Rana Septentrionalis Research Articles

Early Life of the Mink Frog (Rana septentrionalis): From Fertilization to Metamorphosis

Mink Frogs (Rana septentrionalis) are a unique ranid species restricted to Canada and the northern edge of the United States, from northern Minnesota to northern Maine. They are a member of the Aquarana clade that includes Green Frogs (R. clamitans), American Bullfrogs (R. catesbeiana), and four other species. Despite being relatively common where present, the biology of this species has been poorly studied and little in particular is known about its breeding and development from fertilization through overwintering as larvae. Critically, the species' representation in museum collections is limited in general, but particularly at early life stages. Here we report on our initial efforts to describe larval Mink Frog development by inducing breeding of wild-caught adults in the laboratory, then sampling tadpoles from fertilization until the subsequent spring. Specimens, including tissue samples, adult specimens of both sexes, an entire laboratory-induced egg mass, and a captive-bred larval series are available in the Yale Peabody Museum of Natural History. Our approach here demonstrates that Mink Frogs can be captive bred for use in laboratory experiments, and our work provides a novel larval series from egg mass to metamorphosis for this secretive, understudied species.

Terrestrial dispersal of juvenile Mink Frog (<i>Lithobates septentrionalis</i>) in Algonquin Provincial Park, Ontario

Dispersal following metamorphosis is critical for sustaining anuran metapopulations. Mink Frog (Lithobates septentrionalis) is a primarily aquatic species that is common in eastern Canada. The species is not well studied, and little is known about the terrestrial dispersal of recently metamorphosed individuals. Here we present our observations on the phenology of terrestrial activity in recently metamorphosed Mink Frogs in Algonquin Provincial Park, Ontario, Canada. Despite a sampling effort of over 26 000 trap nights over two years (2010 and 2011) in an area with a known population of Mink Frogs, we observed only 35 individuals, all of which were recent metamorphs, in late summer 2011, suggesting annual variability of recruitment. Because all Mink Frogs were observed in a riparian area, it is likely that this species uses riparian corridors to disperse toward other wetlands, thus avoiding forested areas.

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 frog's 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 ...

Initial Dispersal and Breeding Habitat Use of Newly Introduced Mink Frogs in Western Newfoundland, Canada

Insular Newfoundland has no native amphibians. While global amphibian populations are declining at alarming rates, populations of introduced anurans (frogs and toads) continue to expand in western Newfoundland, Canada. We expected the establishment and dispersal of the most recently introduced species, Mink Frog (Lithobates septentrionalis), to be influenced by the presence in the region of the previously introduced and ecologically similar Green Frog (L. clamitans). We used a combination of anuran calling surveys and pond-edge visual surveys to assess the ongoing dispersal, relative regional distribution, and local habitat use for these two species in western Newfoundland. The recently established Mink Frog has dispersed ∼3.8 km/year northeast from the original (2001) discovery location and ∼2.6 km/year southwest; co-occurrence analyses revealed that this population displayed habitat and spatial separation from long-established Green Frog populations, at both landscape and local scales. Although cause an...

Identification and Expression of a Putative Facilitative Urea Transporter in Three Species of True Frogs (Ranidae): Implications for Terrestrial Adaptation

Urea transporters (UTs) help mediate the transmembrane movement of urea and therefore are likely important in amphibian osmoregulation. Although UTs contribute to urea reabsorption in anuran excretory organs, little is known about the protein’s distribution and functions in other tissues, and their importance in the evolutionary adaptation of amphibians to their environment remains unclear. To address these questions, we obtained a partial sequence of a putative UT and examined relative abundance of this protein in tissues of the wood frog (Rana sylvatica), leopard frog (R. pipiens), and mink frog (R. septentrionalis), closely related species that are adapted to different habitats. Using immunoblotting techniques, we found the protein to be abundant in the osmoregulatory organs but also present in visceral organs, suggesting that UTs play both osmoregulatory and nonosmoregulatory roles in amphibians. UT abundance seems to relate to the species’ habitat preference, as levels of the protein were higher in the terrestrialR. sylvatica, intermediate in the semiaquaticR. pipiens, and quite low in the aquaticR. septentrionalis. These findings suggest that, in amphibians, UTs are involved in various physiological processes, including solute and water dynamics, and that they have played a role in adaptation to the osmotic challenges of terrestrial environments.

