Toe Clips Research Articles

Dynamics of Amphibian Pathogen Detection Using Extended Museum Specimens.

Natural history collections have long served as the foundation for understanding our planet's biodiversity, yet they remain a largely untapped resource for wildlife disease studies. Extended specimens include multiple data types and specimen preparations that capture the phenotype and genotype of an organism and its symbionts-but preserved tissues may not always be optimized for downstream detection of various pathogens. Frogs are infected by an array of pathogens including Batrachochytrium dendrobatidis (Bd), Ranavirus (Rv), and Amphibian Perkinsea (Pr), which provides the opportunity to study differences in detection dynamics across tissue types. We used quantitative PCR protocols to screen two tissue types commonly deposited in museum collections, toe clips and liver, from two closely related host species, Rana catesbeiana and Rana clamitans. We compared Bd, Rv, and Pr infection prevalence and intensity between species and tissue types and found no significant difference in prevalence between species, but Bd intensity was higher in R. clamitans than R. catesbeiana. Toe tissue exhibited significantly higher Bd infection loads and was more useful than liver for detecting Bd infections. In contrast, Rv was detected from more liver than toe tissues, but the difference was not statistically significant. Our results support the use of extended specimen collections in amphibian disease studies and demonstrate that broader tissue sampling at the time of specimen preparation can maximize their utility for downstream multipathogen detection.

Comparing skin swabs, buccal swabs, and toe clips for amphibian genetic sampling, a case study with a small anuran (Acris blanchardi).

Multiple methods for collecting genetic samples from amphibians exist, each with their own implications for study design, animal welfare, and costs. Toe clipping is one common method, but there is ongoing debate regarding its potential detriment. Less invasive methods should be implemented, if efficacious, as amphibians are a particularly vulnerable vertebrate group. Skin and buccal swabbing are less invasive methods for genetic sampling, but the potential for contamination and a lower yield of DNA may exist. To compare these methods, we gathered skin swabs, buccal swabs, and toe clips from the same individuals of a relatively small anuran species, Blanchard's Cricket Frog (Acris blanchardi). We then compared DNA yield, DNA purity, amplification success rate, and genotypic data quality among sample types. We found toe clips and buccal swabs generated similar DNA yield and purity, with skin swabs yielding significantly less DNA of significantly lower purity than the other sample types. Amplification success rate was significantly higher using toe clips compared to the other sample types, though buccal swab samples amplified more readily than skin swabs. Genotypic data from toe clips and buccal swabs did not differ significantly in quality, but skin swab data quality was significantly lowest among sample types. Thus, skin swabbing could produce erroneous data in some situations, but buccal swabbing is likely an effective substitute to toe clipping, even for small species. Our results can help future researchers select which genetic sampling method might best suit their research needs.

Non-Lethal Detection of Frog Virus 3-Like (RUK13) and Common Midwife Toad Virus-Like (PDE18) Ranaviruses in Two UK-Native Amphibian Species.

Ranaviruses have been involved in amphibian mass mortality events worldwide. Effective screening to control this pathogen is essential; however, current sampling methods are unsuitable for the detection of subclinical infections. Non-lethal screening is needed to prevent both further spread of ranavirus and losses of at-risk species. To assess non-lethal sampling methods, we conducted two experiments: bath exposing common frogs to RUK13 ranavirus at three concentrations, and exposing common toads to RUK13 or PDE18. Non-lethal sampling included buccal, digit, body and tank swabs, along with toe clips and stool taken across three time-points post-exposure. The presence/load of ranavirus was examined using quantitative PCR in 11 different tissues obtained from the same euthanised animals (incl. liver, gastro-intestinal tract and kidney). Buccal swab screening had the highest virus detection rate in both species (62% frogs; 71% toads) and produced consistently high virus levels compared to other non-lethal assays. The buccal swab was effective across multiple stages of infection and differing infection intensities, though low levels of infection were more difficult to detect. Buccal swab assays competed with, and even outperformed, lethal sampling in frogs and toads, respectively. Successful virus detection in the absence of clinical signs was observed (33% frogs; 50% toads); we found no difference in detectability for RUK13 and PDE18. Our results suggest that buccal swabbing could replace lethal sampling for screening and be introduced as standard practice for ranavirus surveillance.

