbred and outbred lines we used the ‘block’ design proposed by Roff
 (Roff,1998; Fox & Reed, 2011; Vega-Trejo et al., 2015; Joseph et
 al., 2016; Marsh et al., 2017; Hooper & Bonduriansky 2022). A
 block consisted of two (A + B) randomly paired families from the 32
 F<sub>p</sub> pairs we had available, therefore creating 16
 blocks. From each family we haphazardly chose two females and two males;
 we crossed offspring from the two families to create two outbred lines
 (♀A/♂B & ♀B/♂A) and crossed offspring within each family to create
 two inbred lines (♀A/♂A & ♀B/♂B). This design ensured that inbred
 and outbred families were created from the same set of alleles providing
 an advantage over other experimental designs (Fox, 2005). Male-female
 pairs were allowed to mate freely, and ripe fruit was provided as needed.
 Eggs from all the pairs were extracted and set up separately, using the
 same methodology as in the <em>Parental pairs </em>section
 (see also Allen et al., 2018 for more details). Within each block, we
 obtained 30 eggs per cross (new F<sub>1</sub>
 families). <strong>The inbred/outbred generation
 (<em>F1</em>):</strong>  From each block (N=16) we
 obtained 60 inbred nymphs (30/cross) and 60 outbred nymphs (30/cross).
 After molting to the 2nd instar, nymphs from each cross were separated
 equally into 2-nymph (5 cups) and 10-nymph groups (2 cups). Two-nymph
 groups got one fruit/cup and 10-nymph groups five fruits/cup. After
 individuals molted into adults and reached sexual maturity (two weeks
 after molting) they were frozen. <strong>Insect
 measurements</strong>: The measuring protocol follows the same
 methodology used by Procter et al. (2012). A digital camera (Canon EOS
 50D) attached to a dissecting microscope (Leica M165 C) was used to
 photograph all the extremities and body; the software ImageJ (Rasband,
 2011) was used for the linear and area measurements. The morphological
 traits chosen were beak length (=rostrum [mouthparts]), head length,
 pronotum width, front femur length, hind femur length, and hind femur
 width. Legs and body were separated to facilitate the measuring procedure.
 Area measurements were taken of the hind tibia and femur, as these are the
 most enlarged traits part of the male weapon, and the most
 condition-dependent traits (Allen & Miller, 2017). We used
 pronotum width as a proxy for body size; in this species, it is highly
 correlated with overall body size (Gillespie et al., 2014; Miller et al.,
 2016; Allen & Miller, 2017). Where applicable, means of paired
 body parts were used for analyses. For the purposes of this study, all
 male hind leg traits were considered to be sexually selected traits
 because of their involvement in signaling and fighting (Procter et al.,
 2012; Nolen et al., 2017); in contrast, female hind leg traits were
 referred to as the female homologous traits.
description: # Inbreeding depression in a sexually selected weapon and the homologue in
 females
 [https://doi.org/10.5061/dryad.2ngf1vhvw](https://doi.org/10.5061/dryad.2ngf1vhvw) In this study, we aimed to determine the effects of inbreeding and environmental stress (in the form of reduced group size during development) on traits with heightened condition-dependence versus other morphological traits. For this purpose, we used a true bug (Hemiptera) from the family Coreidae, *Narnia femorata*. As all hemipterans do, this insect has a nymphal juvenile stage in which small instars (tiny versions of adult) hatch out of the egg and then growth through molting processes between instars until they reach the winged adult stage. The data presented here are the morphological measurements for all the adult insects used in this study, all 560 insects for which we collected all 8 morphological measurements, all measurements are in **millimeters**. The experimental treatments represent the environment that the nymphs experienced from the 2<sup>nd</sup> instar until they became adults, and their exoskeleton became fully sclerotized (hardened). #### DATA\_inb560 Here are the column descriptions, all the **hind leg traits**\* are part of the male weapon or the female homologue, and they all display higher levels of condition-dependence that the other morphological trait.   Here are the column descriptions: | Data column title | Description | | ----------------- | ----------- | | Rep | treatment repetition within the same treatment combination in the same block (e.g., inbred/. Goes from 1-5 in 2-nymph treatments, and from 1-2 in the 10-nymph treatments. Each individual Rep represents a unique rearing cup. | | Num | Unique bug number assigned to each adult insect used in the experiment | | Sex | F – female or M – male | | Genetic | inbred or outbred, genetic treatment | | Block | Unique block designation, from A through P. Missing blocks E and K due to small sample sizes. | | Family | family number within each block, from 1 to 4, 1 and 2 are the inbred families, and 3 and 4 are the outbred families. | | Den | 2 or 10 nymphs, group size during juvenile development, density treatment. Low density is considered a more stressful environment as survival is lower in this treatment across different experiments, including this one. | | **HFL\*** | **hind femur length** | | **HFW\*** | **hind femur width** | | **HTA\*** | **hind tibia area** | | FFL | front femur length | | HL | head length | | BL | beak length | | **HFA\*** | **hind femur area** | | PW | pronotum width – proxy for overall body size | The 8 treatment combinations used in this experiment are as follow: 1.     Female inbred/2-nymph 2.     Female inbred/10-nymph 3.     Female outbred/2-nymph 4.     Female outbred/10-nymph 5.     Male inbred/2-nymph 6.     Male inbred/10-nymph 7.     male outbred/2-nymph 8.     Male outbred/10-nymph #### Pictures\_of\_all\_the\_measured\_insectsX Within the *Pictures of all the measured insects* folder, you will find 18 folders. Each folder corresponds to a single picture-taking bout by the technician. The name of the folder is the date in 2015 when the pictures were taken, e.g., APRIL\_10(8) = pictures taken April 10th 2015, and this folder includes the pictures for (8) individual insects. Some days include 2 folders, that just means pictures were taken in two different bouts on the same day. In each folder, you will find 4 pictures per insect (A-D; see key below), and an additional picture of the ruler (one per folder) used to calibrate the measuring software, ImageJ. This is the reason why you must use this folder structure to measure the insects, as the ruler only works to measure the insects contained in the same folder. The pictures are labelled as follows: Example: *621\_M\_O\_N\_4\_2\_2\_A* Formula: *Num\_Sex\_Genetic\_Block\_Family\_Den\_Rep\_picture type* * Num = Unique bug number * Sex = F or M * Genetic = inbred or outbred * Block = Unique block designation * Family = family number within each block * Den = 2 or 10 nymphs * Rep = treatment repetition, same family * Picture type = A – hind legs, B – front legs, C – insect body top side, D – insect body underside In the DATA\_inb560.csv file, you will easily find the corresponding measurements by using the Unique bug number (Num). All the pictures taken are included here but we only used those for 560 insects, those for which we could obtain all 8 measurements.