Susceptibility to disease varies with ontogeny and immunocompetence in a threatened amphibian Amalina Abu Bakar, Deborah S. Bower, Michelle P. Stockwell, Simon Clulow, John Clulow & Michael J. Mahony Oecologia ISSN 0029-8549 Volume 181 Number 4 Oecologia (2016) 181:997-1009 DOI 10.1007/s00442-016-3607-4
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Author's personal copy Oecologia (2016) 181:997–1009 DOI 10.1007/s00442-016-3607-4
PHYSIOLOGICAL ECOLOGY - ORIGINAL RESEARCH
Susceptibility to disease varies with ontogeny and immunocompetence in a threatened amphibian Amalina Abu Bakar1 · Deborah S. Bower1 · Michelle P. Stockwell1 · Simon Clulow1 · John Clulow1 · Michael J. Mahony1
Received: 25 February 2015 / Accepted: 9 March 2016 / Published online: 29 March 2016 © Springer-Verlag Berlin Heidelberg 2016
Abstract Ontogenetic changes in disease susceptibility have been demonstrated in many vertebrate taxa, as immature immune systems and limited prior exposure to pathogens can place less developed juveniles at a greater disease risk. By causing the disease chytridiomycosis, Batrachochytrium dendrobatidis (Bd) infection has led to the decline of many amphibian species. Despite increasing knowledge on how Bd varies in its effects among species, little is known on the interaction between susceptibility and development within host species. We compared the ontogenetic susceptibility of post-metamorphic green and golden bell frogs Litoria aurea to chytridiomycosis by simultaneously measuring three host-pathogen responses as indicators of the development of the fungus—infection load, survival rate, and host immunocompetence—following Bd exposure in three life stages (recently metamorphosed juveniles, subadults, adults) over 95 days. Frogs exposed to Bd as recently metamorphosed juveniles acquired higher infection loads and experienced lower immune function and lower survivorship than subadults and adults, indicating an ontogenetic decline in chytridiomycosis susceptibility. By corresponding with an intrinsic developmental maturation in immunocompetence seen in uninfected frogs, we suggest these developmental changes in host susceptibility
Communicated by Raoul Van Damme. Electronic supplementary material The online version of this article (doi:10.1007/s00442-016-3607-4) contains supplementary material, which is available to authorized users. * Amalina Abu Bakar
[email protected] 1
School of Environmental and Life Science, University of Newcastle, Callaghan, NSW 2300, Australia
in L. aurea may be immune mediated. Consequently, the physiological relationship between ontogeny and immunity may affect host population structure and demography through variation in life stage survival, and understanding this can shape management targets for effective amphibian conservation. Keywords Chytrid · Batrachochytrium dendrobatidis · Chytridiomycosis · Litoria aurea · Development
Introduction The impact of disease on population biology is dependent on complex interactions between the host, the etiological pathogen and the environment (Anderson and May 1979), and thus to effectively manage disease in populations, these interactions must be quantified. However, this can be complicated by heterogeneity in host susceptibility between individuals within a population. For example, variation in pathology among hosts of different developmental stages has been widely demonstrated across various taxa including plants (Ficke et al. 2002), invertebrates (Bull et al. 2012), birds (Gavier-Widen et al. 2012), fish (Kelly et al. 2010) and mammals (Burns-Guydish et al. 2005), and often owes to a ‘critical window’ in development during which exposure to the pathogen causes greater disease vulnerability (Johnson et al. 2011). Ontogenetic differences in disease susceptibility can lead to population-level effects such as shifts in population structure and demography, which can ultimately lead to population decline (Muths et al. 2011). For example, exposures of fish (Galaxias anomalus) to trematode cercariae (Telogaster opisthorchis) cause higher frequencies (20–65 %) of spinal and fin deformities in juveniles, which develop during a critical window
