DOI: 10.5061/dryad.f1vhhmh6c
title: Data from: Intestinal nematode infection confers a benefit to a non-declining frog species, while a fungal parasitic infection has sublethal impacts on reproductive investment

description: Emerging infectious disease is a major cause of wildlife decline around
 the world. Understanding the impacts of disease even in non-declining
 populations is important for understanding population-level health and
 resilience to other emerging threats. In this study, we explored the
 sublethal impacts of the fungal pathogen Batrachochytrium dendrobatidis,
 Bd, and a novel intestinal nematode on the non-declining
 Australian frog, Litoria lesueuri. We collected male animals that were
 either infected with the fungal parasite, infected with a nematode
 parasite, infected with both parasites, or uninfected and brought them
 into the lab, and monitored their morphology and fungal pathogen load over
 a 7-week trial. At the end of the experiment, we dissected the animals,
 collected the testes, and identified their nematode prevalence and burden.
 We morphologically and molecularly characterised the intestinal nematode
 as belonging to the genus Parathelandros within the order Oxyuridae. We
 found that this Parathelandros sp. infection was beneficial to the adult
 frogs, where infected animals were large, and had larger forearm width (an
 important trait for mating) when we accounted for animal size. The exact
 mechanisms of this improved condition are unknown and require additional
 research. However, in the Bd infected animals, we found an overall
 negative impact of infection, including reduced forearm width and sperm
 production. Bd infection is prevalent in this species, and there are
 tangible sublethal impacts of Bd infection, indicating that this species
 is affected even if mortality due to disease is low.
description: <strong>Field collection and
 husbandry</strong> Male <em>L.
 lesueuri</em> (n=32) were collected from the wild at two sites in
 Lerderderg State Park (O’Briens Crossing, elevation 453m, -37.496026,
 144.360978; Mackenzies Flat, elevation 33m, -37.615696, 144.424809) in the
 austral summer of December 2022. <em>Litoria lesueuri</em> are
 sexually dimorphic, and males are distinguished by their characteristic
 yellow breeding colouration and their smaller size. The animals were not
 treated for Bd infection on arrival in the lab, and any naturally acquired
 infection from the wild was allowed to develop during the
 experiment.  Once captured in the field, individuals
 were maintained individually for biosecurity to ensure pathogens were not
 spread. Frogs were housed in individual tanks (230× 150×130 mm) on a
 gravel and moss substrate. Animals were maintained in the lab at an
 average temperature of 18°C, with a range between 15 and 23°C. Enclosures
 were flushed daily with filtered water, and frogs were fed small crickets
 ad libitum twice per week. All animals were weighed (digital scale to the
 nearest 0.01g), measured (snout-to-vent length, SVL, using dial callipers
 to the nearest 0.1mm), and swabbed for Bd infection (See methodology
 below) in the two days following their collection from the wild, and
 measurements were then made weekly except for week six. The width of the
 right forearm and the width of the nuptial pad were measured to the
 nearest 0.01mm using digital callipers. We took three repeat readings per
 frog for the nuptial pad and arm width.  Any animals
 showing signs of moderate to severe chytridiomycosis (loss of righting
 reflex, severe erythema of groin and venter, excess skin slough) were
 euthanised with an overdose of MS222 (Tricaine methanesulfonate,
 Sigma-Aldrich), and decapitation was used as a secondary kill procedure.
 The experiment ended after 7 weeks, when all remaining animals were
 euthanised. Testes were removed, and the length of both testes was
 measured using digital callipers (0.01mm). The gonads were placed in
 formalin for histological processing. The whole specimen was thwaslaced in
 buffered formalin.  <strong>Testing for Bd
 infection</strong> We standardised the swabbing
 procedure by performing five repeat swab strokes on the middle of the
 venter, side of the venter, each thigh, and limb of each frog. The swab
 was gently rotated with each stroke to capture the greatest amount of
 fungal DNA from the skin. Swabs were stored at -20°C until processing. The
 DNA from the skin swabs was extracted in 50µL Prepman Ultra (Applied
 Biosystems®, Life Technologies Pty Ltd) and 30–40mg of 0.5mm silica beads
 (Biospec). We homogenised the samples (using a cell homogeniser) for 2min
 at 1400 oscillations per sec, then incubated the samples at 95°C to lyse
 the cells for 10min and collected and diluted the supernatant 6:100 in
 ultra-pure water before directly analysing for pathogen presence and
 quantity using qPCR (Brannelly et al. 2020). The remaining extracted DNA
 was stored at -20°C. With every extraction performed, one B. dendrobatidis
 positive control sample (zoospores from culture) and one negative control
 (swab only) were extracted. The diluted DNA extract
 samples were analysed using standard qPCR (Rotogene, Qiagen) methodology
 (Boyle et al. 2004) to identify and quantify Bd DNA in the samples with
 minor modifications. We ran our reactions at 15µL volume with lo-ROX 2x
 mastermix (SensiFast, Bioline), including BSA to reduce PCR inhibition,
 plus 5 µL of template DNA per reaction well. We ran each sample in
 singlicate for 40 cycles (Roto-Gene Q 2.3.5 software). In each qPCR
 reaction plate, we included a set of seven standards of known Bd
 concentrations (Pisces Molecular) made using plasmid DNA of the Bd ITS
 region to confirm the Bd infection status and load from the skin swab
 samples. On every qPCR reaction plate, we included a no-template control
 (5µL of ultra-pure water to replace the template DNA). 