Can early treatment of twitcher mice with high dose AAVrh10-GALC eliminate the need for BMT?

Introduction: Krabbe disease (KD) is an autosomal recessive disorder caused by mutations in the galactocerebrosidase (GALC) gene resulting in neuro-inflammation and defective myelination in the central and peripheral nervous systems. Most infantile patients present with clinical features before six months of age and die before two years of age. The only treatment available for pre-symptomatic or mildly affected individuals is hematopoietic stem cell transplantation (HSCT). In the animal models, combining bone marrow transplantation (BMT) with gene therapy has shown the best results in disease outcome. In this study, we examine the outcome of gene therapy alone. Methods: Twitcher (twi) mice used in the study, have a W339X mutation in the GALC gene. Genotype identification of the mice was performed shortly after birth or post-natal day 1 (PND1), using polymerase chain reaction on the toe clips followed by restriction enzyme digestion and electrophoresis. Eight or nine-day-old affected mice were used for gene therapy treatment alone or combined with BMT. While iv injection of 4 × 1013 gc/kg of body weight of viral vector was used originally, different viral titers were also used without BMT to evaluate their outcomes. Results: When the standard viral dose was increased four- and ten-fold (4X and 10X) without BMT, the lifespans were increased significantly. Without BMT the affected mice were fertile, had the same weight and appearance as wild type mice and had normal strength and gait. The brains showed no staining for CD68, a marker for activated microglia/macrophages, and less astrogliosis than untreated twi mice. Conclusion: Our results demonstrate that, it may be possible to treat human KD patients with high dose AAVrh10 without blood stem cell transplantation which would eliminate the side effects of HSCT.

The effect of cycling shoes and the shoe-pedal interface on maximal mechanical power output during outdoor sprints

Cyclists and industry professionals believe that cycling shoes with pedal attachment and stiff soles improve performance. However, scientific evidence has demonstrated that cycling shoes have no significant effect on metabolic cost during low-intensity, submaximal, steady-state cycling (50–150 W). Here, we investigated if stiff-soled cycling shoes combined with clip-in pedals provide benefits relevant to sprint cycling. We measured the mechanical power outputs and velocities of twelve healthy male cyclists during maximal sprint cycling. Participants rode outdoors on a paved asphalt road with a steady, uphill gradient of 4.9%. Each participant completed sets of three 100-metre cycling sprints in three conditions: (1) running shoes with flat pedals (no pedal attachment + flexible soles), (2) running shoes with classic aluminium quill pedals with toe clips and straps (pedal attachment + flexible soles), and (3) cycling shoes with clip-in pedals (pedal attachment + stiff soles). When using the running shoes, the toe clip attachment increased maximum sprint power by 9.7 ± 8.7% (p = 1.7E − 03). Maximum sprint power was 16.6 ± 10.2% (p = 3.25E − 06) greater for the stiff-soled cycling shoes combined with clip-in pedals compared to the flexible running shoes with toe clips condition, presumably due to the greater sole stiffness of the cycling shoes. Shoe-pedal attachment and stiff soles each positively improve cycling performance during high-power, uphill sprints.

Investment of both essential fatty and amino acids to immunity varies depending on reproductive stage.

Trade-offs among the key life-history traits of reproduction and immunity have been widely documented. However, the currency in use is not well-understood. We investigated how reproducing female side-blotched lizards, Uta stansburiana, allocate lipids versus proteins when given an immune challenge. We tested whether lizards would invest more in reproduction or immunity depending on reproductive stage. Females were given stable isotopes (15 N-leucine and 13 C-1-palmitic acid), maintained on a regular diet and given either a cutaneous biopsy or a sham biopsy (control). Stable isotopes were monitored and analyzed in feces and uric acid, skin biopsies, eggs, and toe clips. We found that lizards deposited both proteins and lipids into their healing wounds (immune-challenged), skin (control), and eggs (all) and that catabolism of proteins exceeded incorporation into tissue during wound-healing. Specifically, we found that healed biopsies of wounded animals had more leucine and palmitic acid than the nonregrown skin biopsies taken from unwounded control animals. Earlier in reproduction, lizards invested relatively more labeled proteins into healing their wound tissue, but not into unwounded skin of control animals. Thus, reproduction is sometimes favored over self-maintenance, but only in later reproductive stages. Finally, we documented positive relationships among the amount of palmitic acid deposited in the eggs, the amount of food eaten, and the amount of palmitic acid excreted, suggesting higher turnover rates of lipids in lizards investing highly in their eggs.

