DOI: 10.5061/dryad.djh9w0vxx
title: The application of community ecology theory to co-infections in wildlife hosts

description: Priority effect theory, a foundational concept from community ecology,
 states that the order and timing of species arrival during species
 assembly can affect species composition. Although this theory has been
 applied to co-infecting parasite species, it has almost always been with a
 single time lag between co-infecting parasites. Thus, how the timing of
 parasite species arrival affects co-infections and disease remains poorly
 understood. To address this gap in the literature, we exposed
 post-metamorphic Cuban tree frogs (Osteopilus septentrionalis) to
 Ranavirus, the fungus Batrachochytrium dendrobatidis (Bd), a nematode
 Aplectana hamatospicula, or pairs of these parasites either simultaneously
 or sequentially at a range of time lags and quantified load of the
 secondary parasite and host growth, survival and parasite tolerance. Prior
 exposure to Bd or A. hamatospicula significantly increased viral loads
 relative to hosts singly infected with Ranavirus, whereas A. hamatospicula
 loads in hosts were higher when co-exposed to Bd than when co-exposed to
 Ranavirus. There was a significant positive relationship between time
 since Ranavirus infection and Bd load, and prior exposure to A.
 hamatospicula decreased Bd loads compared to simultaneous co-infection
 with these parasites. Infections with Bd and Ranavirus either singly or in
 co-infections decreased host growth and survival. This research reveals
 that time lags between co-infections can affect parasite loads, in line
 with priority effects theory. As co-infections in the field are unlikely
 to be simultaneous, an understanding of when co-infections are impacted by
 time lags between parasite exposures may play a major role in controlling
 problematic co-infections.
description: <i>Amphibian responses to parasites</i>
 To measure the growth of amphibian hosts over the duration of the
 experiment, 24 h after secondary parasite exposure, treefrogs were weighed
 and snout-vent length (SVL) was measured, and these measurements were
 taken weekly for four weeks. Survival and disease signs were assessed
 twice daily. At the time of death, we weighed deceased individuals,
 measured SVL, and assessed infection for the secondary parasite to which
 the individual was exposed. Disease signs were determined to be internal
 hemorrhaging or erythema near the feet for Ranavirus and discoloration,
 sloughing, or thickening (the latter of which was only noted during
 dissections) of the skin for Bd (Cunningham et al. 1996, Kilpatrick et al.
 2010). For Bd- and Ranavirus-exposed individuals, we swabbed the skin and
 conducted qPCR (see below).  For <i>A. hamatospicula
 </i>exposed individuals, we conducted dissections of the gut.
 Twenty-eight days after exposure to the secondary parasite, all surviving
 frogs were euthanized and dissected. Host growth rate was calculated as
 the final mass or snout-vent length (SVL) minus initial mass or SVL
 divided by weeks alive (g wk<sup>-1</sup>).
 <i>Assessing parasite loads</i> To
 assess <i>A. hamatospicula </i>infection, treefrog feces were
 checked weekly for larval worms and amphibian hosts were dissected at time
 of morality and at the end of the experiment to quantify adult <i>A.
 hamatospicula</i> infection load in the gastrointestinal tract. To
 assess Bd and/or viral load, amphibians were swabbed 2, 4, 8, 16, and 24
 days after exposure to the second parasite and at the time of mortality.
 Individuals were swabbed 5 times from hip to toe on both rear legs for Bd
 and around the cloaca and mouth to test for Ranaviral infection. Swabs
 were placed in 2-ml sterile microcentrifuge tubes and stored at -80 °C
 until processing. DNA was extracted (Qiagen DNEasy
 Blood & Tissue Kit) from 2-, 4, 8, and 24-day swabs where
 Ranavirus was the second infecting parasite and from day 4-, 8-, 16-, and
 24-day swabs where Bd was the second infecting parasite. Day 2 swabs were
 not extracted for Bd because Bd DNA from the original inoculum can be
 detected for up to 2 days after dosing, making it impossible to
 differentiate infection from exposure at this time (McMahon et al. 2014).
