Summary
Genetic studies have indicated that some parasite species formerly thought to be generalists are complexes of morphologically similar species, each appearing to specialize on different host species. Studies on such species are needed to obtain ecological and parasitological data to address whether there are fitness costs in parasitizing atypical host species. We examined whether lungworms from two anuran host species, Lithobates sylvaticus and Lithobates pipiens, differed in measures of infection success in L. pipiens recipient hosts. We also determined if the worms from the two host species were sources of genetically resolvable species of morphologically similar nematodes. Sequences of internal transcribed spacer and lsrDNA regions of adult lungworms from each host species indicated that worms from L. sylvaticus matched Rhabdias bakeri, whereas worms from L. pipiens matched Rhabdias ranae. Our work suggested that these morphologically similar species are distant non-sibling taxa. We infected male and female metamorphs experimentally with lungworm larvae of the two species. We observed higher penetration, higher prevalence and higher mean abundance of adult worms in lungs of male and female metamorphs exposed to R. ranae larvae than in lungs of metamorphs exposed to R. bakeri larvae. Furthermore, metamorphs exposed to R. ranae larvae carried larger adult female worms in their lungs. Some variation in infection measures depended on host sex, but only for one parasite species considered. Overall, the differential establishment and reproductive potential of R. ranae and R. bakeri in L. pipiens suggests co-adaptation
Methodology
Frog collection and tank rearing
Portions of four L. pipiens egg masses were collected from Old Chelsea, (39° 50′N, 36° 42′W) on May 1, 2005. Each egg mass was individually housed in aquaria with pond water that was aerated by an airstone in the laboratory. Tadpoles were fed a granular mixture of tadpole food (Ward’s Nat. Sci. Tadpole Food # 88V 6534) and Hagen Fishflakes (Nutrafin Max Complete Flake Food #668672) until they had developed to Gosner stage 25 (Gosner, 1960). At stage 25 they were introduced to outdoor Rubbermaid© tanks. Equal numbers of tadpoles from each egg mass were allocated to each tank to a total of 30 individuals in each of 13 tanks. Tanks were established in a fenced compound on the Carleton University campus (45° 23′N, 75° 42′W). Each tank was filled with 300 L of water and covered with 40% shadecloth to prevent oviposition by invertebrates, eliminate predation of the tadpoles by wildlife, and prevent the escape of the frogs from the tanks. Each tank was seeded with leaf litter collected from a mixed deciduous forest stand and populations of Daphnia spp. collected from a local pond. Water levels were maintained at 300 L by the removal of water after heavy rainfalls.
Frog husbandry
Metamorphs were removed from the tanks when they developed forelimbs (Gosner stage 42) and brought indoors into a quiet room. Details of individual frog husbandry and feeding during the experiment are outlined in Dare and Forbes (2008).
To obtain worm larvae for infection experiments, five wild-caught adult L. pipiens from Ottawa (45° 23′N, 75° 42′W) and five L. sylvatica from Bishops Mills (44° 53′N, 75° 40′W) were housed in the laboratory. We collected worms from different host species from different locations to increase the chances of our finding genetically different worms. We housed each frog individually in 2.5 L glass aquaria in the laboratory. Each aquarium was lined with a damp paper towel and an overturned Petri dish at one end as a dry post. The temperature in the room was maintained between 23 and 25 °C. We fed each frog three to five large crickets daily and dusted the crickets in amphibian multivitamin mix (Herptivite, Rep-Cal Research Labs, CA, USA) every other day. Two additional adult frogs of each species (and locality) were captured and necropsied to obtain adult worms for PCR and sequencing. These worm specimens were preserved in 95% ethanol and stored at −20 °C prior to nucleic acid extraction.