The effects of multiple pathogens on amphibians in the Pacific Northwest

The earth is undergoing a “biodiversity crisis” characterized by loss of populations, species, genetic diversity, and ecosystem services. Part of this crisis consists of population declines, extinctions, and increased incidence of deformities in amphibians. It is unknown whether deformities contribute to these declines. Many cases of population declines in amphibians are associated with infectious disease. Water molds (Oomycota: Oomycetes: Saprolegniales) (e.g. Achlya and Saprolegnia) may contribute to amphibian population declines whereas parasitic flatworms (Ribeiroia) appear to be major causes of amphibian deformities. I found that Saprolegnia can kill larval and newly-metamorphosed juvenile amphibians. In addition, I investigated synergistic effects of Saprolegnia and the environmental contaminant nitrate on survival of amphibian larvae. No synergisms were found. However, there was a less-than-additive interaction affecting one frog species. Saprolegnia killed Rana aurora (red-legged frog) larvae, but only when nitrate was not added, consistent with nitrate preventing Saprolegnia from killing R. aurora. I also tested for possible interactions among the pathogenic water mold A. flagellata, the pathogenic fungus Batrachochytrium dendrobatidis (BD), and the parasitic trematode Ribeiroia. No between-pathogen interactions were found, however, there were main effects of Ribeiroia and BD. Ribeiroia caused mortality and increased frequency of limb deformities. There were differences in the composition of deformity types between my study and previous work, suggesting that the relative frequencies of different deformity types produced by Ribeiroia may be context-dependent. Also, there was an overall effect of delayed development in Ribeiroia-exposed individuals. In addition, individuals that did not die or display limb deformities following Ribeiroia exposure had slower development than controls not exposed to Ribeiroia. Delayed development may contribute to the effects of Ribeiroia on amphibian populations. In contrast, BD sped up development, although there was no evidence of BD infection. These results are consistent with amphibian larvae responding to the presence of BD by increasing the rate of development. Hastened metamorphosis and dispersal from larval habitats may decrease the risk of BD infection or reduce BD infection load. Mortality of post-embryonic life stages from water molds and sublethal effects of Ribeiroia and BD on amphibian development may influence how these pathogens interact with amphibian populations

Effects of the pathogenic water mold Saprolegnia ferax on survival of amphibian larvae

Infectious diseases are a significant threat to worldwide biodiversity. Amphibian declines, a significant part of current biodiversity losses, are in many cases associated with infectious disease. Water molds are one group of pathogens affecting amphibians on a worldwide basis. Although water molds have been studied extensively for their effects on host embryos, little information is available about how they affect post-embryonic amphibians. We tested the effects of one species of water mold, Saprolegnia ferax, in a comparative study of larvae of 4 amphibian species: Pseudacris regilla (Pacific treefrog), Rana cascadae (Cascades frog), Ambystoma macrodactylum (long-toed salamander), and R. aurora (red-legged frog). S. ferax can kill amphibians at the embryonic and juvenile life history stages, depending on the amphibian species. In the present study, a 1 wk exposure to S. ferax killed P. regilla larvae and a 2 wk exposure killed R. aurora larvae. Larvae of the other host species were unaffected after 1 wk of exposure to S. ferax. Our results suggest that S. ferax can kill amphibian larvae and further suggest that evaluation of how pathogens affect amphibians at the population level requires investigation at various life stages

Influence of ultraviolet-B radiation on growth, prevalence of deformities, and susceptibility to predation in Cascades frog (Rana cascadae) larvae

Abstract Ambient levels of ultraviolet-B radiation (UVB) have a variety of detrimental effects on aquatic organisms. These include death and effects on growth, development, physiology, and behavior. Amphibians show all of these effects. However, the effects vary with species, life history stage, and ecological context. Little is known about the implications of the detrimental effects of UVB on ecological dynamics. Our study was designed to test how UVB may affect predator-prey interactions, an important ecological dynamic. Specifically, we tested the effect of UVB on the susceptibility of Cascades frog (Rana cascadae) larvae to predation by roughskinned newts (Taricha granulosa). We also further examined the sublethal effects of UVB on growth and development in Cascades frog larvae. We found no direct effect of UVB exposure on survival. However, UVB-exposed frog larvae displayed decreased growth and increased prevalence of deformities. UVB also caused increased susceptibility to predation, but there was a significant treatment-block interaction. UVB increased susceptibility to predation in two out of five blocks of Cascades frogs. The other three blocks did not show an effect of UVB on susceptibility to predation. Our study suggests that UVB can alter susceptibility to predation in at least one amphibian species. UVB-induced alteration of predator-prey interactions could potentially lead to changes at the population, community, and ecosystem levels

The Fungicide Chlorothalonil Is Nonlinearly Associated with Corticosterone Levels, Immunity, and Mortality in Amphibians

Background: Contaminants have been implicated in declines of amphibians, a taxon with vital systems similar to those of humans. However, many chemicals have not been thoroughly tested on amphibians or do not directly kill them

Complex interactive effects of water mold, herbicide, and the fungus Batrachochytrium dendrobatidis on Pacific treefrog Hyliola regilla hosts