Ranavirus infection dynamics and shedding in American bullfrogs: consequences for spread and detection in trade.

American bullfrogs Lithobates catesbeianus are thought to be important in the global spread of ranaviruses-often lethal viruses of cold-blooded vertebrates-because they are commonly farmed, dominate international trade, and may be 'carriers' of ranavirus infections. However, whether American bullfrogs are easily infected and maintain long-lasting ranavirus infections, or are refractory to or rapidly clear infections, remains unknown. We tracked the dynamics of ranavirus in American bullfrogs through time and with temperature in multiple types of samples and also screened shipments from commercial suppliers to determine whether we could detect subclinical infections. Collectively, we found that tadpoles and juveniles were commonly infected at moderate doses, and while some died, others controlled and appeared to clear their infections. Some individuals, however, harbored subclinical infections for up to 49 d, suggesting that American bullfrogs may be important carriers. Indeed, tadpoles and metamorphosed frogs from 2 of 5 commercial suppliers harbored subclinicial infections. Juveniles at warmer temperatures had less intense but still persistent infections. Because diagnostic performance was strongly related to infection intensity, non-lethal samples (i.e. tail or toe clips, swabs, and environmental DNA) had only a moderate chance of detecting subclinical infections. Even internal tissues may fail to detect subclinical infections. However, viral shedding was correlated with the intensity of infection, so while subclinically infected tadpoles shed virus for 35-49 d, the low levels might lead to little transmission. We suggest that a quantitative focus on virus dynamics within hosts can provide a more nuanced view of ranavirus infections and the risk presented by American bullfrogs in trade.

Road-killed toads as a non-invasive source to study age structure of spring migrating population

Development of road system causes worldwide barriers to wildlife movement and leads to mass road mortality which is a serious threat to the biodiversity, especially globally endangered amphibians. Animal carcasses, however, constitute an important non-invasive material for further research including species life history traits, but there is little known whether the road-killed animals are a representative sample of entire population. The aim of our research was to compare the age structures between road-killed and live individuals which crossed the road successfully during the breeding migration. As a model species, we used the common toad Bufo bufo which is a main amphibian traffic victim in Europe. We collected toe clips of dead and live toads during their breeding migration in four sampling sites located in the lowlands of western Poland. Phalanges were used for age determination by the skeletochronology. There were no significant differences in age distribution between live and dead individuals of the same sex in all populations studied. In compared groups, medians and mean ages were the same or differed marginally and confidence intervals for the mean age almost completely overlapped. Our results suggest that toads are killed on roads regardless of individual age, thus skeletochronological analysis of dead individuals is useful to study age structure of a live population. Our approach allows to avoid mutilation of amphibians and can be used to increase the knowledge of age structure of a given species living near the road as well as to study long-term road effect on amphibian demography. However, further comparative research on different species is required.

Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.

Testing skin swabbing for DNA sampling in dendrobatid frogs.

Skin swabbing, a minimally invasive DNA sampling method recently proposed for adult amphibians, was tested on the dendrobatid frog Allobates femoralis. I compared DNA yield from skin swabs and toe clips by evaluating obtained DNA concentrations and purity of extracts, as well as amplification success using eleven polymorphic microsatellite loci. I also tested whether storing skin swabs for two months at −20°C affected the properties of the extract or microsatellite analysis. Results show that skin swabs of adult A. femoralis suffered from high contamination and yielded significantly lower DNA quality and quantity, resulting in insufficient genotyping success, than DNA obtained from toe clips. The relatively dry skin in dendrobatid frogs may have impeded the collection of sufficient viable cells, and the presence of skin alkaloids and microbiota in the frog mucus may lead to high contamination load of skin swabs.