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of skeletal ossification in the host’s larval stage (Kelly et al. 2010). Following population monitoring of infected fish in New Zealand, peaks in spinal malformation during this critical window were associated with peaks in mortality and a population crash, and consequential restrictions to recruitment may be contributing to the overall decline of the species (Kelly et al. 2010). In light of this, understanding the interaction between host development and infection outcome is an important precursor to managing disease in populations by identifying susceptible life stages as conservation targets. This is particularly crucial for amphibian conservation, where disease has contributed to the global decline of amphibian species (Skerratt et al. 2007). Chytridiomycosis, a disease caused by the fungus Batrachochytrium dendrobatidis (Bd), is one of the largest threats to amphibian conservation (Hussain and Pandit 2012), linked to the extirpation of numerous amphibian species (e.g. Crawford et al. 2010; Gillespie et al. 2015; Skerratt et al. 2007). Bd infects the keratinised outer epidermal layers of amphibian skin (Berger et al. 2005) where it can release lethal toxins (Blaustein et al. 2005) and cause death by inhibiting normal osmoregulatory function eventuating in circulatory collapse (Voyles et al. 2007, 2009). Whilst Bd has infected over 500 species (Olson et al. 2013), it does not have lethal consequences for every host. Interspecific variation in Bd susceptibility, even among sympatric species, has been widely demonstrated both in the laboratory and in wild amphibian communities (e.g. Kriger et al. 2007; Ortiz-Santaliestra et al. 2013; Searle et al. 2011; Woodhams et al. 2007a, b). This is thought to be mediated by differences in resistance—determined by the efficacy of the host species’ innate immune defences involving the abundance and diversity of cutaneous antimicrobial peptides, bacterial microflora, and circulating granulocytes, which inhibit the establishment and proliferation of Bd on the skin (Rollins-Smith et al. 2002, 2011; Woodhams et al. 2007a, b); and the skin architecture of the host species, involving the thickness, distribution, and abundance of keratin (Greenspan et al. 2012; Reeder et al. 2012). Within species, the effect of ontogeny on susceptibility to chytridiomycosis further creates individual-level variation in infection outcome. For many amphibian species, the larval stage is less susceptible to mortality following exposure to Bd, presumably because keratinised tissue is restricted to the mouthparts of tadpoles (Berger et al. 2005; Fellers et al. 2001). However, within the terrestrial post-metamorphic life stage, the impact of ontogeny on chytridiomycosis susceptibility is complicated, and appears to vary with species and corresponding life history (Searle et al. 2011). When considering the mode of Bd transmission, fully developed adult amphibians may be more susceptible to chytridiomycosis than juvenile post-metamorphic conspecifics. Their larger body size (and larger infective surface area), greater amounts
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of keratin in the integument, and behavioural attributes (e.g. mating, anuraphagy) not only increase the probability of zoospore contact rates, but also infer a greater capacity for higher pathogen loads and thus infection intensity (Garner et al. 2009; Kuris et al. 1980; Searle et al. 2011). However, if congruent with the critical development window of many vertebrates, newly metamorphosed and less developed juvenile post-metamorphic amphibians may instead possess greater disease risk due to their immature immune systems (Rollins-Smith 1998) and limited exposure to pathogens which would stimulate adaptive immunity (Fisher et al. 2009; Solomon 1978)—yet, this remains untested. As new lineages of Bd spread to new areas (James et al. 2015), identifying how chytridiomycosis interacts with developmental changes in susceptible species is vital for understanding the role of Bd in amphibian decline, to inform amphibian conservation strategies. Our objective was to identify the relationship between ontogeny and physiological susceptibility to chytridiomycosis in postmetamorphic individuals of a threatened frog species (the green and golden bell frog Litoria aurea). Specifically, we aimed to determine the effect of post-metamorphic life stage (recently metamorphosed juveniles, subadults and adults) at exposure to Bd on host response, as indicated by three factors of disease progression—survivorship, immunocompetence, and infection intensity. By identifying host responses to Bd under laboratory conditions, we aim to disentangle external and environmental influences from baseline physiological host susceptibility—a necessary precursor to more complex studies accounting for multiple interacting factors on host susceptibility and pathogen dynamics. We hypothesised that in the absence of environmental variables, an intrinsic developmental increase in immunocompetence in the host species would correspond to an ontogenetic decline in physiological susceptibility to chytridiomycosis, reflected by an increase in survival rate, increase in immune function, and decrease in infection load with maturing post-metamorphic life stage.
