DOI: 10.5061/dryad.j3tx95xjq
title: Identity and density of parasite exposures alter the outcome of co-infections: Implications for management

description: 1) Although research has focused on density-dependent responses of hosts
 to single-parasite infections, hosts are exposed to numerous parasites
 simultaneously under natural conditions and if these exposures lead to
 infections, they can threaten host populations and ecosystem stability.
 Moreover, spatiotemporal variation in abundance of co-occurring parasites
 might influence host infection intensity. If interactions are consistent
 between different co-infecting parasites, then these patterns could give
 managers another tool to control disease spread and even predict
 problematic disease emergences. 2) We investigated how parasite density
 and identity alter within-host co-infection dynamics. To test this, we
 simultaneously exposed Cuban treefrogs (Osteopilus septentrionalis) as a
 model amphibian species to all pairwise combinations of three problematic
 parasites that commonly co-infect amphibians: the fungus Batrachochytrium
 dendrobatidis (Bd), the nematode Aplectana hamatospicula, and Ranavirus.
 Hosts were exposed to one parasite at a fixed dose and another parasite at
 a range of five doses. 3) Higher doses of Bd decreased Ranaviral and A.
 hamatospicula loads, but Bd load was not influenced by the dose of either
 parasite. Ranaviral load was negatively associated with A. hamatospicula
 dose, but A. hamatospicula load was not affected by Ranaviral dose. We
 found that all the pairwise co-infections were dependent on parasite
 density and that pairwise interactions were highly asymmetric – strong in
 one direction and weak in the other – consistent with interactions
 dominating food webs. 4) Synthesis and applications: We also revealed that
 the exposure dose of A. hamatospicula was positively associated with host
 tolerance to Bd infection and negatively associated with Ranaviral load in
 hosts. Ranavirus and Bd cause mass die-offs in amphibians, but A.
 hamatospicula does not. Therefore, in systems where these parasites
 coexist, maintaining or increasing densities of A. hamatospicula could
 reduce the negative effects of Bd and Ranavirus infections. Additionally,
 if these asymmetric and density dependent patterns from community ecology
 are applicable to other amphibian co-infections or co-infections in other
 systems, this should allow conservation organizations and resource
 managers to predict outbreaks and manage host declines associated with
 deadly parasites by modifying the abundance of co-infecting parasites that
 might be easier to manage.
description: Cuban treefrog tadpoles (Osteopilus septentrionalis) were
 collected from kiddy pools filled with water (140 L) in the University of
 South Florida Botanical Gardens (Tampa, FL, USA) in August 2016. All
 animal husbandry throughout the experiment was carried out according to
 IUCAC protocol #W IS00002203. A. hamatospicula were collected from the
 gastrointestinal tract of euthanized Cuban treefrogs from Flatwoods
 Wilderness Park (Tampa, FL, USA). Identical Petri dishes, but with
 gastrointestinal content from uninfected frogs were used as a sham
 treatment. Ranavirus (FV3) was cultured in fathead minnow (Pimephales
 promelas) cells and maintained at -80°C in minimal essential medium (MEM).
 MEM without Ranavirus was used as a sham. Bd (SRS-JEL 212 strain) was
 grown in a 1% tryptone solution. SRS-JEL212 was chosen as it was isolated
 from the Southwestern US, where the experimental frogs were collected.