Infectious diseases pose a serious threat to global biodiversity. However, their ecological impacts are not independent of environmental conditions. For example, the pathogenic fungus Batrachochytrium dendrobatidis (Bd), which has contributed to population declines and extinctions in many amphibian species, interacts with several environmental factors to influence its hosts, but potential interactions with other pathogens and environmental contaminants are understudied. We examined the combined effects of Bd, a water mold (Achlya sp.), and the herbicide Roundup® Regular (hereafter, Roundup®) on larval Pacific treefrog Hyliola regilla hosts. We employed a 2 wk, fully factorial laboratory experiment with 3 ecologically realistic levels (0, 1, and 2 mg l-1 of active ingredient) of field-formulated Roundup®, 2 Achlya treatments (present and absent), and 2 Bd treatments (present and absent). Our results were consistent with sublethal interactive effects involving all 3 experimental factors. When Roundup® was absent, the proportion of Bd-exposed larvae infected with Bd was elevated in the presence of Achlya, consistent with Achlya acting as a synergistic cofactor that facilitated the establishment of Bd infection. However, this Achlya effect became nonsignificant at 1 mg l-1 of the active ingredient of Roundup® and disappeared at the highest Roundup® concentration. In addition, Roundup® decreased Bd loads among Bd-exposed larvae. Our study suggests complex interactive effects of a water mold and a contaminant on Bd infection in amphibian hosts. Achlya and Roundup® were both correlated with altered patterns of Bd infection, but in different ways, and Roundup® appeared to remove the influence of Achlya on Bd

Evaluating the links between climate, disease spread, and amphibian declines

Human alteration of the environment has arguably propelled the Earth into its sixth mass extinction event and amphibians, the most threatened of all vertebrate taxa, are at the forefront. Many of the worldwide amphibian declines have been caused by the chytrid fungus, Batrachochytrium dendrobatidis (Bd), and two contrasting hypotheses have been proposed to explain these declines. Positive correlations between global warming and Bd-related declines sparked the chytrid-thermal-optimum hypothesis, which proposes that global warming increased cloud cover in warm years that drove the convergence of daytime and nighttime temperatures toward the thermal optimum for Bd growth. In contrast, the spatiotemporal-spread hypothesis states that Bd-related declines are caused by the introduction and spread of Bd, independent of climate change. We provide a rigorous test of these hypotheses by evaluating (i) whether cloud cover, temperature convergence, and predicted temperature-dependent Bd growth are significant positive predictors of amphibian extinctions in the genus Atelopus and (ii) whether spatial structure in the timing of these extinctions can be detected without making assumptions about the location, timing, or number of Bd emergences. We show that there is spatial structure to the timing of Atelopus spp. extinctions but that the cause of this structure remains equivocal, emphasizing the need for further molecular characterization of Bd. We also show that the reported positive multi-decade correlation between Atelopus spp. extinctions and mean tropical air temperature in the previous year is indeed robust, but the evidence that it is causal is weak because numerous other variables, including regional banana and beer production, were better predictors of these extinctions. Finally, almost all of our findings were opposite to the predictions of the chytrid-thermal-optimum hypothesis. Although climate change is likely to play an important role in worldwide amphibian declines, more convincing evidence is needed of a causal link

Individual and combined effects of multiple pathogens on Pacific treefrogs

In nature, individual hosts often encounter multiple pathogens simultaneously, which can lead to additive, antagonistic, or synergistic effects on hosts. Synergistic effects on infection prevalence or severity could greatly affect host populations. However, ecologists and managers often overlook the influence of pathogen combinations on hosts. This is especially true in amphibian conservation, even though multiple pathogens coexist within amphibian populations, and several pathogens have been implicated in amphibian population declines and extinctions. Using an amphibian host, Pseudacris regilla (Pacific treefrog), we experimentally investigated interactive effects among three pathogens: the trematode Ribeiroia sp. (hereafter, Ribeiroia), the fungus Batrachochytrium dendrobatidis (hereafter, BD), and the water mold Achlya flagellata. We detected no effects of A. flagellata, but did find effects of Ribeiroia and BD that varied depending on context. Low doses of Ribeiroia caused relatively few malformations, while higher Ribeiroia doses caused numerous deformities dominated by missing and reduced limbs and limb elements. Exposure to low doses of BD accelerated larval host development, despite there being no detectable BD infections, while exposure to higher BD doses caused infection but did not alter developmental rate. Hosts exposed to both Ribeiroia and BD exhibited the highest mortality, although overall evidence of interactive effects of multiple pathogens was limited. We suggest further research on the influence of multi-pathogen assemblages on amphibians, particularly under a variety of ecological conditions and with a wider diversity of hosts and pathogens

Isolation and maintenance of Batrachochytrium salamandrivorans cultures

Discovered in 2013, the chytrid fungus Batrachochytrium salamandrivorans (Bsal) is an emerging amphibian pathogen that causes ulcerative skin lesions and multifocal erosion. A closely related pathogen, B. dendrobatidis (Bd), has devastated amphibian populations worldwide, suggesting that Bsal poses a significant threat to global salamander biodiversity. To expedite research into this emerging threat, we seek to standardize protocols across the field so that results of laboratory studies are reproducible and comparable. We have collated data and experience from multiple labs to standardize culturing practices of Bsal. Here we outline common culture practices including a medium for standardized Bsal growth, standard culturing protocols, and a method for isolating Bsal from infected tissue