Janet Koprivnikar

MicroCT scanner performance and considerations for vascular specimen imaging

Obtaining three-dimensional geometrical data of vascular systems is of major importance to a number of research areas in medicine and biology. Examples are the characterization of tumor vasculature, modeling blood flow, or genetic effects on vascular development. The performance of the General Electric Medical Systems MS8 microCT scanner is examined in the context of these applications. The system is designed to acquire high-resolution images of specimens up to 5 cm in diameter. A maximum resolution of 38 lp/mm at the 10% modulation transfer function level or 22 microm full width at half maximum of the plane spread function can be achieved with 8.5 microm voxels and a 17 mm field of view. Three different contrast agents are discussed and applied for imaging of small animal vasculature: corrosion casting material Batsons No. 17 with an added lead pigment, silicon rubber MICROFIL MV122, and a suspension of barium sulfate (Baritop) in gelatin. Contrast for all of these agents was highly variable in different vessels as well as within the same vessel. Imaging of PMMA tubing filled with MICROFIL shows that even vessels below 20 microm in diameter are detectable and that diameter estimation of vessels based on thresholding is possible with a precision of 2-3 pixels.

ENVIRONMENTAL FACTORS INFLUENCING TREMATODE PREVALENCE IN GREY TREE FROG (HYLA VERSICOLOR) TADPOLES IN SOUTHERN ONTARIO

The emergence or increased prevalence of various parasites may be linked to alterations in host—parasite interactions caused by environmental changes. We investigated prevalence of trematode infections in grey tree frog (Hyla versicolor) tadpoles from ponds in nonagricultural settings versus ponds adjacent to cornfields in southern Ontario. We found that agricultural activity was a significant factor in determining the percentage of tadpoles infected by 1 or more trematodes from 1 or more species (combined trematode infection). However, we found no associations between combined trematode infection and forest cover; pond size; road density; and measures of water quality, such as nitrate level and the presence of the herbicide atrazine. Although combined trematode prevalence was associated only with agricultural activity, prevalence of Alaria species showed a positive association with forest cover. This latter result probably reflects the importance of habitat suitability for the canid definitive hosts of this trematode species.

Macroparasite Infections of Amphibians: What Can They Tell Us?

Understanding linkages between environmental changes and disease emergence in human and wildlife populations represents one of the greatest challenges to ecologists and parasitologists. While there is considerable interest in drivers of amphibian microparasite infections and the resulting consequences, comparatively little research has addressed such questions for amphibian macroparasites. What work has been done in this area has largely focused on nematodes of the genus Rhabdias and on two genera of trematodes (Ribeiroia and Echinostoma). Here, we provide a synopsis of amphibian macroparasites, explore how macroparasites may affect amphibian hosts and populations, and evaluate the significance of these parasites in larger community and ecosystem contexts. In addition, we consider environmental influences on amphibian–macroparasite interactions by exploring contemporary ecological factors known or hypothesized to affect patterns of infection. While some macroparasites of amphibians have direct negative effects on individual hosts, no studies have explicitly examined whether such infections can affect amphibian populations. Moreover, due to their complex life cycles and varying degrees of host specificity, amphibian macroparasites have rich potential as bioindicators of environmental modifications, especially providing insights into changes in food webs. Because of their documented pathologies and value as bioindicators, we emphasize the need for broader investigation of this understudied group, noting that ecological drivers affecting these parasites may also influence disease patterns in other aquatic fauna.

Contaminant effects on host‐parasite interactions: atrazine, frogs, and trematodes

The effects of contaminants on multispecies interactions can be difficult to predict. The herbicide atrazine is commonly used in North America for corn crops, runs off into wetlands, and has been implicated in the increasing susceptibility of larval frogs to trematode parasites. Using experimental challenges with free-living stages of trematodes (cercariae), it was found that Rana sylvatica tadpoles exposed to 30 microg/L of atrazine had significantly higher intensity of parasitism than did larval frogs either not exposed or exposed to 3 microg/L of atrazine. This result could not be explained by high concentrations of atrazine diminishing antiparasite behavior of tadpoles. Furthermore, when tadpoles and cercariae both were exposed to the same concentration of atrazine, either 3 or 30 microg/L, the abundance of formed cysts was not different from the condition in which both were housed at 0 microg/L of atrazine. Atrazine appears to be debilitating to both free-living cercariae and tadpoles. Studies examining relations between parasitism and contaminant levels must account for such combined effects as well as influences on other interacting species (e.g., first intermediate snail hosts).