 Additionally, this strain successfully infects Cuban treefrogs. Identical
 plates, but with a sterile 1% tryptone solution were used as a sham. See
 supplemental methods for more details. Experimental
 Design To examine how the dose of parasite exposures
 affects host-parasite dynamics, we exposed a total of 174 frogs to one of
 28 total parasite treatments. Treatments included exposure to Ranavirus,
 Bd, or A. hamatospicula alone, simultaneous infections with all pairwise
 combinations (6 total pairwise combinations) of these parasites at a range
 of densities (all n=6), and controls (no exposure; n=12). In each of the
 pairwise co-infection treatments hosts were exposed to one parasite at an
 intermediate density and the second parasite at one of a range of
 densities (one of four potential densities) in all possible combinations
 (Table 1). Parasite exposures Two
 days before parasite exposure, individuals were moved to a 17°C
 environmental chamber because Cuban treefrogs can clear Bd at higher
 temperatures (McMahon et al. 2014, Cohen et al. 2017). In the co-infection
 treatments, the host was exposed to one parasite at an intermediate, fixed
 dose and simultaneously to the other parasite at a dose that ranged from
 low to high for each of the tested parasites (Garner et al. 2009,
 Echaubard et al. 2010, Hoverman et al. 2010, Gervasi et al. 2013, Knutie
 et al. 2017a, Knutie et al. 2017c). For the microparasites Bd and
 Ranavirus, doses ranged from 103 to 105 zoospores or plaque forming units
 (PFU), respectively, and the intermediate dose was 104. For the
 macroparasite A. hamatospicula, doses ranged from 15 to 60 J3 larvae and
 the intermediate dose was 30 (Table 1). Doses were applied to frogs held
 in Petri dishes (25 x 100 mm) for 24h. For Bd and A. hamatospicula
 exposures, 1-mL of DI water containing Bd zoospores or A. hamatospicula
 larvae was pipetted directly onto the backs of the hosts. This simulates
 exposure through water or soil contact (Kilpatrick et al. 2010, Roznik et
 al. 2021). For Ranavirus exposures, a 69 µl aliquot of MEM with Ranavirus
 was applied directly into the host’s mouth to mimic fecal-oral or
 cannibalistic transmission that is common in natural settings (Hoverman et
 al. 2010). All hosts also received sham exposures for parasites to which
 they were not exposed. For example, hosts co-exposed to Bd and A.
 hamatospicula also received the Ranavirus sham treatment. Control
 individuals received sham treatments for all three parasites.
 Assessing parasite load and host health To
 assess loads of Ranavirus, hosts were swabbed five times around the mouth
 and cloaca on day 4 after exposure. To assess Bd load, hosts were swabbed
 five times from hip to toe on both rear legs on day 16 after exposure.
 These time points were chosen to measure load differences as they
 represent times where each parasite would have had time to establish, but
 not create high levels of mortality (Gray et al. 2009, Voyles et al. 2009,
 Knutie et al. 2017c). Swabs were stored at -80°C for later processing. DNA
 from each swab was extracted using a Qiagen DNEasy Blood & Tissue
 Kit and analyzed using quantitative polymerase chain reaction (qPCR; Boyle
 et al. 2004, Picco et al. 2007).  A. hamatospicula loads were assessed by
 counting adult worms in amphibian gastrointestinal tracts when they
 experienced mortality or at the end of the experiment.
 To measure growth, frogs were weighed weekly for four weeks.
 Individuals were also checked twice daily for mortality. If mortality
 occurred, frogs were weighed and swabbed and/or dissected, depending on
 their treatment group. All surviving frogs were euthanized and dissected
 28 days after initial parasite exposure.          
 Statistical Analyses All analyses were run
 with R version 3.6.1 (R Core Team 2019). Plots were created using the
 visreg package and visreg function (Breheny and Burchett 2019). The
 survival plot was created using the survminer package and ggsurvplot
 function (Kassambara et al. 2019). To test how the
 interaction between the identity of a co-infecting parasite and its dose
 altered the load of Ranavirus or Bd, we conducted a generalized linear
 model with a negative binomial error distribution. For the A.
 hamatospicula load model, we used a binomial error distribution, and the
 dependent variable was defined as the proportion of larvae that
 successfully reached maturity (i.e., using the cbind function on
 “successes” and “failures”). The independent variables were dose, identity
 of the changing-dose parasite, and their interaction. Dose was expressed
 as a log 10-transformed proportion of the highest possible dose hosts were
 exposed to for each parasite. This allowed us to make comparisons across
 doses even though the exposure dose varied widely for macro- and
 microparasites. To test how the interaction between the
 identity of a co-infecting parasite and its dose affect host weight, a
 generalized linear model was run with growth rate as the dependent
 variable. Growth rates were calculated as the final mass minus the initial
 mass divided by weeks spent alive (g/wk). To address changes in host
 survival, we conducted a survival analysis using the survival package and
 the coxph function (Therneau and Lumley 2019), with host survival as the
 dependent variable. The identity of the fixed dose parasite, the identity
 of the changing dose parasite, and the dose of the changing dose parasite
 were used as interacting independent variables in both host health
 analyses. To test how these same factors affected tolerance (measured as
 host growth rate or survival given a parasite burden), the above-described
 models were rerun, but the interacting predictor variables were the
 identity of the changing dose parasite, exposure dose, and parasite load
 of the fixed-dose parasite. For all analyses Tukey Post-hoc tests were run
 to compare among parasite identities when the variables were significant
 (multcomp package and glht function (Hothorn 2010). Table S1 outlines all
 above-described analyses and error distributions.