Materials and methods Study species The green and golden bell frog (Litoria aurea; Anura: Hylidae) is a native Australian ground-dwelling frog. Once common throughout eastern Australia, L. aurea is now considered threatened following a 90 % decline of historically recorded populations to 40 small, fragmented remnant populations with a restricted coastal distribution (Mahony et al. 2013). It is postulated that Bd has contributed to this decline, given that many local extinctions were rapid, and occurred at high altitude and inland regions where cooler temperatures
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favour the virulence of the fungus (Mahony et al. 2013). It is therefore of great conservation value to understand the interactions between Bd and L. aurea. Metamorphic and postmetamorphic L. aurea are highly susceptible to Bd under laboratory conditions (Stockwell et al. 2010) as well as in the field (Stockwell et al. 2008). Their skin secretions (as part of their innate immune defences against Bd) show very limited antifungal activity (Apponyi et al. 2004), and the fungus has been identified in dead and dying frogs in the wild (Penman 2008). However, whether this susceptibility further varies between individuals is unknown. Animal rearing and husbandry In this experiment we used 96 captive-bred L. aurea where development and metamorphosis occurred without exposure to Bd, to eliminate any adaptive responses elicited by previous Bd exposure as a confounding effect and to ensure our study reflected baseline physiological host susceptibility driven by intrinsic host traits. Experimental individuals were obtained from a single generation, bred in captivity using four adult breeding pairs (four egg clutches produced over 4 months). Breeding pairs were secondgeneration captive-breeding stock, with original breeding stock obtained from Kooragang Island, New South Wales, Australia (−32.86, 151.74). Tadpoles were reared in their separate clutches to allow us to account for clutch effects, and each clutch metamorphosed over a period of 9 months (considered natural variation that is commonly observed in the field) (Pyke and White 2001). As L. aurea exhibit rapid but non-exponential growth and development toward adulthood in the first 6–12 months following metamorphosis (with the species able to reach adult sizes within 2 months following metamorphosis) (Pyke and White 2001), this produced post-metamorphic individuals across a range of developmental stages in each clutch. L. aurea stock were kept in semi-natural mesocosm environments and were reared as tadpoles on a diet of boiled lettuce, and as frogs on a diet of brown crickets (Acheta domestica). Determination of life stage Frogs were separated into three distinct groups based on their developmental life stage—recently metamorphosed juveniles, subadults, and adults—determined primarily by the time since tail absorption during metamorphosis and the presence of secondary reproductive characteristics, and as amphibians have indeterminate growth, secondarily confirmed through body size (snout-vent length; SVL) which was measured using dial callipers (accurate to the nearest 0.1 mm; Swiss Precision Instruments, Switzerland) and compared to single-size thresholds from literature [field surveys (Hamer 1998; Hamer and Mahony 2007; Pyke and White
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2001)]. Recently metamorphosed juveniles were individuals which had absorbed their tails up to 2 weeks prior to experimentation, lacked reproductive traits, and were ≤32 mm SVL. Adults exhibited sexually reproductive characteristics such as nuptial pads in males and/or were ≥45 mm in SVL. Subadults were those lacking in reproductive traits, but had absorbed their tails over 4 weeks prior to experimentation. Characterised by when the majority of growth and development occurs, frogs in subadult life stage ranged from 32.1 to 44.9 mm SVL. Due to varying rates of metamorphosis among tadpoles in each clutch, each developmental life stage consisted of individuals from each of the four egg clutches, avoiding confounding clutch effects. Bd cultivation Bd (strain Gibbo River-Llesueuri-00-LB-1, passage number 4) was derived from stock held at the University of Newcastle, New South Wales, and cultivated in vitro. This was conducted by seeding agar plates (16 g tryptone, 4 g gelatine hydrolysate, 2 g lactose and 10 g bacteriological agar in 1000 ml distilled water; TGhL) with 2 ml of a 1-weekold actively growing Bd broth. Agar plates were then sealed with parafilm (Parafilm M laboratory film; Pechiney Plastic Packaging), incubated at room temperature for 5–7 days, and checked at 24-h intervals until growth of Bd was sufficient for