Fatty Acid Composition of Muscle Tissue Measured in Amphibians Living in Radiologically Contaminated and Non-Contaminated Environments

Fatty acid composition was identified as a potential biological indicator of the effects of environmental exposure to radiological contaminants. This end point was measured in muscle tissues of Mink frogs ( Rana septentrionalis ) obtained from a radiologically contaminated pond and from a non-contaminated pond. It was also measured after the frogs obtained from both ponds were exposed to a 4 Gy (60)Co γ radiation dose delivered in vivo at a dose rate of approximately 8 Gy/min. Statistically significant differences for the increase of a couple of polyunsaturated omega-3 fatty acid residues and the decrease of a polyunsaturated omega-6 fatty acid residue were observed between radiologically contaminated and non-contaminated frogs, indicating a partial remodeling of muscle lipids in response to a chronic low-dose tritium exposure. The effects of an acute high-dose exposure to (60)Co γ radiation, either for the radiologically contaminated or non-contaminated frogs indicated fast post-irradiation fatty acid changes with an increase of polyunsaturated and decrease of saturated fatty acid contents. Fatty acid composition was found to be a sensitive marker that may be useful to study and monitor biota health in environments that are radiologically contaminated, as well as for understanding the differences between low chronic and high acute stress responses.

Evaluation of Listener-Based Anuran Surveys with Automated Audio Recording Devices

Volunteer-based audio surveys are used to document long-term trends in anuran community composition and abundance. Current sampling protocols, however, are not region- or species-specific and may not detect relatively rare or audibly cryptic species. We used automated audio recording devices to record calling anurans during 2006–2009 at wetlands in Maine, USA. We identified species calling, chorus intensity, time of day, and environmental variables when each species was calling and developed logistic and generalized mixed models to determine the time interval and environmental variables that optimize detection of each species during peak calling periods. We detected eight of nine anurans documented in Maine. Individual recordings selected from the sampling period (0.5 h past sunset to 0100 h) described in the North American Amphibian Monitoring Program (NAAMP) detected fewer species than were detected in recordings from 30 min past sunset until sunrise. Time of maximum detection of presence and full chorusing for three species (green frogs, mink frogs, pickerel frogs) occurred after the NAAMP sampling end time (0100 h). The NAAMP protocol’s sampling period may result in omissions and misclassifications of chorus sizes for certain species. These potential errors should be considered when interpreting trends generated from standardized anuran audio surveys.

Prey detection of aquatic predators: Assessing the identity of chemical cues eliciting prey behavioral plasticity

Chemical cues transmitted through the environment are thought to underlie many prey responses to predation risk, but despite the known ecological and evolutionary significance of such cues, their basic composition are poorly understood. Using anuran tadpoles (prey) and dragonfly larvae (predators), we identified chemical cues associated with predation risk via solid phase extraction and mass spectrometry of the extracts. We found that dragonfly larvae predators consistently produced a negative ion, m/z 501.3, when they fed on bullfrog ( Rana catesbeiana) and mink frog ( Rana septentrionalis) tadpoles, but this ion was absent when dragonflies were fasted or fed invertebrate prey. When tadpole behavioral responses to dragonfly chemical cues were examined, tadpoles reduced their activity, particularly in response to dragonflies feeding on tadpoles. Furthermore, a negative correlation was noted between the level of tadpole activity and the concentration of the m/z 501.3 compound in dragonfly feeding trials, indicating that this ion was possibly responsible for tadpole anti-predator behavior.