EFFECTS OF ATRAZINE ON CERCARIAL LONGEVITY, ACTIVITY, AND INFECTIVITY

Susceptibility of free-living infective stages of parasites to contaminants is relatively understudied compared with independent effects on measures of host health or immunity, but may be important in affecting prevalence and intensity of parasite infections. We investigated whether atrazine, an herbicide commonly used in North America, affected the cercariae of 4 different species of digenetic trematodes, and found that effects of atrazine concentration on mortality and activity of cercariae varied among species. Mortality of Echinostoma trivolvis increased in a 200 μg/L atrazine solution, and a species of Alaria showed both decreased activity and increased mortality. We also examined whether the ability of E. trivolvis to infect the second intermediate host, larval amphibians, was compromised by atrazine exposure. Longevity and prevalence of E. trivolvis cercariae was affected at 200 μg/L atrazine, whereas intensity of infection in Rana clamitans tadpoles was reduced at both 20 μg/L and 200 μg/L atrazine. Our results indicate that the viability of cercariae of some species is compromised by exposure to atrazine, emphasizing the importance of considering the influence of contaminants on free-living stages of parasites in addressing how environmental degradation may relate to host parasitism.

Infectious personalities: behavioural syndromes and disease risk in larval amphibians

Behavioural consistency or predictability through time and/or different contexts (‘syndromes’ or ‘personality types’) is likely to have substantial influence on animal life histories and fitness. Consequently, there is much interest in the forces driving and maintaining various syndromes. Individual host behaviours have been associated with susceptibility to parasitism, yet the role of pre-existing personality types in acquiring infections has not been investigated experimentally. Using a larval amphibian–trematode parasite model system, we report that tadpoles generally showed consistency in their activity level in response to both novel food and parasite exposure. Not only were individual activity level and exploration in the novel food context correlated with each other and with anti-parasite behaviour, all three were significant predictors of host parasite load. This is the first empirical demonstration that host behaviours in other contexts are related to behaviours mitigating infection risk and, ultimately, host parasite load. We suggest that this system illustrates how reliably high levels of activity and exploratory behaviour in different contexts might maximize both energy acquisition and resistance to trematode parasites. Such benefits could drive selection for the behavioural syndrome seen here owing to the life histories and ecological circumstances typical of wood frog (Lithobates sylvaticus) larvae.

Interactions of environmental stressors impact survival and development of parasitized larval amphibians

Infected hosts are exposed to many environmental stressors that must be taken into account in order to determine the importance of disease, as various combinations can interact in unpredictable ways. Here, northern leopard frog (Rana pipiens) tadpoles, a species in decline, were exposed to stressors singly or in combination. Stressors included infection by Echinostoma trivolvis (a trematode parasite), exposure to predator chemical cues (larval dragonflies), and exposure to varying concentrations of the herbicide atrazine. Parasitism decreased survival only in combination with exposure to 3 microg/L atrazine, with a negative interaction observed for mass as well. Similarly, a negative interaction of parasitism and predation on survival occurred. However, atrazine exposure alone negatively affected the survival, mass, and developmental stage of tadpoles. These results indicate that certain stressor combinations are particularly deleterious for young parasitized tadpoles. Notably, very common low-intensity parasite infection can be particularly harmful in certain situations. Such negative impacts on larval amphibians in certain scenarios may contribute to ongoing amphibian population declines, emphasizing that the combination of environmental stressors must be considered when evaluating the general role of disease in species extinctions.

Larval amphibian growth and development under varying density: are parasitized individuals poor competitors?

