Nutrition affects larval survival and the development of morphological traits in male and female flour beetles, but genital size and shape remain canalized

The caloric content and macronutrient ratio of the diet consumed is a major source of phenotypic variation in most animal populations. While these nutritional effects have been well-documented for a variety of life-history and morphological traits, the effects of nutrition on male genitals are poorly understood but genitals are thought to be more canalised than general morphology and hence less susceptible to variation in nutrition. Even less is known about the effects of nutrition on female genital form, which to our knowledge, have never been investigated. Here we tested for the effects of juvenile dietary macronutrients (protein and carbohydrate) on larval survival, and adult morphology, including genital size and shape in male and female flour beetles (Tribolium castaneum). We found there was nutritionally induced plasticity in larval survival and morphology, although the latter effect was variable, with body size being most responsive to dietary macronutrients and genital size and shape being least responsive. Functionally equivalent morphological traits in the sexes responded similarly to nutrition. Previously, we showed that the genitalia of male and female T. castaneum are subject to strong stabilizing sexual selection, and our current findings suggest that developmental mechanisms reduce the nutritional sensitivity of male and female genitals, possibly to ensure matching during mating.

Methods
The size of the male and female beetles was a simple, linear measurement of body and elytra size. The size and shape (4 relative warps) of male and female genitalia were extracted using geometric morphometrics. The data was z-score transformed before analysis and the details of the analysis are provided in the manuscript. The survival data had a binomial distribution, so a binary GLMM fitted in the MCMCglmm package of R (version 3.1.2; Hadfield 2010) to test the statistical significance of these coefficients. The full details for these analyses are provided in the Supplementary Info, Text S2.

The genitalia were dissected using a dissecting microscope (Leica125, Leica Microsystems Ltd., Bletchley, UK), and an image was captured using a digital microscope camera (Pixelink M20-CYL). These images are unavailable (the student completed her studies in 2016 and has employment elsewhere). The details for the morphometrics analysis are provided in the manuscript but we also include 1) the links to the SB Morphometrics website (see below) and the co-ordinate data for each male and female genital image. To open these files (.NTS files), go to the SB Morphometrics website. In "Software:" -> "Thin-plate spline" -> "Relative warps analysis" -> download the program tpsRelw32 program. Within tpsRelw32 open the Data - NGFemAllDiets OR NGMaleAllDiets and in 'File' select 'Open sliders file' - these are notepads that specify which landmarks are fixed or sliding landmarks. Compute the "Consensus", "Partial warps" and "Relative warps". Finally, visualize the "Consensus" shape and "Relative warps" in the "Display"