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.

Field collection and husbandry

Male L. lesueuri (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. Litoria lesueuri 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. 

Testing for Bd infection

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 >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.

Testes preparation and morphology 

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). 

Parasitic nematode collection

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.