Matthew J. Parris

FUNGAL PATHOGEN CAUSES COMPETITIVE AND DEVELOPMENTAL STRESS IN LARVAL AMPHIBIAN COMMUNITIES

Ecologists increasingly recognize pathogens as playing significant roles in community dynamics, yet few experimental studies have documented patterns of disease impacts on natural systems. We studied the effects of a pathogenic fungus (Batrachochy- trium dendrobatidis) on community interactions between two sympatric North American anurans: Fowlers toad, Bufofowleri, and the gray treefrog, Hyla chrysoscelis. Recent studies have identified chytridiomycosis as an emerging infectious disease, whose widespread oc- currence poses a significant risk for amphibian populations. We manipulated the presence of B. dendrobatidis in replicated experimental tanks and examined if exposure to this fungus during development could influence recruitment and competitive interactions between larval Bufo and Hyla. Tanks were followed throughout larval development, and we recorded survival, time to metamorphosis, and body mass at metamorphosis. We also tested the effects of competitive and pathogen-mediated stress on developmental stability by mea- suring fluctuating asymmetry (FA) of surviving metamorphs. B. dendrobatidis had no sig- nificant effect on larval survival, but the pathogen reduced metamorphic body mass and increased larval-period length. B. dendrobatidis had different effects on larval growth depending on the community structure; in the presence of the pathogen, both Bufo and Hyla metamorphosed at smaller body masses when reared together compared to when reared separately. Bufo also had strong negative effects on Hyla development, but only in the presence of B. dendrobatidis. FA of hind-limb length was significantly higher for meta- morphs from pathogen tanks, but was not associated with density or competitive environ- ment. Our results suggest that chytridiomycosis may impact Bufo and Hyla directly by reducing life-history performance and increasing developmental instability, and indirectly by inducing interspecific competitive effects in mixed-species environments.

Chytridiomycosis impacts predator-prey interactions in larval amphibian communities

Despite ecologists increasingly recognizing pathogens as playing significant roles in community dynamics, few experimental studies have quantified patterns of disease impacts on natural systems. Amphibians are experiencing population declines, and a fungal pathogen (Batrachochytrium dendrobatidis; Chytridiomycota) is a suspected causal agent in many declines. We studied the effects of a pathogenic fungus on community interactions between the gray treefrog, Hyla chrysoscelis, and eastern newts, Notophthalmus viridescens. Recent studies have characterized chytridiomycosis as an emerging infectious disease, whose suspected rapid range expansion and widespread occurrence pose a significant risk for amphibian populations worldwide. We reared larvae in outdoor polyethylene experimental tanks and tested the effects of initial larval density, predator presence, and fungal exposure on Hyla recruitment and predator-prey interactions between Hyla and Notophthalmus. Newts reduced treefrog survival, and high intraspecific density decreased metamorphic body mass independent of B. dendrobatidis. The presence of fungi reduced treefrog body mass at metamorphosis by 34%, but had no significant main effect on survival or larval period length. B. dendrobatidis differentially affected larval development in the presence of predators; Hyla developed slower when reared with the pathogen, but only when newts were present. This significant predator-by-pathogen interaction suggests that the impact of chytridiomycosis on larval amphibians may be exacerbated in complex communities. Our data suggest that B. dendrobatidis effects on host life history may be complex and indirect. Direct measurements of the community-level effects of pathogens offer an important opportunity to understand a significant threat to global biodiversity—declining amphibian populations.

Dietary protein restriction impairs growth, immunity, and disease resistance in southern leopard frog tadpoles.

The immune system is a necessary, but potentially costly, defense against infectious diseases. When nutrition is limited, immune activity may consume a significant amount of an organism’s energy budget. Levels of dietary protein affect immune system function; high levels can enhance disease resistance. We exposed southern leopard frog [Lithobates sphenocephalus (=Rana sphenocephala)] tadpoles to high and low protein diets crossed with the presence or absence of the pathogenic amphibian chytrid fungus (Batrachochytrium dendrobatidis; Bd) and quantified: (1) tadpole resistance to Bd; (2) tadpole skin-swelling in response to phytohaemagglutinin (PHA) injection (a measure of the T cell-mediated response of the immune system); (3) bacterial killing ability (BKA) of tadpole blood (a measure of the complement-mediated cytotoxicity of the innate immune system); and (4) tadpole growth and development. Tadpoles raised on a low-protein diet were smaller and less developed than tadpoles on a high-protein diet. When controlled for developmental stage, tadpoles raised on a low-protein diet had reduced PHA and BKA responses relative to tadpoles on a high-protein diet, but these immune responses were independent of Bd exposure. High dietary protein significantly increased resistance to Bd. Our results support the general hypothesis that host condition can strongly affect disease resistance; in particular, fluctuations in dietary protein availability may change how diseases affect populations in the field.