harvest. This was signified by identifiable colonies of zoosporangia and free-swimming zoospores, detected using an inverted light microscope. Bd exposure treatments Frogs were individually housed in 20 × 12 × 12-cm plastic tanks labelled with a unique identification number. Each tank was partly filled with an autoclaved gravel substrate sloping into 150 ml of distilled water uniformly distributed throughout the tank, and provided with one piece of 25-mm-diameter polyvinyl chloride pipe elbow shelter. Half of the frogs in each life stage were allocated to the treatment group exposed to Bd, and the remaining half allocated as the exposure control group (not exposed to Bd). To limit any confounding effects of body size on exposure treatments within each life stage, individuals were paired by increasing SVL and individuals of each pair were then randomly allocated to either exposure treatment group. Inoculation dosage, timing and duration of inoculate exposure were identical across treatment groups, ensuring variation in infection outcomes were functions of intrinsic host traits. Individuals exposed to Bd were inoculated with 1 ml Bd suspension of approximately 9.05 × 106 zoospores (determined using a haemocytometer) diluted into 150 ml of distilled water in their tanks. Zoospores were acquired by flooding actively growing TGhL agar plates
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with deionised water, and extracting the supernatant. Individuals in the control group were inoculated with 1 ml of a sham suspension, acquired by flooding sterile TGhL agar plates with deionised water, diluted into 150 ml of distilled water in their tanks. Immediately following inoculation, tanks were randomly positioned within a temperature cabinet optimised for Bd growth (mean temperature = 20.1 °C) (Longcore et al. 1999; Piotrowski et al. 2004), and set to an automated 12-h light/12-h dark regime simulating light conditions in the wild. Throughout the experiment, husbandry procedures were standardised with water in each tank drained and changed twice a week, and all frogs provided with eight small crickets twice a week using strict hygiene and sterilisation procedures. Measures of chytridiomycosis susceptibility Three response variables were measured during the experiment as indicators of chytridiomycosis progression: the phytohaemagglutinin swelling response (PHA), survivorship and infection load. Phytohaemagglutinin swelling response The PHA assay was used as a functional immune challenge representing the capacity of individuals to mount a dynamic innate and adaptive immune response, determined through the level of localised inflammation (measured by tissue swelling) caused by subcutaneous injection of the lectin PHA into the skin (Brown et al. 2011; Clulow et al. 2015). Inflammation can depend on multiple innate and adaptive immune mechanisms such as rapid inflammatory skin responses to PHA as a large antigenic molecule capable of causing tissue damage (Kennedy and Nager 2006); and cell-mediated responses to the mitogenic capabilities of PHA including recruitment of phagocytes, granulocytes and lymphocytes (Clulow et al. 2015; Fites et al. 2014). As such, the extent of inflammation (indicated by swelling) following injection is directly proportional to host immunocompetence. Assays were performed 30 days following inoculation of Bd exposure treatments. Prior to injection with PHA, the thickness of each frog’s right and left lower leg (below the knee) was measured in triplicate rapid succession (readings taken at 1-s intervals) using a custom-built set-force Peacock G-1A dial thickness micrometer modified to 0.4 N of force (accurate to the nearest 0.005 mm; Ozaki Manufacturing, Japan). A subcutaneous injection of PHA [40 µg delivered in 40 µl of sterile phosphate-buffered saline (PBS)] was then made into the anterior lateral surface of the lower leg (where the previous measurement had occurred) in one leg using a 0.3-mL, 31 G insulin syringe, and a control injection (40 µl PBS) was injected into the same position on the contralateral leg
