DOI: 10.5061/dryad.f1vhhmgxf
title: Male reproductive effort might be evolving in the face of devastating disease in a threatened amphibian

description: The devastating infectious disease chytridiomycosis has caused declines of
 amphibians across the globe, yet some populations are persisting and even
 recovering. One understudied effect of wildlife disease is changes in
 reproductive effort. Here we aimed to understand if disease has plastic
 effects on reproduction and if reproductive effort could evolve with
 disease endemism. We compared the effects of experimental pathogen
 exposure (trait plasticity) and population-level disease history
 (evolution in trait baseline) on reproductive effort using gametogenesis
 as a proxy in the declining and endangered frog Litoria verreauxii alpina.
 We found that unexposed males from disease-endemic populations had higher
 reproductive effort, which is consistent with an evolutionary response to
 chytridiomycosis. We also found evidence of trait plasticity, where males
 and females were affected differently by infection: pathogen exposed males
 had higher reproductive effort (larger testes), whereas females had
 reduced reproductive effort (smaller and fewer developed eggs) regardless
 of the population of origin. Infectious disease can cause plastic changes
 in reproductive effort at an individual level, and population-level
 disease exposure can result in changes to baseline reproductive effort;
 therefore, individual- and population-level effects of disease should be
 considered when designing management and conservation programs for
 threatened and declining species. --
description: <b><i>Experimental
 design</i></b> We conducted a
 common garden experiment in which individuals from four populations (one
 disease-naïve: Grey Mare; and three<i> </i>disease-endemic:
 Eucumbene, Kiandra and Ogilvies) were collected as eggs from the wild,
 brought into captivity, and raised in a bio-secure, pathogen-free
 environment (Bataille et al. 2015; Grogan et al. 2018a,b,c). At
 approximately eight months after metamorphosis, we experimentally exposed
 the frogs to 750,000 <i>B. dendrobatidis </i>zoospores in 25mL
 of dilute salt solution for 18hrs, or mock exposed (exposed n=215,
 unexposed-controls n=39, see Table S1). Sample size between exposed and
 unexposed-control groups are not even because these animals were part of a
 larger clinical infection experiment (Bataille et al. 2015; Grogan et al.
 2018a,b,c). We tested the animals for <i>B. dendrobatidis
 </i>infection every 14 days using quantitative PCR until the end of
 the experiment (first signs of morbidity or day 86 after inoculation) (see
 Text S1 husbandry details). 
 <b><i>Sample fixation and processing
 </i></b> On the day that animals first
 showed clinical signs of infection (from day 19 onwards after inoculation)
 or at the end of the experiment if they remained healthy (day 86 after
 inoculation), we swabbed the animals for <i>B.
 </i><i>dendrobatidis </i>infection, weighed them to the
 nearest 0.01g, humanely euthanized them with buffered Tricaine
 Methanesulfonate (MS-222), and fixed them in neutral buffered formalin. We
 used mass as a proxy for animal size (Text S1), and measured mass on the
 day of experimental pathogen exposure and on the day of euthanasia for all
 individuals. We dissected the formalin fixed animals
 and removed the left gonad. We conducted visual gross examination blind to
 experimental pathogen exposure and population of origin. All males
 dissected had well developed testes, which indicated sexual maturity. We
 assigned females to one of three developmental categories based on gross
 examination of the ovaries: 1) small, underdeveloped ovaries, 2) mid-sized
 almost developed ovaries with some developed eggs, and 3) ovaries with
 developed eggs. We processed a subset of those
 dissected individuals (16 unexposed females, 16 unexposed males, 21
 exposed females and 23 exposed males, see Table S1) for histological
 examination following standard methods (Woods and Ellis 1994). We analysed
 the histological samples blinded to experimental exposure and population
 of origin.   <b><i>Gonad and gamete
 characteristics</i></b> We assessed six
 histosections per male individual to determine testis area, number of
 seminiferous tubules, density of seminiferous tubules, and germinal
 epithelium depth. We measured the largest tubule size and identified
 spermatogenesis cell clusters to stage (spermatogonia, spermatocytes,
 spermatids, spermatozoa) following Brannelly et al.
 (2016<i>c</i>), (see Text S1). For most of the characteristics
 measured, larger depth, area, or higher count indicates higher
 reproductive output. However, a lower density of seminiferous tubules and
 a lower proportion of spermatogonia indicates higher reproductive output:
 lower tubule density per unit of testis area allows for more
 spermatogenesis to occur due to increased intra-tubule space (i.e., more
 space inside the testis devoted to spermatogenesis) and is therefore
 associated with higher reproductive effort when spermatogenesis is active
 (Marion 1982). Because spermatogonia are an early stage of
 spermatogenesis, a lower proportion of spermatogonia cell clusters (i.e.,
 more cells in later stages of development) are indicative of higher
 spermatogenesis effort (Delgado et al. 1992; Rastogi et al.
 2005). We assessed six histosections per female
 individual to determine ovary size, number of eggs, size of the largest
 eggs, oviduct width, density of eggs within the ovary, and oogenesis
 stages (Stage I – pre-vitellogenic; Stage II-V – vitellogenic; Stage VI –
 post-vitellogenic) (Text S1). Similar to the interpretation of the male
 gonad/gamete characteristic analyses, for females, a lower density of eggs
 per ovary correlates with larger average egg size, which indicates higher
 reproductive effort because larger egg cells are associated with later
 oogenesis. A smaller proportion of pre-vitellogenic eggs indicates an
 overall higher proportion of combined vitellogenic and post-vitellogenic
 eggs (i.e., eggs that have yolk development), which we interpreted as
 higher reproductive effort. In both males and females, we analysed gonad
 size relative to body size, rather than unscaled gonad size.