 We calculated infection load as Bd ITS DNA copies present on the
 whole swab. We considered the sample to be positive if &gt;2 ITS DNA
 copies were present in the reaction well. Animals were considered infected
 if they had at least one positive swab sample throughout the experiment.
 Animals were considered cleared of infection if they had at least two
 consecutive negative swab results and remained negative to the end of the
 experiment. In some cases, animals came in with a negative swab result,
 but over the weeks in the lab they r, they returned positive swab samples
 (n=8). We considered these individuals as positive for infection because
 they likely came into the experiment with a low/undetectable infection
 load, which gradually grew over time in captivity. While qPCR as a
 diagnostic tool is sensitive, low infection loads are often missed
 (Brannelly et al. 2020; Hollanders and Royle 2022). We believe these
 animals had a low infection when they entered the facility, rather than
 contamination, because our biosecurity protocols were rigorous and
 individuals were maintained in individual enclosures.
 <strong>Testes preparation and
 morphology</strong>  There is a developing
 understanding of how reproductive effort is being influenced by
 chytridiomycosis in amphibians, evaluated via proxies for gametogenesis
 such as gonad size and morphology, as well as secondary sex
 characteristics including colour and calling behaviour. Testis size in
 amphibians is closely tied to reproductive success, with an increase in
 size typically corresponding to greater sperm production (McCallum and
 Trauth 2007; Brannelly et al. 2016, 2021), where increases in seminiferous
 tubule size (or reduced tubule density) indicate greater reproductive
 output – potentially due to an increase in intra-tubule space
 accommodating more mature spermatozoa (McCallum and Trauth 2007; Brannelly
 et al. 2016, 2021). Germinal epithelium depth of seminiferous tubules has
 been used to indicate changes in reproductive effort in amphibians, where
 increased germinal epithelium depth correlates with increased effort by
 increasing the space for spermatogenesis to occur (McCallum and Trauth
 2007; Brannelly et al. 2016, 2021). During the testes and sperm staging
 procedures, we were blinded to the experimental pathogen exposure status
 of the sample.  The formalin-fixed left testis (n=30)
 was analysed for testis morphology and spermatogenesis. Testes were
 dehydrated in a graded series of ethanol, cleared with xylene, and
 embedded in paraffin. Two sequential 5µm transections of tissue were taken
 from six random locations (total of 12 histosections) along the length of
 the testis between the pole and centre. The sections were mounted on glass
 slides, stained with haematoxylin and eosin stain, and cover-slipped.
 Sequential histosections (one histosection randomly chosen from the pairs
 of sequential histosections) were examined for testis morphology for each
 animal. Photographs were taken of the whole histosection to calculate the
 histosection area and count the number of seminiferous tubules at 40
 magnification. In each histosection analysed, the two largest seminiferous
 tubules were identified, and a photograph at 200 magnification, where
 tubule area was measured, and b, the maximum and minimum germinal
 epithelium depth was measured within each of the largest tubules. The
 photos were analysed using ImageJ. To assess
 spermatogenesis activity within the testis, we analysed the number of cell
 clusters of each sperm stage within the testis. We chose four
 non-sequential histosections, and within each of those four histosections,
 we identified the two largest seminiferous tubules. At 400 magnification,
 we took photos that included the germinal epithelium of each of the two
 largest seminiferous tubules. We then identified the number of cell
 clusters within that field of view that belonged to each of the four
 distinct stages of spermatogenesis: spermatogonia, spermatocytes,
 spermatid, and spermatozoa (Brannelly et al. 2016, 2021). 