Antimicrobial Properties of Brevinin‐2‐Related Peptide and its Analogs: Efficacy Against Multidrug‐Resistant Acinetobacter baumannii

Brevinin-2 related peptide (B2RP; GIWDTIKSMG(10)KVFAGKILQN(20)L.NH(2)), first isolated from skin secretions of the mink frog Lithobates septentrionalis, shows broad-spectrum antimicrobial activity but its therapeutic potential is limited by moderate hemolytic activity. The peptide adopts an alpha-helical conformation in a membrane-mimetic solvent but amphipathicity is low. Increasing amphipathicity together with hydrophobicity by the substitutions Lys(16)-->Leu and Lys(16)-->Ala increased hemolytic activity approximately fivefold without increasing antimicrobial potency. The substitution Leu(18)-->Lys increased both cationicity and amphipathicity but produced decreases in both antimicrobial potency and hemolytic activity. In contrast, increasing cationicity of B2RP without changing amphipathicity by the substitution Asp(4)-->Lys resulted in a fourfold increase in potency against Escherichia coli [minimal inhibitory concentration (MIC) = 6 microm) and twofold increases in potency against Staphylococcus aureus (MIC = 12.5 microm) and Candida albicans (MIC = 6 microm) without changing significantly hemolytic activity against human erythrocytes (LC(50) = 95 microm). The emergence of antibiotic-resistant strains of the Gram-negative bacterium Acinetobacter baumannii constitutes a serious risk to public health. B2RP (MIC = 3-6 microm) and [Lys(4)]B2RP (MIC = 1.5-3 microm) potently inhibited the growth of nosocomial isolates of multidrug-resistant Acinetobacter baumannii. Although the analogs [Lys(4), Lys(18)]B2RP and [Lys(4), Ala(16), Lys(18)]B2RP showed reduced potency against Staphylococcus aureus, they retained activity against Acinetobacter baumannii (MIC = 3-6 microm) and had very low hemolytic activity (LC(50) > 200 microm).

Parasites of the Mink Frog (Rana septentrionalis) from Minnesota, U.S.A

Twenty-two mink frogs, Rana septentrionalis, collected from two locations in Minnesota, United States, were examined for helminth and protozoan blood parasites in July 1999. A total of 16 parasite taxa were recovered including 5 larval digenean trematodes, 7 adult digenean trematodes, 3 nematodes, and 1 Trypanosoma species. Infracommunities were dominated by the digeneans in terms of richness and abundance. In particular, echinostomatid metacercariae in the kidneys of frogs were the most common parasites found, infecting 100% of the frogs and consisting of about 90% of all helminth individuals recovered. Gorgodera amplicava, Gorgoderina multilobata, Haematoloechus parviplexus, Haematoloechus breviplexus, Cosmocercoides dukae, and Oswaldocruzia pipiens represent new host records. The survey presented here represents the second known helminth survey of mink frogs conducted in North America. A summary of metazoan parasites reported from mink frogs is included.

Interactions between climate, beaver activity, and pond occupancy by the cold-adapted mink frog in New York State, USA

Mink frogs ( Rana septentrionalis) are cold-adapted, climate-sensitive amphibians occurring above the 43rd parallel in North America. We conducted repeated night call surveys at 46 ponds near the southern edge of the species’ range to collect presence/absence data and used information-theoretic models to relate pond occupancy to historical climate and habitat factors. Mink frog occurrence was strongly and positively influenced by pond size (>1.5 ha) and the presence of beavers ( Castor canadensis). Presence of calling mink frogs decreased sharply at sites with historical mean July air temperatures >19.5 °C. Our results suggest that predicted changes in temperature over the next century might reduce the range of mink frogs in New York, yet we hypothesize that any such reductions could be mitigated by generation of suitable breeding habitat associated with future expansion of beaver populations.

Predator diet and prey adaptive responses: Can tadpoles distinguish between predators feeding on congeneric vs. conspecific prey?