Effect of Changes in Cycle Ergometer Settings on Bioelectrical Activity in Selected Muscles of the Lower Limbs

Abstract Introduction . The aim of this study was to measure the duration of biopotentials in selected muscles of the lower limbs, evaluate the time of elevated bioelectrical activity in these muscles, and identify similarities and differences in electrical phenomena that occur in the muscles for various external settings of a cycle ergometer. Material and methods . The study examined 10 healthy people (5 women and 5 men) aged from 20 to 30 years. A cycle ergometer and EMG apparatus were used in the experiment. The bioelectrical activity of six muscles of the lower limbs (rectus femoris, vastus medialis, tibialis anterior, biceps femoris, gastrocnemius caput mediale, and gastrocnemius caput laterale) was recorded for four different settings of the cycle ergometer (variable saddle height and method of foot attachment to pedals). The EMG records were presented with reference to the bicycle crankset rotation cycle. Conclusions . The study found that changing the height of the saddle of the cycle ergometer and the use of toe clips in the pedals caused changes in bioelectrical activity in the muscles. The adjustment of saddle height affected the duration of potentials more noticeably than the use of toe clips. Furthermore, only one period of elevated electrical activity in the muscles of the lower limbs was found in the pedalling cycle. The longest time of the presence of action potentials was recorded for the m. gastrocnemius caput laterale, whereas the shortest time was observed in the m. vastus medialis.

Effects of shoe type and shoe–pedal interface on the metabolic cost of bicycling

Cyclists, coaches, and equipment manufacturers claim that cycling-specific shoes coupled with clipless pedals are ‘more efficient’. However, scientific evidence supporting or refuting these claims is lacking. We measured the metabolic cost of cycling at sub-maximal power outputs and tested the null hypothesis that there would be no differences between three different shoe-pedal combinations. Eleven healthy subjects participated (six males, four females, age 24.9 ± 6.84 yr, mass 69.98 ± 9.37 kg). We compared: (1) Nike Free 3.0 running shoes with flat rubber pedals, (2) Nike Free 3.0 running shoes with classic aluminium quill pedals, toe clips, and straps, and (3) the cyclists' own rigid-soled, cleated cycling shoes with corresponding clipless pedals. With the three different shoe-pedal conditions in random order, subjects completed three sequential 5-min cycling trials (50, 100, and 150W all at 90RPM) on a custom pan-loaded cycle ergometer equipped with a standard Monark flywheel. Subjects remained seated with both hands placed on the tops of the ergometer's racing-style handlebars. A 5-min rest period separated each of the three sets of trials. We analysed each subject's expired gases and from the rates of oxygen consumption and carbon dioxide production, we calculated metabolic power in watts (W). As hypothesized, there were no significant differences (p > 0.57) in the metabolic power consumed for pedaling at 50,100, and 150 W: Nike Free and flat pedals: 445.7, 619.0, and 817.9 W; Nike Free and quill pedals with toe clips: 428.7,600.7, and 818.0 W and cycling shoes with clipless pedals: 441.6, 612.3, and 806.4 W, respectively. Though cycling shoes may have comfort or safety benefits, they do not enhance efficiency.

