Catherine R. Bevier

Purification and characterization of antimicrobial peptides from the skin secretions of the carpenter frog Rana virgatipes (Ranidae, Aquarana).

The members of the Aquarana (or Rana catesbeiana species group) form a well-supported monophyletic clade but phylogenetic relationships between species within the group are incompletely understood. Peptides that differentially inhibited the growth of bacteria were purified from electrically stimulated skin secretions of the carpenter frog Rana virgatipes. Structural characterization identified members of the ranatuerin-2 (3 peptides) and temporin (3-peptides) families, previously found in the skins of R. catesbeiana, R. clamitans, R. grylio and R. septentrionalis. Ranalexin, a peptide previously found only in the Aquarana, was isolated together with a variant (FFGLHNLVPSMLCVVRKKC) that lacks the propensity to adopt an alpha-helical conformation and so was devoid of antimicrobial activity. Two C-terminally alpha-amidated peptides belonging to the brevinin-2 family were isolated from the skin secretions that, like an ortholog from R. septentrionalis, lacked the C-terminal cyclic heptapeptide domain associated with members of this family. Ranatuerin-1, previously isolated from R. catesbeiana, R. clamitans and R. grylio but absent from R. septentrionalis, was also not identified in R. virgatipes. Synthetic replicates of temporin-1Va (FLSSIGKILGNLL.NH2), temporin-IVb (FLSIIAKVLGSLF.NH2) and temporin-1Vc (FLPLVTMLLGKLF.NH2) potently inhibited growth of Gram-positive bacteria (including methicillin-resistant Staphylococcus aureus). Temporin-1Va was also active against Gram-negative bacteria and the opportunistic yeast pathogen Candida albicans and had relatively weak hemolytic activity (LD50=120 microM) and may therefore represent a candidate for drug development. Our data support the placement of R. virgatipes in the Aquarana and indicate a closer phylogenetic relationship of R. virgatipes with R. septentrionalis than with R. catesbeiana, R. clamitans and R. grylio.

Peptidomic analysis of skin secretions from Rana heckscheri and Rana okaloosae provides insight into phylogenetic relationships among frogs of the Aquarana species group.

The members of the Aquarana (or Rana catesbeiana species group) form a monophyletic group comprising seven species: R. catesbeiana, Rana clamitans, Rana grylio, Rana virgatipes, Rana septentrionalis, Rana heckscheri and Rana okaloosae. Previous work has led to structural characterization of the antimicrobial peptides present in electrically-stimulated skin secretions from the first five species listed and this study presents the primary structures of orthologs from the river frog R. heckscheri and the Florida bog frog R. okaloosae. Peptidomic analysis of R. heckscheri and R. okaloosae skin secretions led to the identification of peptides with antimicrobial activity belonging to the ranalexin, ranatuerin-2, and temporin families. In addition, a peptide (GFLDIIKDTGKDFAVKILNNLKCKLAGGCPR) was isolated from R. okaloosae whose primary structure identified it as a member of the palustrin-2 family. Consistent with previous data based upon morphological analysis and comparisons of the nucleotide sequences of mitochondrial and ribosomal genes, cladistic analysis based upon a comparison of the amino acid sequences of antimicrobial peptides indicates a sister-group relationship between R. heckscheri and R. grylio and a close, but less well defined, phylogenetic relationship between R. okaloosae and R. clamitans.

Peptidomic analysis of skin secretions supports separate species status for the tailed frogs, Ascaphus truei and Ascaphus montanus

The tailed frog Ascaphus truei Stejneger, 1899 is the most primitive extant anuran and the sister taxon to the clade of all other living frogs. The species occupies two disjunct ranges in the Northwest region of North America: the Cascade Mountains and coastal area from British Columbia to Northern California, and an inland range in the northern Rocky Mountains and the Blue and Wallowa mountains. A previous study led to the isolation of eight peptides with antimicrobial activity (termed the ascaphins) from skin secretions of A. truei from the coastal range. The present study has used peptidomic analysis to identify the products of orthologous ascaphin genes in electrically-stimulated skin secretions from inland range specimens. Structural characterization of the peptides demonstrated that ascaphins from the inland range contained the following amino acid substitutions compared with orthologs from the coastal range frogs: ascaphin-1 (Ala(12)-->Glu), ascaphin-3 (Asp(4)-->Glu), ascaphin-4 (Ala(19)-->Ser), ascaphin-5 (Lys(12)-->Thr), and ascaphin-7 (Gly(8)-->Ser and Ser(20)-->Asn). Orthologs of ascaphins-2, -6, and -8 were not identified but a paralog of ascaphin-5, identical to ascaphin-5 from coastal range frogs, was found. The data support the claims, derived from analysis of the nucleotide sequences of mitochondrial genes, that the inland populations of the tailed frog should be recognized as a distinct species, the Rocky Mountain tailed frog Ascaphus montanus and that the divergence of the species from A. truei probably occurred in the late Miocene (approximately 10 Mya).