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(Clulow et al. 2015). Legs were allocated randomly for injection with PHA or the PBS control solution. Thirty-five hours following injection [the optimal interval for maximum swelling with recruitment of lymphocytes, neutrophils and macrophages in L. aurea (Clulow et al. 2015)] the thickness of both lower legs at the injection sites was remeasured and the differences in leg thickness from time zero (swelling response) were recorded. Survivorship Time until the onset of terminal chytridiomycosis was used to assess the survivorship of L. aurea. Terminal illness was identified by observing and testing the severity of notable signs of morbidity which indicated mortality was likely in the next 48 h (as per Stockwell et al. 2010). Each day following inoculation, all frogs were monitored for lethargy (e.g. absence of hunting behaviour during feeding, unresponsiveness to opening tank lids during water changes and feeding events), skin abnormalities (e.g. excessive sloughing, discolouration, redness in webbing), and abnormal posture (Berger et al. 2005; Stockwell et al. 2010). If signs were observed, the individual’s righting time was tested immediately with a stopwatch, thereby quantitatively detecting chytridiomycosis in its terminal stages. Righting tests were repeated up to four times. Prior to the experiment commencing, the righting time of all frogs was measured and in healthy animals shown to take up to 4 s when repeated up to four times. If the righting time of lethargic frogs was greater than 8 s (i.e. at least twice the healthy response time) for any of the four righting tests, the frog was closely monitored, with righting time tested again after 1 and 6 h. If there was no reduction, the frog was confirmed as terminally diseased with death expected to follow within 48 h (Berger et al. 2005). When this occurred, the number of days since Bd exposure was recorded, and the frog euthanised by immersion in 4 % tricaine methanesulfonate (MS-222) solution to avoid pain. Infection load The progression of infection intensity was followed by repeated skin swabs at 0 days (i.e. prior to Bd exposure treatments), 5, 14, 30, 60 and 90 days following initial exposure to Bd or control inoculates. A standardised swabbing technique was used across all life stages employing sterile fine-tipped swabs (Tubed Sterile Dryswab MW100; Medical Wire, UK). This involved swabbing firstly each frog’s ventral surface of the fore and hind feet twice, and then the right and left sides of the frog’s ventral surface with four 20-mm strokes. By standardising stroke length at 20 mm, the swabbing process was uniform across SVLs, avoiding bias of higher Bd infection loads for larger individuals arising from their larger ventral surface length. Following each
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swabbing event, all swabs and one sterile swab (negative template control; NTC) were analysed using quantitative polymerase chain reaction (qPCR). Extraction and quantification of Bd from swabs were performed following standard protocols for a qPCR Taqman assay (Boyle et al. 2004) employing DNA amplification using a Rotor Gene 6000 real time system (Corbett Life Science, Sydney). Samples from each swab were analysed in triplicate using a 1/10 dilution prepared from the extracted DNA. Following amplification, the number of Bd genomic equivalents (GE) at a standardised cycle threshold was averaged across all three replicates as a geometric mean. Mean GE values were only generated if amplification occurred in at least two replicates. Samples were considered negative for the presence of Bd if the process of amplification did not occur in any of the replicates (i.e. 0 GE mean value). To avoid false negatives, samples were tested for inhibition by inclusion of an internal positive control in one replicate of each sample (including the NTC). Following qPCR, the cycle number of the internal positive control crossing a threshold set midway along the amplification curve was examined, and if the sample crossed the threshold more than five cycles after the negative control, it was considered inhibited. Where inhibition was detected, a 1/100 dilution of the original extracted DNA was prepared to dilute inhibitory agents and the amplification process repeated. If the NTC revealed no contamination and the sample was negative for inhibition, the mean value generated for that sample was recorded, reflecting the relative measures of the infection load of the respective host frog. For any previously inhibited samples, the mean value was multiplied by 10 to account for the additional dilution step. Absence of infection in all frogs before experimentation (0 days) was confirmed prior to inoculating tanks with Bd exposure treatments, ensuring subsequent infection loads were a result of first exposure to the fungus. Incidental variables During experimentation, an unintentional infection was detected in the study species and recorded. Thirty-six frogs had signs indicating infection by the Gammaproteobacteria, Pseudomonas sp. (confirmed by histopathology and microbiology; Vetnostics, Specialist Diagnostic Services, Newcastle, NSW), despite no inoculation of frogs with the bacteria in the course of the study. Incidence of a secondary infection may have confounded the response variables we measured, particularly as infection by some species of Pseudomonas can be fatal in amphibians (Brodkin et al. 1992), and some Pseudomonas species can inhibit Bd growth and activity (Flechas et al. 2012; Harris et al. 2009). Pseudomonas sp.-diseased frogs were identified through observation (signs included protuberant eyes and discolouration of the dorsal surface and inner hind legs of frogs)
