DOI: 10.5061/dryad.44j0zpck7
title: Ontogeny of immunity and potential implications for co-infection

description: Immunity changes through ontogeny and can mediate facilitative and
 inhibitory interactions among co-infecting parasite species. In
 amphibians, most immune memory is not carried through metamorphosis,
 leading to variation in the complexity of immune responses across life
 stages. To test if the ontogeny of host immunity might drive interactions
 among co-infecting parasites, we simultaneously exposed Cuban treefrogs
 (Osteopilus septentrionalis) to a fungus (Bactrachochytrium dendrobaditis)
 and a nematode (Aplectana hamatospicula) at tadpole, metamorphic, and
 post-metamorphic life stages. We measured metrics of host immunity, host
 health, and parasite abundance. We predicted facilitative interactions
 between co-infecting parasites as the different immune responses hosts
 mount to combat these infectious are energetically challenging to mount
 simultaneously. We found ontogenetic differences in IgY levels and
 cellular immunity but no evidence that metamorphic frogs were more
 immunosuppressed than tadpoles. There was also little evidence that these
 parasites facilitated one another and no evidence that A. hamatospicula
 infection altered host immunity or health. However, Bd, which is known to
 be immunosuppressive, decreased immunity in metamorphic frogs. This made
 metamorphic frogs both less resistant and less tolerant of Bd infection
 than the other life stages. These findings indicate that changes in
 immunity altered host responses to parasite exposures throughout ontogeny.
description: <strong>Animal Husbandry</strong>
 Cuban treefrog (<em>Osteopilus septentrionalis</em>)
 tadpoles were collected from 140-L pools filled with rainwater at the
 University of South Florida botanical gardens (Tampa, FL, USA) in the
 summer of 2017. Individuals collected early in the season (May), midseason
 (July), and later in the summer (August) were all reared to
 post-metamorphic, metamorphic, and tadpole life stages respectively. See
 supplemental methods for additional details.
 <strong>Experimental Design</strong>
 To examine how the life stage of amphibians affect host-parasite
 dynamics, we exposed <em>O. septenrionalis</em> to one of four
 treatments at each of the three amphibian life-stages. Treatments included
 no parasite exposure, and exposures to either Bd alone, <em>A.
 hamatospicula </em>alone, or Bd and <em>A.
 hamatospicula</em> together. These four treatments were applied at
 the tadpole (Gosner development stage 28-35; Gosner 1960), metamorphic
 (stage 42-45 frogs), and post-metamorphic stages. In total, there were
 5,8, and 9 control individuals for tadpole, metamorphic, and
 post-metamorphic frogs, respectively, and 18 individuals for each of 9
 treatment groups (3 life stages and 3 treatment groups), for a total of
 184 individuals. Bd (SRS-JEL 212 strain) was cultured
 in a 1% tryptone solution and <em>A. hamatopsicula</em> larvae
 were harvested from adult worms and developed in Petri dishes (see
 supplement for more details). Exposures were applied to post- and
 mid-metamorphic frogs in Petri dishes (25 x 100 mm) with 1 mL and 10 mL of
 ASW water, respectively, for 24 h. Tadpoles were exposed in cups
 containing 30 mL of ASW water. Hosts were exposed to 1 mL of DI water
 containing either 30 J3 <em>A. hamatopsicula</em> larvae or
 105 zoospores of Bd. All frogs received sham exposures for parasites to
 which they were not exposed. <strong>Assessing
 amphibian responses to parasites</strong> To
 assess Bd abundance, hosts were swabbed eight days after treatment
 exposures because it is late enough for parasite establishment, but early
 enough so few hosts have experienced parasite-induced mortality (McMahon
 et al. 2014). Frogs were either swabbed 5 times from hip to toe on rear
 legs (if frogs had rear legs) or around the mouth for tadpoles. DNA was
 extracted from swabs (Qiagen DNEasy Blood & Tissue Kit) and
 analyzed using quantitative polymerase chain reaction (qPCR) to quantify
 Bd abundance (Boyle et al. 2004). <em>A. hamatospicula
 </em>abundance were assessed by counting adult worms in the
 gastrointestinal tract of the host after either mortality or euthanasia
 (for individuals surviving the entire experiment).