Chorus Attendance and Site Fidelity in the Mink Frog, Rana septentrionalis: Are Males Territorial?

Abstract The Mink Frog, Rana septentrionalis, is closely related and behaviorally similar to the Green Frog, Rana clamitans, and the Bullfrog, Rana catesbeiana, but has a much smaller geographical range and is not as well known. We used mark-recapture and observation techniques to estimate male chorus attendance and site fidelity during the breeding season. We captured, measured, and individually marked 139 males in 2002 and 85 males in 2003. Each males initial and subsequent positions were recorded using a Global Positioning System. About half the males were never recaptured in either year. Most of the remaining males were recaptured only four times; two males were recaptured more than 10 times. Males were recaptured from subsequent positions as far apart as 90 m and as close as 0.6 m. Males recaptured on consecutive observation nights were in positions less than 15 m apart on average. Although aggressive calling, chasing, and fighting occur when males encounter each other, there is no evidence that male Mink Frogs defend territories over prolonged periods as in Green Frogs and Bullfrogs.

The Effect of Parasite Infection on Phonotactic Response in the Mink Frog, Lithobates septentrionalis

Abstract The detrimental effects of parasite infection include modulation of behaviors important to host fitness. The Mink Frog, Lithobates septentrionalis, is the final host of the digenean flatworm, Halipegus eccentricus, which inhabits the eustachian tube. Extreme infection results in complete occlusion of the eustachian tubes and could adversely affect a frogs hearing. The tympanic membranes are coupled internally through the buccal cavity via open eustachian tubes, making them vulnerable to pressure changes induced by obstruction from the presence of H. eccentricus, accumulated necrotic tissue, and mucus resulting from the infection. We tested phonotactic response in male L. septentrionalis to determine whether acoustic response is affected by infection of H. eccentricus. We placed frogs, in turn, in a floating choice arena, gave them 10 min to acclimate and 10 min to respond to a conspecific advertisement call broadcast from a speaker. We tracked the frogs and recorded positive phonotaxis when the frog approached the broadcast speaker. We measured individual frogs, checked them noninvasively for infection, and released them at the capture site. Infection rates were moderate: 43% of males had at least one H. eccentricus in one or both sides. Parasite infection significantly affected phonotaxis response; parasite-free males exhibited significantly more, and parasitized males significantly fewer, positive responses than expected. Although response measures, including time to response, did not differ significantly between the two groups of males, this behavioral assay provides good evidence that infection by H. eccentricus affects a frogs ability to localize and respond to a conspecific call.

Assessing LakeSmart, a community-based lake protection program

Anthropogenic impacts, particularly shoreline development and related nutrient runoff, threaten Maine lakes. The LakeSmart program was created in response to these threats to promote the use of lake-friendly landscaping practices. We used 237 surveys and 8 stakeholder interviews to investigate motivations that drive conservation behavior among lakeshore residents, to explore the effectiveness of criteria used for LakeSmart evaluations and to identify potential areas for improvement of its structural design and marketing strategies. LakeSmart participants were more likely than non-participants to recognize the threat of declining water quality, to adopt or enhance existing lake-friendly landscaping best management practices, and to help foster a strong sense of community. We conclude that the LakeSmart program could be strengthened with enhanced training for inspectors through increased “job-shadowing” opportunities with experienced inspectors, expanded mentoring for owners whose properties do not pass the initial LakeSmart evaluation, and by eliminating ambiguous wording in the property evaluation form. Partnerships with member-rich groups and identifying ways to reduce costs of recommended mitigation actions could increase program participation. The defined parameters of the property evaluation, along with recommendations to strengthen the evaluation process, can result in greater citizen participation and ultimately improved lake ecosystem health measured by fewer algal blooms and greater water clarity. LakeSmart could serve as a model for other conservation programs attempting to transform knowledge to action.

Ecology and Evolution in Tropical Herpetofauna

Donnelly, Maureen A., Brian I. Crother, Craig Guyer, Marvalee H. Wake, and Mary E. White, editors. 2005. Ecology and evolution in the tropics: a herpetological perspective. The University of Chicago Press, Chicago, Illinois. xv + 675 p.

Purification and characterization of antimicrobial peptides from the skin secretions of the mink frog (Rana septentrionalis).

95.00 (cloth), ISBN: 0-226-15657-5 (alk. paper);

Antimicrobial properties of two purified skin peptides from the mink frog (Rana septentrionalis) against bacteria isolated from the natural habitat.

45.00 (paper), ISBN: 0-226-15658-3 (alk. paper).