Mountain Biking

A somewhat recent introduction to the world of fitness is mountain biking. The good news is that an actual mountain is not generally needed, so an “off road,” an old hiking trail, and even gravel roads can substitute as a mountain biking excursion. For more fit and experienced riders, however, only a true mountain will do. Either way, it allows an outdoor “getaway” and can be a great supplement to traditional workouts. BENEFITS OF MOUNTAIN BIKING The varying terrain allows for a full body workout. For example, going uphill challenges the leg muscles, whereas riding downhill engages the muscles of the arm, shoulder, chest, back, and abdomen. The uphill and downhill training also makes for a natural interval workout. Ascending will get the heart rate up, sometimes substantially — and often for several minutes if the incline is long, whereas descending or returning to level ground allows the heart rate to recover. Like all forms of biking, it induces a lower impact on the skeletal system than most activities — at least for the lower limbs. Also, because of ever-changing terrain and grade, mountain bike rides can improve coordination and proprioception and challenge decision-making skills. Most important, it’s fun. The combination of good aerobic (and anaerobic) workout, being outside, and the euphoria of traveling at fast speeds makes mountain biking a favorite activity for many. GETTING STARTED First, get fitted for a mountain bike made by a company that has a reputation of making a durable product. Don’t hesitate to buy a used bike to save some money. Next, get a helmet. And for now, don’t worry about getting the special pedals and clipless shoes that many riders have. Simply use toe clips. Because water bottles are more difficult to access during a ride, consider the hands-free drinking option that Camelbak and others provide. Once you’ve acquired the appropriate gear, team up with other riders who can show you some trails and give basic pointers along the way. Initially consider mountain biking once every week or two because commuting time and organizing a ride with others can be a laborious process. Also, start on easy trails to get used to your bike — including gear changing — and avoid extended rides that may lead to muscle soreness. MOUNTAIN BIKE TIPS Relax the upper body — Rigid and extended arms increase the chance of joint injury in the shoulder, elbow, and wrists. Allow your arms and hands to act as shock absorbers. Uphill — The focus should be on the right gear to allow a challenging, yet lighter, intensity. The goal is to make it up the hill, so pace yourself. Downhill — The main goal here is safety. One wrong turn or a fallen branch can lead to an injurious fall. Be patient — Mountain biking is a skill as much as a great form of exercise, and both take time to acquire. Stay safe — follow the safety tips presented below. Mountain biking presents a great opportunity for enjoyment, but riders must ease into it. Preparation, appropriate equipment, and good decision making can make it one of the most enjoyable activities in your fitness routine. SAFETY TIPS Start and progress slowly — Because two distinct dynamic qualities are required — fitness and skill — error on the side of caution. A combination of too much speed and some shifty terrain under the tires can make for a dangerous ride. Stay focused — A genuine respect for this activity is needed. Even a minor lapse in attention can lead to an accident on the trail. Partner up — For two main reasons. First, an unknown trail is more likely to cause an injury via a fall, so have the more experienced rider go first so he or she can give warning of upcoming obstacles and potentially dangerous turns. Second, getting lost is a real possibility. Fatigue and onsetting darkness can amplify this situation. Know the weather and trail conditions — Nothing makes a trip seem more wasted than to run into a bad storm on the trail. Furthermore, slick trails simply increase the probability of a fall.

Influence of dietary zinc and copper on apparent mineral retention and serum biochemical indicators in young male mink (Mustela vison).

An experiment was conducted in a 3 × 3 (Cu × Zn) factorial experiment based on a completely randomized design to evaluate the effects of dietary copper and zinc on apparent mineral retention and serum biochemical indicators in young male mink on a corn-fish meal based diet. Animals were fed basal diets supplemented with Cu from copper sulfate (CuSO4) and Zn from zinc sulfate (ZnSO4). Supplemental Cu levels were 0, 15, and 30 mg/kg copper, respectively, while supplemental Zn levels were 0, 150, and 300 mg/kg, respectively. A metabolism trial of 4 days was conducted during the last week of experimental feeding. Blood samples were collected via the toe clip to determine blood hematology and blood metabolites. Copper excretion, retention, and digestibility were influenced by dietary copper (P < 0.05), however, there was also a zinc-copper interaction. Copper digestibility and retention were substantially reduced when zinc was added to the low copper diet but showed little change with zinc supplementation of the high copper diet. Both plasma Cu and plasma Zn were influenced by dietary level of the respective mineral (P < 0.05). There was no influence of dietary copper on plasma zinc; however, high Zn in the diet reduced plasma Cu concentrations. There was a zinc-copper interaction for plasma Cu (P = 0.053). Cu-Zn superoxide dismutase (Cu-Zn SOD) activity tended to be influenced by dietary zinc (P = 0.065) and dietary copper (P = 0.035). Dietary copper had a significant effect on ceruloplasmin (CER) and triglyceride (TG) concentrations (P < 0.05). Our results show that moderately high Zn in the diet (Zn:Cu ratio of 40:1) significantly reduce the apparent Cu digestibility. Our results also show that moderately high Cu in the diet increases Cu retention, but not reduces Zn absorption, and moderately high Zn in the diet reduced plasma Cu concentrations and CER activity.

Copper bioavailability, blood parameters, and nutrient balance in mink.