 Subsets of experimental animals were euthanized on day 11, 19 or
 28 to test host immunity during early, but detectable, Bd infections and
 the peak immune response for <em>A. hamatospicula</em> and Bd,
 respectively. Blood was drawn using micro-hematocrit capillary tubes
 (Fisher Scientific, Pittsburgh, PA) and used to create blood smears. Blood
 smears were stained using Camco Quik Stain II (Cambridge Diagnostic
 Products, Inc, Fort Lauderdale, FL). Three separate researchers counted
 2000 cells on each slide and identified white blood cell types and the
 results were averaged (Heatley and Johnson 2009). Enzyme-linked
 immunosorbent assays (ELISA) from remaining plasma were used to detect IgY
 antibody presence (adapted from Knutie et al. 2017b; see supplemental
 methods). Individuals were measured (snout-vent length
 [SVL]) and weighed weekly for four weeks. Amphibians were also checked
 twice daily for mortality. If mortality occurred, frogs were weighed,
 measured, swabbed and dissected as described above. Twenty-eight days
 after treatment exposures, all the remaining frogs were euthanized. Growth
 rates were calculated as the final mass or SVL minus the initial mass or
 SVL divided by weeks alive (g or mm/w).
 <strong>Statistical Analyses</strong>
 All analyses were run with R version 3.6.1 (R Core Team 2019) and
 figures were created using the <em>visreg</em> function and
 package (Breheny and Burchett 2019) or the ggplot function and
 <em>ggplot2</em> package (Wickham et al. 2020). For all models
 host life stage, exposure to Bd, and exposure to <em>A.
 hamatospicula </em>were included as interacting independent
 variables unless stated otherwise. Tukey’s post-hoc tests to compare
 across life stages were run using the <em>multcomp</em>
 package and using the glht function (Hothorn 2010). To
 investigate how host immune response and parasite abundance are affected
 by host life stage and co-infection, we ran models with all types of white
 blood cells counted (i.e., lymphocytes, neutrophils, basophils, monocytes,
 thrombocytes, and eosinophils), total white blood cell count, neutrophil
 to lymphocyte ratio, IgY antibody level, and Bd or <em>A.
 hamatospicula</em> abundance as separate dependent variables.
 Neutrophil, monocyte, eosinophil, and thrombocyte counts and Bd abundance
 were analyzed with negative binomial error distributions. The proportion
 of J3 larvae that hosts were exposed to that successfully penetrated the
 hosts’ skin and the proportion of J3 larvae that penetrated the hosts’
 skin that successfully established as an adult worm in the amphibian guts
 were run as separate analyses with binomial distributions error. Weight
 was also used as a predictor variable in these models to control for
 differences in host size.  All other immune responses were analyzed as
 generalized linear models with normal error distributions. Life stage was
 based on the Gosner stage of the amphibian when immune metrics or parasite
 abundance were quantified. We also investigated how
 co-infections and host life stage alter host health. We conducted a
 survival analysis using the <em>survival</em> package and the
 coxph function (Therneau and Lumley 2019). Individuals that survived to
 the end of the experiment were right-censored. Total development and
 growth rate (g or mm) were divided by four weeks or time to metamorphosis
 for development (e.g., g/w) and used as dependent variables in separate
 models. To test how host life stage and identity of the co-infecting
 parasite affected host tolerance, all the above-described models with host
 health and immunity were rerun with the addition of parasite abundance (Bd
 and <em>A. hamatospicula </em>abundance were run separately)
 as a fourth, interacting independent variable. Thus, tolerance is being
 measured as the slope of survival and growth against parasite
 abundance.