Inbreeding depression in a sexually selected weapon and the homologue in females

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.

Methods
Parental generation (Fp): 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 (Opuntia mesacantha) 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.

Parental pairs (Fp): 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 (1st instar nymphs do not feed). We selected the first 15 eggs from each of 32 females over a two-week period.

Founding generation nymphs (F0): Once the eggs hatched and nymphs reached the 2nd 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.

Founding generation adults (F0): 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 Fp 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 Parental pairs section (see also Allen et al., 2018 for more details). Within each block, we obtained 30 eggs per cross (new F1 families).

The inbred/outbred generation (F1):  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.

Insect measurements: 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.