FUNGAL PATHOGEN CHANGES THE FEEDING KINEMATICS OF LARVAL ANURANS

Abstract Pathogens can alter host life-history traits by affecting host feeding activities. In anuran tadpoles, keratinized mouthparts (teeth and jaw sheaths) are essential for feeding. Batrachochytrium dendrobatidis (Bd) is a pathogenic fungus of amphibians that can infect these mouthparts and reduce tadpole survival. However, the precise way that Bd-induced changes in tadpole mouthparts impact tadpole feeding is unknown. We use high-speed (500 frames/sec) videography to study how Bd-induced mouthpart deformities affect the feeding kinematics of Fowlers toad (Anaxyrus [ =  Bufo] fowleri) and grey tree frog (Hyla chrysoscelis) tadpoles. We tested for species-specific patterns of Bd-induced mouthpart deformities to assess how deformations to specific areas of tadpole mouthparts alter feeding kinematics. The teeth of tadpoles from the Bd-exposed treatment slipped off of surfaces on which tadpoles graze and were in contact with an algal-covered substratum for a shorter duration in each gape cycle compared to teeth of control tadpoles. We also found that the jaw sheaths had significantly more deformations than labial teeth; however, how this relates to feeding kinematics is unclear. Our data show explicitly how Bd infection reduces foraging efficiency of anuran tadpoles by altering feeding kinematics and elucidate a mechanistic link between the pathogen infection and reduced host fitness.

Unlikely Remedy: Fungicide Clears Infection from Pathogenic Fungus in Larval Southern Leopard Frogs (Lithobates sphenocephalus)

Amphibians are often exposed to a wide variety of perturbations. Two of these, pesticides and pathogens, are linked to declines in both amphibian health and population viability. Many studies have examined the separate effects of such perturbations; however, few have examined the effects of simultaneous exposure of both to amphibians. In this study, we exposed larval southern leopard frog tadpoles (Lithobates sphenocephalus) to the chytrid fungus Batrachochytrium dendrobatidis and the fungicide thiophanate-methyl (TM) at 0.6 mg/L under laboratory conditions. The experiment was continued until all larvae completed metamorphosis or died. Overall, TM facilitated increases in tadpole mass and length. Additionally, individuals exposed to both TM and Bd were heavier and larger, compared to all other treatments. TM also cleared Bd in infected larvae. We conclude that TM affects larval anurans to facilitate growth and development while clearing Bd infection. Our findings highlight the need for more research into multiple perturbations, specifically pesticides and disease, to further promote amphibian heath.

How Does a Change in Labial Tooth Row Number Affect Feeding Kinematics and Foraging Performance of a Ranid Tadpole (Lithobates sphenocephalus)

Recent studies have explored feeding kinematics in tadpoles with intact labial teeth; however, it is unknown how missing teeth impacts foraging. We explored the impact of missing labial teeth on the feeding mechanics and foraging performance of Southern leopard frog (Lithobates sphenocephalus [= Rana sphenocephala]) tadpoles by controlling the pattern of labial tooth loss; that is, by surgically removing one row of labial teeth. We then used high-speed (500 frames/second) videography to test the hypothesis that tooth loss reduces the time that tadpoles attach to and graze upon an algal-covered substrate. We next conducted trials of foraging efficiency and foraging activity to test the hypothesis that tadpoles with fewer teeth forage less effectively than control tadpoles. The teeth of tadpoles from the surgery treatment slipped while closing and were in contact with an algal-covered substrate for a shorter duration compared to control tadpoles. Surprisingly, tadpoles with missing labial teeth obtained similar amounts of food and were as active as tadpoles with intact mouthparts. However, tadpoles with missing teeth completed about 25% more gape cycles per unit time than control tadpoles. Our data suggest that tadpoles with missing teeth compensate for inferior feeding kinematics during mouth closing in each gape cycle by increasing the number of gape cycles per unit time.