Population density and infection with parasites often are important factors affecting the growth and development of individuals. How these factors co-occur and interact in nature should have important consequences for individual fitness and higher-order phenomena, such as population dynamics of hosts and their interactions with other species. However, few studies have examined the joint effects of density and parasitism on host growth and development. We examined the co-influences of rearing density and parasitism, by the trematode Echinostoma trivolvis, on the growth and development of larval frogs, Rana (=Lithobates) pipiens. We also examined the potential role of parasite-mediated intraspecific competition by observing how unparasitized individuals performed when housed with other unparasitized tadpoles, versus housing with a combination of unparasitized and parasitized hosts. Mean mass and mean developmental stage were reduced under high rearing densities. The presence of parasitized conspecifics had no significant effect, but there was a significant interaction of density and parasitism presence on host mass, due to the fact that parasitized conspecifics grew poorly at high densities. Unparasitized individuals reared with parasitized and unparasitized conspecifics fared no better than unparasitized individuals reared only with one another. This result indicates that infected hosts compete as much as uninfected hosts for resources, even though infected individuals have reduced mass under high-density conditions. Resource acquisition and resource allocation are different processes, and parasitism, if it only affects the latter, might not have a discernible impact on competitive interactions.

Development and application of an eDNA method to detect and quantify a pathogenic parasite in aquatic ecosystems.

Approaches based on organismal DNA found in the environment (eDNA) have become increasingly utilized for ecological studies and biodiversity inventories as an alternative to traditional field survey methods. Such DNA-based techniques have largely been used to establish the presence of free-living organisms, but have much potential for detecting and quantifying infectious agents in the environment, which is necessary to evaluate disease risk. We developed an eDNA method to examine the distribution and abundance of the trematode Ribeiroia ondatrae, a pathogenic parasite known to cause malformations in North American amphibians. In addition to comparing this eDNA approach to classical host necropsy, we examined the detectability of R. ondatrae in water samples subject to different degradation conditions (time and temperature). Our test exhibited high specificity and sensitivity to R. ondatrae, capable of detecting as little as 14 fg (femtograms) of this parasites DNA (1/2500th of a single infectious stage) from field water samples. Compared to our results from amphibian host necropsy, quantitative PCR was -90% concordant with respect to R. ondatrae detection from 15 field sites and was also a significant predictor of host infection abundance. DNA was still detectable in lab samples after 21 days at 25°C, indicating that our method is robust to field conditions. By comparing the advantages and disadvantages of eDNA vs. traditional survey methods for determining pathogen presence and abundance in the field, we found that the lower cost and effort associated with eDNA approaches provide many advantages. The development of alternative tools is critical for disease ecology, as wildlife management and conservation efforts require reliable establishment and monitoring of pathogens.

AGRICULTURAL EFFECTS ON AMPHIBIAN PARASITISM: IMPORTANCE OF GENERAL HABITAT PERTURBATIONS AND PARASITE LIFE CYCLES

Agricultural activity can alter host-parasite interactions through associated contaminants and habitat perturbations. It is critical to determine whether agricultural effects are widespread or limited to specific types of agriculture. We examined influences of soybean agriculture on trematode parasitism of larval amphibians (grey tree frogs; Hyla versicolor) to assess the potential effects of a commonly applied pesticide (glyphosate) and landscape factors relative to previous field studies focusing on the herbicide atrazine. Overall, trematode parasite infection did not differ between soybean-adjacent and nonagricultural ponds (87.7% and 72.6% mean infection, respectively). However, host-generalist echinostome species were more common in tadpoles from soybean-associated ponds (86.3% mean infection versus 36.2% in nonagricultural ponds) as well as sites with large or short average distances to forest cover and roads, respectively. In contrast, the occurrence of a host-specialist (Alaria sp.) group was greater in nonagricultural ponds (50.3% mean infection versus 9.8% in soybean-associated ponds) and increased with shorter distances to the closest forest patch and smaller average forest distance. Because glyphosate was not detected at any site and landscape influences were parasite-specific, we suggest that agriculture may have broad effects on wildlife diseases through habitat alterations that affect pathogen transmission via host habitat suitability. Notably, nonagricultural ponds had a lower mean distance to the nearest forest patch and lower mean forest distance compared with soybean-adjacent ponds. As a result, we emphasize the need for wider investigations of habitat perturbations generally associated with agriculture for host-pathogen interactions, and consequently, wildlife conservation and management strategies.