Predator diet can play an important role in facilitating detection of predation risk among prospective prey, and such detection should have adaptive significance in reducing mortality in environments where not all predators confer similar risk. In the laboratory, we tested behavioural and morphological responses of tadpoles from two congeneric frog species (bullfrog ( Rana catesbeiana Shaw, 1802) and mink frog ( Rana septentrionalis Baird, 1854)) to cues from an odonate predator (genus Aeshna Fabricius, 1775). In a separate experiment we found that both frog species had similar baseline vulnerability to Aeshna predation, implying that species’ responses to predators feeding on conspecific vs. congeneric prey also would be similar. Both species reduced their activity in the presence of predators feeding on tadpoles of either species vs. those fed invertebrates (Libellulidae) or not subjected to predators (controls). Bullfrog tadpoles grew bigger than controls when exposed to predators fed mink frog tadpoles only, whereas mink frogs failed to show a comparable response. Neither species exhibited changes in shape that were attributable to predator diet. Our results suggest that closely related frog species do not distinguish between predators feeding on conspecific vs. congeneric prey, implying that selection favours generalized antipredator responses when prey species are subject to similar predation risk.

Are Anurans of Great Lakes Coastal Wetlands Reliable Indicators of Ecological Condition?

Frogs and toads (anurans) are sensitive to a variety of anthropogenic stressors and are widely suggested as indicators of ecological condition. We surveyed 220 coastal wetlands along the U.S. shores of the Laurentian Great Lakes and quantified relationships between presence of anuran species and degree of anthropogenic disturbance. Results were used to derive explicit, functional relationships between environmental condition and anuran occurrences. These functions were subsequently used to calculate a multi-species indicator of ecological condition at other (novel) wetlands. Of 14 anuran species observed, spring peeper (Pseudacris crucifer) exhibited the strongest and most consistent relationship with environmental condition across the entire study area. Other species exhibited significant relationships with the environmental gradient, but the direction of association varied geographically or the overall species abundance was very low (e.g., mink frog, Rana septentrionalis). Even if applied to separate ecological provinces (Laurentian Mixed Forest or Eastern Deciduous Forest), multi-species estimates of wetland condition based on anurans are not much better indicators of environmental condition based on human disturbance than are indices based solely on occurrence of spring peeper. Nevertheless, indicators grounded in explicit relationships with environmental stress are superior to traditional measures (e.g., species richness) that combine species with different responses to the stress gradient. At least one anuran species (spring peeper) can contribute meaningfully to the assessment of ecological condition in Great Lakes coastal wetlands; its value as an indicator will be improved if it can be combined with information from other wetland species such as birds, fishes, and vascular plants.

Ambient Solar UV Radiation Causes Mortality in Larvae of Three Species of Rana Under Controlled Exposure Conditions†¶

Recent reports concerning the lethal effects of solar ultraviolet-B (UV-B) (290–320 nm) radiation on amphibians suggest that this stressor has the potential to impact some amphibian populations. In this study embryos and larvae of three anuran species, Rana pipiens,Rana clamitans and Rana septentrionalis, were exposed to full-spectrum solar radiation and solar radiation filtered to attenuate UV-B radiation or UV-B and ultraviolet-A (UV-A) (290–380 nm) radiation to determine the effects of each wavelength range on embryo and larval survival. Ambient levels of solar radiation were found to be lethal to all three species under exposure conditions that eliminated shade and refuge. Lethality was ameliorated by filtration of UV-B radiation alone, demonstrating that ambient UV-B radiation is sufficient to cause mortality. Although several studies have qualitatively demonstrated the lethality of UV-B to early life stage amphibians this study demonstrates that the larval life stages of the three species tested are more sensitive than the embryonic stages. This suggests that previous reports that have not included the larval life stage may underestimate the risk posed to some anuran populations by increasing UV-B exposure. Furthermore, this study reports quantitative UV-B dosimetry data, collected in conjunction with the exposures, which can be used to begin the assessment of the impact of environmental changes which increase UV-B exposure of these anurans.

