Host-consumed resources increase pathogen load of endoparasites more than integument-infecting parasites.

. MATERIALS AND METHODS2.1. Animal husbandryLarval and adult Cuban tree frogs <i>Osteopilus septentrionalis</i> were collected from the University of South Florida Botanical Gardens (Tampa, Florida, USA) in November 2015 and August 2017, respectively. Both life stages were housed in 946 ml sterilized plastic deli cups, with larvae placed in 800 ml of artificial spring water (ASW, recipe from Cohen et al. 1980) and adults placed on unbleached paper towels wet with ASW. All individuals were kept at 20°C with a 12 h photoperiod. Tadpoles and adults were used in ranavirus and <i>Bd</i> experiments, respectively.2.2. Experimental design2.2.1. Ranavirus experimentTen Cuban tree frog tadpoles were assigned randomly to the 6 treatment groups (2 × 3 experiment), for a total of 60 tadpoles individually housed in deli cups with 100 ml of ASW. The 6 treatment groups were either exposed or not to ranavirus and 3 levels of nutrient treatments. There were no significant differences across treatment groups in starting frog mass (<i>F</i>_5,54 = 1.15, p = 0.35). Ranavirus (frog virus 3 [FV3]-like) was cultured in fathead minnow <i>Pimephales promelas</i> cells, quantified by plaque assay, and maintained at –80°C in MEM. Tadpoles received either 77 µl (105 plaque-forming units [PFUs]) of ranavirus (FV3-like) in MEM or 77 µl of MEM without virus as a sham treatment, pipetted into the 100 ml of ASW. Exposure to ranavirus often occurs through exposure from the environment, so the experimental method used mimics a natural exposure route (Hoverman et al. 2010). Previous studies found that doses between 10² and 10⁶ PFUs cause sublethal effects and morbidity in tadpoles (Hoverman et al. 2010). After 24 h, 700 ml of ASW was added to each cup, for a total of 800 ml tadpole^–1. Each tadpole was then provided either one, two, or three 0.6 × 1 cm cylindrical plugs (measured using the first centimeter of a disposable micropipette tip) of a mix of fish flakes and <i>Spirulina</i> (1:1) in 1% agar, according to their randomly assigned low, medium, or high food treatment. The food treatments were determined from personal observation of this frog species. It was found that all tadpoles can consume 1.5 to 2 plugs a day, whereas only a few tadpoles are likely to consume 3 plugs a day. Therefore, 1 plug represents nutrient restriction, 2 plugs function for most tadpoles as a normal diet, and 3 plugs represent excess.2.2.2. Bd experimentEighteen adult Cuban tree frogs of equal mass were assigned randomly to the 4 treatments (2 × 2 experiment), for a total of 72 individually housed frogs. Frogs were either exposed or not to <i>Bd</i> and produced either a normal or restricted diet. All individuals then received 1 ml of deionized water, pipetted onto their dorsal surface, that was from rinsed and pooled Petri dishes (15 × 150 mm; 1% agar, 1% tryptone medium) that either were (<i>Bd</i> treatment; n = 36) or were not (control; n = 36) inoculated with <i>Bd</i> (SRS 812 isolate). <i>Bd</i> was grown at 25°C until zoospores were active (~5 d). Each <i>Bd</i>-exposed frog received 10⁵ zoospores, quantified via hemocytometer (Gervasi et al. 2013). After <i>Bd</i> or sham exposure, individuals were placed on either an ad libitum diet, where they were provided with 10 small house crickets <i>Acheta domestica</i> 3 times wk^–1, or a restricted diet, where they were provided 3 crickets 3 times wk^–1. These food quantities were based on prior experience feeding this species, where it was found that for the restricted diet, frogs almost always ate 3 crickets within a couple of hours of being fed, whereas with the ad libitum diet, frogs often had 1 to 2 crickets still remaining at the time of the subsequent feed.2.3. General methodsAll amphibians were weighed and staged (if tadpoles; Gosner 1960) at the beginning of the experiment and every week thereafter. Clean containers were provided weekly. To quantify ranaviral infections, the mouth and cloaca of each tadpole were swabbed 5 times at the end of the experiment. To quantify <i>Bd</i> infections, adult frogs were swabbed weekly on their abdomen from snout to vent and hind limbs from hip to toe 5 times each. All swabs were placed in 1 ml microcentrifuge tubes and stored at –20°C for later processing. After the experiment concluded, DNA was extracted from the swabs and quantitative PCR (qPCR) was used to quantify ranavirus load (Hoverman et al. 2010) and <i>Bd</i> load (Boyle et al. 2004). We confirmed that no unexposed control individuals were infected via qPCR. Any individuals surviving to the end of the experiment (2 wk for ranavirus and 3 wk for <i>Bd</i>) were euthanized with a 0.1% solution of buffered MS-222.For the <i>Bd</i> experiment, blood from euthanized frogs was extracted using microcapillary tubes to collect plasma. We then conducted ELISAs on the plasma to quantify immunoglobulin Y (IgY) antibody levels (adapted from Knutie et al. 2017; see Appendix for details). We chose to focus on IgY levels because this is the most common immunoglobulin and is used to combat <i>Bd</i> (Grogan et al. 2018).<b>Metadata</b>There are three sheets with data. The Bd sheet includes a summary of the Bd experiment across all the tested weeks. Bd-all weeks includes Bd experiment data on a week by week basis. The Ranavirus tab includes data from the ranavirus experimentBd VariablesNumber - individual animal numberTreatment - whether or not an individual was exposed to Bd and the level of resources the individual had access to. L=low food resources, H=high food resources, + = exposed to Bd, - = not exposed to Bd.Resource- whether an individual had access to high or low levels of food resources.exposure- whether an individual was exposed or not to Bd.antibody - measure of immune responsepremass - weight of frog at the start of the experiment (g).postmass- weight of frog at the end of the experiment (g).dmass- change in mass over the course of the experiment (g).infected - whether the individual was infected (y) or not (n) with Bd.Bd- all weeksNumber - individual animal numberResource- whether an individual had access to high or low levels of food resources.exposure- whether an individual was exposed or not to Bd.load - load of Bd (ZE)date - week number (1-3).infected - whether the individual was infected (y) or not (n) with Bd.Ranavirustreatment- whether an individual was exposed to ranavirus (r) or not (c) and the level of resources the individual had access to (h-high, m-medium, l-low).Resource- whether an individual had access to high, medium, or low levels of food resources.exposure- whether an individual was exposed or not to ranavirus.Number - individual animal numberweight 0d - weight of individual at the start of the experiment (g).weight 7d - weight of individual at the end of week one (g).weight 14d - weight of individual at the end of week two (g).weight change - difference in weight from start to the end of the experiment (g).length 0d- length of individual at the start of the experiment (cm).length 7d - length of individual at the end of week one (cm).length 14d-length of individual at the end of week two (cm).length change - difference in length from start to end of experiment (cm).stage 0d- developmental stage of individual at the start of the experiment (Gosner).stage 7d - developmental stage at the end of week one (Gosner).stage 14d- developmental stage of the end of week two (Gosner).stage change - difference in developmental stages between the start and the end o fthe experiment.Date of death - If an individual experienced mortality during the experiment, the date is recorded. If an individual did not, the date is the last day of the experiment (6/9/2017).dpi death - days post infection to death (d).survival - whether or not an individual survived to the end of the experiment (survived =0, died =1).ln ranavirus load- the natural log of ranavirus load (PFU).infected - whether or not an exposed individual was infected with ranavirus over the course of the experiment (y- infected, n-not).