 <strong>Parasitic nematode
 collection</strong> The presence of
 gastrointestinal nematodes within the colony was identified during initial
 dissection. During dissection, nematode samples were taken from two male
 frogs, and these samples were preserved in 95% ethanol and stored at
 -20°C. Approximately 6 months after euthanasia (n= 32), dissection and
 fixation in formalin, we dissected each frog to remove the lungs and
 intestine. The lungs were placed on a glass slide and observed under a
 light microscope (Leica LED2000) to examine for the presence of lungworm.
 No lungworm was found in any of the samples. We then removed the large
 intestine and analysed the faeces for intestinal nematodes. We removed the
 faeces by making an incision at the distal end of the large intestine and
 applying pressure to compress the large intestine. We made one or two
 faecalsmears, depending on the volume of faeces removed from the frog. The
 faecal smears were observed under a light microscope for the presence of
 adult female nematodes. If adult nematodes were found, they were counted
 and photographed. Because the frogs were fixed in formalin, only animals
 with adult nematodes were considered infected with intestinal nematodes.
 While nematode eggs can be identified using a faecal floatation test, this
 is not possible once samples have been fixed in formalin. However, because
 we analysed all the faeces within the intestine, we can be confident that
 if an adult female nematode was present, then we detected it.
description: # Data from: Intestinal nematode infection confers a benefit to a
 non-declining frog species, while a fungal parasitic infection has
 sublethal impacts on body condition and reproductive investment
 [https://doi.org/10.5061/dryad.f1vhhmh6c](https://doi.org/10.5061/dryad.f1vhhmh6c) ## Description of the data and file structure **Wallace Data1 coinfection**: This data file includes metadata on the frogs as well as infection data for each individual over time. The week is the week of the experiment, starting at 0, which was when the individuals were collected from the wild. FrogID is the individual ID for each frog. Tank is f, rog ID but in numeric form. Sex is male for all animals. SVLmm is the snout to venter length in mm, and Frogmass is the mass of the frog in g.Arm1, 2,2, and 3 are the width of the arm in mm, thumb1, 2, and are the length of the nuptial pad on the thumb in mm. Bd status week is whether the individual swabbed for each week for Bd infection was positive (1) or negative (0). Load is the number of Bd DNA copies detected on the swab.  Bd infection is the category that we gave each individual for whether they were positive for Bd at the end of the experiment, clear, or negative throughout for Bd infection. Date collected and Date euthanised are the dates for when the individual was collected from the wild and then euthanized in the lab. Parasite status is infected with intestinal nematodes (1) or not infected (0). Parasite count is the number of intestinal nematodes counted. Survival status is whether the individual survived to the end of the experiment (0) or was euthanized before the experiment (1). Many of these column headings are carried across data sheets.  **Wallace Data2 survival**: The survival and euthanasia records for each individual. Julian Colled is the date of collection from the wild and brought into captivity. Julian euthanised the animals that day. Days survived is the number of days the individual survived in captivity. **Wallace Data3 testis**: The morphological data on the testes for each individual. Right.testis.length.mm is the length of the right testis in mm. Left.testis.length.mm is the length of the left testis in mm. Magnification is the lens of the light microscope used to take photographs of the testis histosections. Histosection is the unique label we gave to each histosection that was cut, probed, and stained per individual. Photo ID is the name of each photo taken of the histological sections that we used to measure testis parameters. Tubule count is the number of tubules present within the histosection. Testis hμmght um is the height of the testis histosection inμmm. Testis μmdth um is the width of the testis histosectiμm in μm. Testis area um2 is the area of the testis histosectionμm²ared. Testis area mm² is the area of the testis histosection inmm²d. Areas per tube is the area μm²red of the total histosection area divided by the number of tubules for the average area per tubule.  **Wallace Data4 testis tubule**: The morphological data of the testis histosection. Tubule height is the height of the largest tubule within the photograph. Tubulebule width μm is the width of the largest tubule within the photograph. Tubuleubule area um2 is the area of the largest tubule within the photograph in um squared. GE deep um is the largest depth of the germinal epithelium within the largest tubule in the photograph in um. GE shallow um is the shallowest depth of the germinal epithelium within the largest tubule in the photograph in um.  **Wallace Data5 spermstage**: The spermatogenesis stages of cell clusters within the testis per field of view. Spermatogonia is the number of spermatogonia cell clusters within the field of view. The number of spermatocytes is the number of spermatocyte cell clusters within the field of view. Spermatid: the number of spermatid cell clusters within the field of view. Spermatozoa is the number of spermatozoa cell clusters within the field of view.  Note: Any cell that has an NA instead of a number means that the data was not collected for that individual data point.