Development of antimicrobial peptide defenses of southern leopard frogs, Rana sphenocephala, against the pathogenic chytrid fungus, Batrachochytrium dendrobatidis

Amphibian species face the growing threat of extinction due to the emerging fungal pathogen Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis. Antimicrobial peptides (AMPs) produced in granular glands of the skin are an important defense against this pathogen. Little is known about the ontogeny of AMP production or the impact of AMPs on potentially beneficial symbiotic skin bacteria. We show here that Rana (Lithobates) sphenocephala produces a mixture of four AMPs with activity against B. dendrobatidis, and we report the minimum inhibitory concentration (MIC) of synthesized replicates of these four AMPs tested against B. dendrobatidis. Using mass spectrometry and protein quantification assays, we observed that R. sphenocephala does not secrete a mature suite of AMPs until approximately 12 weeks post-metamorphosis, and geographically disparate populations produce a different suite of peptides. Use of norepinephrine to induce maximal secretion significantly reduced levels of culturable skin bacteria.

The Impact of Pesticides on the Pathogen Batrachochytrium dendrobatidis Independent of Potential Hosts

Amphibians around the world are experiencing the greatest organismal decline in recent history. Xenobiotics, such as pesticides, and pathogenic biotic perturbations, including the fungus Batrachochytrium dendrobatidis (Bd), have played major roles in amphibian decreases. We conducted laboratory culture studies to determine the effects of three pesticides {carbaryl, glyphosate, and thiophanate-methyl [TM; Topsin-M(R) (Cerexagri-Nisso LLC)]} on Bd zoospore production and zoosporangia growth. We applied Bd to pesticides mixed in an agar culture to simulate pathogen introduction to a system with pre-existing pesticides (Bd addition). Alternatively, pesticides were applied to pre-established Bd to simulate pesticide introduction after Bd establishment (pesticide addition). We then measured Bd zoosporangia and zoospore production. All pesticides significantly inhibited zoospore production; however, glyphosate and TM were more effective at doing so than carbaryl. In addition, only carbaryl and glyphosate inhibited zoosporangia production. Our data suggest that carbaryl and glyphosate are equally effective at inhibiting both zoosporangia and zoospore production; however, TM is selectively toxic to zoospores but not zoosporangia. One possible explanation for this observation could be that TM is toxic to zoospores but not the protective zoosporangia. In the case of pesticides applied to established Bd cultures, all pesticides caused significant mortality in both zoosporangia and zoospores, and no differences were found among pesticides. We conclude that examining pesticide and pathogen interactions independent of hosts provides mechanistic understanding of such interactions before and after host infection or contamination.

Influence of priority effects and pond location on invaded larval amphibian communities

In Florida, the Cuban Treefrog (Osteopilus septentrionalis) is a superb colonist and appears to be a significant driver of amphibian community dynamics. Decline of native anurans has been linked to possible competition with adult O. septentrionalis but interactions during the larval stage are largely unknown. Rearing O. septentrionalis tadpoles along with two native anurans, the Squirrel Treefrog (Hyla squirella) and the Southern Toad (Bufo terrestris) in both experimental artificial ponds and laboratory aquaria, the role of competition as the mechanism driving the dynamics of invaded amphibian communities in Florida was examined. Also examined was the role of priority effects and variation between pond locations in altering interactions between O. septentrionalis and native anuran larvae. Interspecific competition was strong during the larval stage; the presence of O. septentrionalis reduced larval performance and survival of native anurans. Pond location alone had little effect on interspecific interactions, but priority effects were strong. Pond location and priority effects acted together to influence species interactions. The selective influence of different interaction modifiers acted to increase or decrease the impacts of exotic species on native taxa.

Can differences in host behavior drive patterns of disease prevalence in tadpoles

Differences in host behavior and resistance to disease can influence the outcome of host-pathogen interactions. We capitalized on the variation in aggregation behavior of Fowlers toads (Anaxyrus [ = Bufo] fowleri) and grey treefrogs (Hyla versicolor) tadpoles and tested for differences in transmission of Batrachochytrium dendrobatidis (Bd) and host-specific fitness consequences (i.e., life history traits that imply fitness) of infection in single-species amphibian mesocosms. On average, A. fowleri mesocosms supported higher Bd prevalences and infection intensities relative to H. versicolor mesocosms. Higher Bd prevalence in A. fowleri mesocosms may result, in part, from higher intraspecific transmission due to the aggregation of tadpoles raised in Bd treatments. We also found that, independent of species, tadpoles raised in the presence of Bd were smaller and less developed than tadpoles raised in disease-free conditions. Our results indicate that aggregation behavior might increase Bd prevalence and that A. fowleri tadpoles carry heavier infections relative to H. versicolor tadpoles. However, our results demonstrate that Bd appears to negatively impact larval growth and developmental rates of A. fowleri and H. versicolor similarly, even in the absence of high Bd prevalence.