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and exposed to a 5-day treatment of an antibiotic, thereby preventing Pseudomonas sp.-induced mortality. Treatment involved replacing water in the tanks of affected frogs with a solution of 2:100 ml of enrofloxacin in deionised water, which was drained and changed in conjunction with the routine water change for standardisation purposes. To preserve experimental replicates and avoid reducing sample size, treated frogs remained in experiments after treatment, with an additional factor incorporated in data analysis. Due to the symptomatic detection of Pseudomonas sp., infection duration and intensity were not quantified and any lowlevel infections may have been missed, and data analysis is limited to animals showing disease with the bacterium. A shortage of antibiotic availability in the given time frame of the experiment also meant we were restricted to treating visibly infected animals, and thus any effects of Pseudomonas sp. on the response variables are confounded with any effect of the antibiotic. Data analysis Data analyses were conducted separately for each indicator. Immunocompetence and survivorship data were analysed using JMP version 10 (SAS Institute 1989–2013), whilst infection load data were analysed using SPSS version 21 (IBM 2012). Immunocompetence The swelling response following PHA injections for each frog was calculated as the difference in the proportional increase in leg thickness 35 h after injection from preinjection leg thickness between the control and PHAinjected legs [i.e. the change in swelling (%) in the PBS leg was subtracted from the change in swelling (%) in the PHA leg following injections]. These data were assessed for normality using a normal probability plot of residuals with outliers detected using a Studentised residual plot, and analysed as tests of immunocompetence. A three-way ANOVA incorporating Pseudomonas sp. infection (present/ absent), Bd exposure treatment and life stage at exposure to Bd as factors was conducted to determine differences in PHA-induced leg swelling between life stages following Bd exposure, and account for any effects of Pseudomonas sp. infection on the immunocompetence of frogs. Tukey’s tests were used post hoc to determine which life stages significantly differed in swelling response. Survivorship At the end of the experiment, individuals were assigned a survival status (alive/dead), yielding binary data. As such, we used a generalised linear model with a binomial error
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distribution to assess the effect of the Bd exposure treatment, the life stage of frogs at exposure to treatment inoculations, and incidental Pseudomonas sp. infection on the proportion of individuals that survived the duration of the experiment. Infection loads The effect of Bd exposure treatment on the infection loads (mean GE value of Bd zoospores) acquired by L. aurea over time was not determinable due to zero inflation owing to the absence of infection in all control frogs. Analyses were therefore restricted to treated frogs exposed to Bd. Individuals were included for analysis until euthanasia, when they were then removed from the dataset for swab events. Infection load data were normalized with logarithmic transformations, and variation over swabbing events (days since exposure to Bd) was investigated using a repeatedmeasures general linear model with pairwise interactions. Models were constructed using life stage at exposure to Bd, time since exposure to Bd and incidental Pseudomonas sp. infection as fixed effects to test the variation in infection loads among life stages over time and account for any confounding effects of Pseudomonas sp.
Results Immunocompetence Effect of Pseudomonas sp. infection Pseudomonas sp. infection had no significant effect on the proportion of leg swelling in L. aurea induced by PHA
Effect of exposure to Bd The mean proportion of swelling induced by PHA injections was significantly greater in control L. aurea (x̄ = 5.45 ± 0.76 %) than in frogs exposed to Bd (x̄ = 2.61 ± 0.75 %) [F(1, 82) = 5.93, P = 0.02; Fig. 1]. Comparison of swelling response between life stages The life stage of frogs at exposure to Bd had a significant effect on the proportion of leg swelling induced by PHA injections [F(2, 80) = 8.97, P