A 3 × 3 + 1 factorial experiment was conducted based on a completely randomized design to evaluate the effects of different sources of copper on plasma metabolites, nutrient digestibility, relative copper bioavailability, and retention of some minerals in male mink. Animals in the control group were fed a basal diet, which mainly consisted of corn, fish meal, meat and bone meal, and soybean oil, with no copper supplementation. Mink in the other 9 treatments were fed the basal diet supplemented with Cu from reagent-grade copper sulfate (CuSO4), tribasic copper chloride (TBCC), or copper methionine (CuMet). Copper concentrations of the experimental diets were 50, 100, and 150 mg Cu/kg DM. Blood samples were collected via the toe clip at the end of study (d 42) to determine blood hematology and blood metabolites. A metabolism trial of 4 d was conducted during the last week of experimental feeding. There was a linear (P < 0.01) effect of dose of Cu on plasma Cu concentrations, ceruloplasmin concentration, and Cu-Zn superoxide dismutase activity. A linear response to Cu dose was noted for fat (P < 0.05) digestibility. Supplemental dose of Cu linearly increased (P < 0.05) liver Cu and decreased (P < 0.05) liver Zn level but did not alter liver Fe. The concentration of liver Cu of the mink fed with TBCC and CuMet diets was greater (P < 0.05) than that fed CuSO4. Compared with CuSO4 (100%), relative bioavailability values of TBCC were 104 and 104%, based on serum ceruloplasmin and liver copper, respectively, and relative bioavailability values of CuMet were 130 and 111%. CuMet and TBCC are more bioavailable than CuSO4. In conclusion, the relative bioavailability of CuMet obtained in this study was greater than that of CuSO4 and TBCC. Dose of Cu had an important effect on the regulating ceruloplasmin concentration, Cu-Zn superoxide dismutase activity, and the digestion of dietary fat in mink.

Effects of 6 weeks of aerobic exercise combined with conjugated linoleic acid on the physical working capacity at fatigue threshold.

The purpose of this study was to examine the effects of conjugated linoleic acid (CLA) supplementation in conjunction with 6 weeks of aerobic exercise training on the physical working capacity at the fatigue threshold (PWCFT), timed sit-ups, and the standing long jump. Thirty-three untrained to moderately trained men (mean ± SD; age = 21.6 ± 2.8 years) participated in this double-blind, placebo-controlled study and were randomly assigned to either a CLA (Clarinol A-80; n = 17) or placebo (PLA; sunflower oil; n = 16) group. Before and after 6 weeks of aerobic training (50% VO2peak for 30 minutes, twice per week) and supplementation (8 ml CLA or PLA per day), each subject completed an incremental cycle ergometer test to exhaustion to determine the PWCFT, maximal number of sit-ups in 1 minute, and the standing long jump. There were no differences (all p ≥ 0.23) between the CLA and PLA groups for the analysis of covariance-adjusted posttest mean values for PWCFT, sit-ups, or standing long jump. The PWCFT increased from pre- to posttraining in the CLA (p = 0.003) and PLA (p = 0.003) groups. There were no differences (p > 0.05) from pre- to posttraining for sit-ups and standing long jump in either the CLA or PLA groups. There was no effect of CLA on the training-induced increases in PWCFT, nor were there any effects of CLA or aerobic training on the maximum number of sit-ups or standing long jump. Thus, CLA had no ergogenic benefits on this model of aerobic training-induced improvements in neuromuscular fatigue, or on field tests of muscle endurance and power.

LOW DETECTION OF RANAVIRUS DNA IN WILD POSTMETAMORPHIC GREEN FROGS, RANA (LITHOBATES) CLAMITANS, DESPITE PREVIOUS OR CONCURRENT TADPOLE MORTALITY

Ranavirus (Iridoviridae) infection is a significant cause of mortality in amphibians. Detection of infected individuals, particularly carriers, is necessary to prevent and control outbreaks. Recently, the use of toe clips to detect ranavirus infection through PCR was proposed as an alternative to the more frequently used lethal liver sampling in green frogs (Rana [Lithobates] clamitans). We attempted reevaluate the use of toe clips, evaluate the potential use of blood onto filter paper and hepatic fine needle aspirates (FNAs) as further alternatives, and explore the adequacy of using green frogs as a target-sampling species when searching for ranavirus infection in the wild. Samples were obtained from 190 postmetamorphic (≥1-yr-old) green frogs from five ponds on Prince Edward Island (PEI), Canada. Three of the ponds had contemporary or recent tadpole mortalities due to Frog Virus 3 (FV3) ranavirus. PCR testing for ranavirus DNA was performed on 190 toe clips, 188 blood samples, 72 hepatic FNAs, and 72 liver tissue samples. Only two frogs were ranavirus-positive: liver and toe clip were positive in one, liver only was positive in the other; all blood and FNAs, including those from the two positive frogs, were negative. Results did not yield a definitive answer on the efficacy of testing each type of sample, but resemble what is found in salamanders infected with Ambystoma tigrinum (rana)virus. Findings indicate a low prevalence of FV3 in postmetamorphic green frogs on PEI (≤2.78%) and suggest that green frogs are poor reservoirs (carriers) for the virus.