 Bd and Ranavirus swabs were analyzed using quantitative polymerase chain
 reaction (qPCR) to quantify parasite load (Boyle et al. 2004, Picco et al.
 2007; see supplement methods for more details).
 <i>Assessing amphibian immune response</i>
 We conducted a separate experiment that included 48 additional
 Cuban tree frogs to quantify amphibian antibody responses to single and
 simultaneous co-infections with the parasites that we tested above. Four
 individuals were tested for each of the three single infection treatments,
 for a total of twelve singly infected individuals. Eight individuals were
 tested for each of the three, pairwise, co-infection treatments and half
 of the individuals in each pairwise co-infection treatment were tested for
 one parasite and the other half were tested for the other. Finally, twelve
 individuals were tested as controls, with four individuals tested to
 compare with each parasite (Appendix S1: Table S4). To measure the immune
 response for each parasite, frogs were euthanized as described above and
 all available blood (approximately 100-300uL depending on the frog size)
 was extracted directly from frog hearts with microcapillary tubes 11, 19,
 and 28 days after exposure to Ranavirus, <i>A.
 hamatospicula</i>, and Bd, respectively. These times were chosen to
 reflect when antibody immune responses to each parasite should be
 detectable (Gantress et al. 2003, Ramsey et al. 2010, Knutie et al. 2017).
 While we acknowledge that immunity is a complex, multifactorial response,
 we chose to focus on IgY antibodies because IgY is a metric of acquired
 immunity, is the most common immunoglobulin, and is used to combat the
 parasites we tested (Gantress et al. 2003, Maniero et al. 2006, Knutie et
 al. 2017, Grogan et al. 2018).  General IgY measurements cannot
 differentiate between Th1 and Th2 immune responses (Bretscher 2014, Menon
 et al. 2018). However, assuming co-infected individuals would mount higher
 IgY levels than singly infected individuals to combat both pathogens, no
 significant increase in IgY levels for co-infected individuals could
 suggest that a Th1/Th2 tradeoff is limiting the hosts’ ability to mount a
 significantly higher immune response.  Blood plasma was
 used in enzyme-linked immunosorbent assays (ELISA) to detect IgY antibody
 presence. Individual frog serum was first diluted at 1:100 in carbonate
 coating buffer (0.05 M, pH 9.60) and then added to ninety-six well plates
 with 100 μL/well. Each individual sample was run in triplicate. After
 serum was added, plates were incubated at 4 °C overnight.  Plates were
 washed five times with 300 μL/well of a Tris-buffered saline wash
 solution. The plates were then filled with 200 μL/well of bovine serum
 albumin (BSA) blocking buffer and incubated for 2 h at room temperature on
 an orbital table. Plates were washed again five times, loaded with 100
 μL/well of primary detection antibody (Goat-αAlligator-IgY, diluted
 1:1000; Bethyl), placed on an orbital table for 1 h, washed again (five
 times), loaded with 100 μL/well of secondary detection antibody
 (Rabbit-αGoat-IgG, diluted 1:5000; Bethyl) for 1 h, and then washed for
 the final time (five times). Plates were then loaded with 100 μL/well of
 tetramethylbenzidine (TMB: Bethyl Laboratories) and incubated for 30 mins
 before the reaction was stopped with 100 μL/well of stop solution (Bethyl
 Laboratories). Finally, optical density (OD) of the wells was measured
 with a spectrophotometer (BioTek, PowerWave HT, 450-nanometer filter).
  All samples were run in triplicate.
description: There are two provided data sheets with the Priority Effects
 Ecology spreadsheet. The first sheet (All Swab Days) was used to analyze
 parasite loads over time since co-infection. The metadata for this
 spreadsheet is included and titled Metadata-All Swab Days. The second
 sheet (Focal Swab Days) was used to analyze how the time lags between
 infections altered disease progression. The metadata for this spreadsheet
 is also included and titled Metadata-Focal Swab Days.
 There is an additional excel file for the separate immune
 experiment titled, Priority Effects ELISAs Ecology. Metadata is
 included.