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

Numerous peptides exhibiting antimicrobial properties have been isolated from the skins of many amphibian species. These peptides offer an innate chemical defense system against various microbial agents that exist in the amphibian's environment. Amphibian skin peptides are typically tested for antimicrobial activity against microbial strains that are pathogenic to humans, but not on potential pathogenic or opportunistic bacteria that exist in the organism's habitat. Two peptides, a brevinin-2-related peptide and temporin-1SPb previously isolated from secretions of the mink frog, Rana septentrionalis, were tested for antimicrobial activity on bacterial isolates endemic to the frog's habitat. Ten isolates were identified, using 16S rRNA gene sequencing techniques, in the genera Pseudomonas, Serratia, Bacillus, Aeromonas, Burkholderia, Microbacterium, and Delftia. Bacterial isolates were tested with peptides at concentrations ranging from 0.8 μM to 1000 μM to determine the minimum inhibitory concentration (MIC) to inhibit growth. Growth of four of the isolates was inhibited by temporin-1SPb at the concentrations used, but all of the isolates were inhibited by the brevinin-2-related within the range of peptide concentrations used. This demonstrates the efficacy of both peptides as a component of the frog's innate chemical defense system.

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.

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

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 male's 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.

Calling periods for frogs and toads near Pinawa, Manitoba, with an update on mink frog and green frog distribution

Calling periods for frogs and toads near Pinawa, Manitoba, with an update on mink frog and green frog distribution

Effects of the synthetic estrogen ethinylestradiol on early life stages of mink frogs and green frogs in the wild and in situ

Estrogenic contaminants are known to disrupt growth and development in amphibians. Field-based research is needed to elucidate their potential impacts on wild populations. Hatch success, larval growth and development rates, and gonad development were examined in native amphibians exposed to low ng/L concentrations of 17alpha-ethinylestradiol (EE2) in a whole-lake addition experiment at the Experimental Lakes Area, northwestern Ontario, Canada. Egg masses were reared in situ in the EE2-amended lake and in two reference lakes in 2001 and 2002. Hatching success was reduced significantly in green frogs (Rana clamitans) but not in mink frogs (Rana septentrionalis) exposed to EE2. Ethinylestradiol had no consistent effect on mass or development stage of hatchlings in the early larval stages of the caging study. Ethinylestradiol had no effect on sex ratios of either species in situ, and no intersex gonads were observed in exposed or reference green frog tadpoles or in reference mink frog tadpoles. However, 5.6% (total n = 18) and 12.5% (total n = 56) of EE2-exposed mink frog tadpoles were intersex in the 2001 and 2002 caging studies, respectively. Wild mink frog tadpoles also were examined, and EE2 had no effect on sex ratios. No intersex gonads were observed in reference lake tadpoles or in tadpoles from the experimental lake prior to EE2 additions; however, 2.4, 0, and 28.6% of wild EE2-exposed first-year tadpoles had intersex gonads (2001, 2002, and 2003, respectively). These results indicate that exposure to EE2 in the wild and in situ at concentrations comparable to those detected in effluents and, occasionally, in surface waters can impact gonad development and hatch success in native amphibians.

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

Skin secretions were obtained from male, female, and juvenile specimens of the mink frog ( Rana septentrionalis) by electric stimulation and shown to contain 10 peptides that differentially inhibited the growth of microorganisms. The elution profiles of secretions from the three groups following reverse-phase HPLC were almost identical indicating that there were no major sexual or developmental differences in chemical composition. Four peptides of the brevinin-1 family, with potent antimicrobial activity and strong hemolytic activity, two members of ranatuerin-2 family and three members of the temporin family, were purified and characterized structurally. A 21-amino-acid C-terminally α-amidated peptide (GIWDTIKSMGKVFAGKILQNL.NH 2) with broad-spectrum antimicrobial activity was also isolated from the skin secretions. This peptide shows limited structural similarity with the N-terminal region of brevinin-2 peptides previously isolated from R. temporaria skin but lacks the C-terminal cyclic heptapeptide domain associated with this family. Molecular and morphological data support the placement of R. septentrionalis in the R. catesbeiana species group, but analysis based upon the distribution of the molecular forms of the antimicrobial peptides is indicative of a closer phylogenetic relationship between R. septentrionalis and the frogs of the R. pipiens and R. boylii groups.