The measurement of maximal (anaerobic) power output on a cycle ergometer: a critical review.

The interests and limits of the different methods and protocols of maximal (anaerobic) power (P max) assessment are reviewed: single all-out tests versus force-velocity tests, isokinetic ergometers versus friction-loaded ergometers, measure of P max during the acceleration phase or at peak velocity. The effects of training, athletic practice, diet and pharmacological substances upon the production of maximal mechanical power are not discussed in this review mainly focused on the technical (ergometer, crank length, toe clips), methodological (protocols) and biological factors (muscle volume, muscle fiber type, age, gender, growth, temperature, chronobiology and fatigue) limiting P max in cycling. Although the validity of the Wingate test is questionable, a large part of the review is dedicated to this test which is currently the all-out cycling test the most often used. The biomechanical characteristics specific of maximal and high speed cycling, the bioenergetics of the all-out cycling exercises and the influence of biochemical factors (acidosis and alkalosis, phosphate ions…) are recalled at the beginning of the paper. The basic knowledge concerning the consequences of the force-velocity relationship upon power output, the biomechanics of sub-maximal cycling exercises and the study on the force-velocity relationship in cycling by Dickinson in 1928 are presented in Appendices.

Clip or Snap? An Evaluation of Toe-Clipping and Photo-Identification Methods for Identifying Individual Southern Red-Bellied Toads,Melanophryniscus cambaraensis

4 Corresponding author. ABSTRACT. The most common method for identifying individual amphibians is toe-clipping (TC), whereby captured individuals are marked by a unique combination of amputated phalanges that corresponds to a unique alphanumeric code. However, ethical and methodological objections to this method have been raised and there is broad interest in developing alternative methods. One alternative is to use photo-identification methods (PIMs) to identify individuals based on their natural markings. We tested the ef- ficacy of TC and two PIMs — visual matching (VM) and computer-assisted matching (CAM) using the software Wild-ID — in identifying individual adults of the endangered southern red-bellied toad, Melanophryniscus cambaraensis. We collected data over 5 mo at Floresta Nacional de Sao Francisco de Paula, Rio Grande do Sul, Brazil. All specimens were toe-clipped and photographed. The total dataset included 492 captures of 147 individuals. VM was most accurate (99.4%), followed by TC (95.3%) and CAM (90.9%); VM was significantly more accurate than TC and CAM and TC was significantly more accurate than CAM. CAM ac- curacy diminished as dataset size increased but was considerably faster than VM. All CAM and VM errors were false negatives but involved different images; all TC errors were cross-identifications. Given that misidentifications occurred using both PIMs and TC, our results suggest that studies that require high accuracy should employ at least two methods to allow cross-validation. The performance of each method and the impacts of different kinds and rates of error on inferences depend on the organisms, field conditions, dataset sizes, and study questions. As such, researchers must carefully evaluate the trade-offs of each method before investing significant time and resources in collecting field data.

Use of toe clips as a nonlethal index of mercury accumulation and maternal transfer in amphibians

Nonlethal indices of contaminant exposure can facilitate research on the accumulation and effects of contaminants in wildlife. Here, we tested the efficacy of using amputated toes ("toe clips"), a common byproduct when marking amphibians in population and genetic studies, to determine mercury (Hg) concentrations in amphibians. We examined total mercury (THg) concentrations in American toads (Bufo americanus) collected along a contamination gradient at a Hg-contaminated field site. We found significant positive correlations between toe THg and blood THg concentrations in adult males and females collected in two different years. We also found that blood and toe clips could be used to predict maternal transfer of Hg, an important mechanism of reproductive toxicity in wildlife. Maternal toe THg concentrations were more highly correlated with egg THg concentrations than were maternal blood THg concentrations. Our results indicate that amputated toes are effective for identifying Hg concentrations in amphibians.