Multilocus Genotyping of Sympatric Hepatozoon Species Infecting the Blood of Ontario Ranid Frogs Reinforces Species Differentiation and Identifies an Unnamed Hepatozoon Species.

Intraerythrocytic gamonts of at least 2 named Hepatozoon species have been reported to infect the erythrocytes of ranid frogs in Ontario, Canada. Although gamonts of both species are morphometrically similar, the cytopathological changes that 1 of these species, Hepatozoon clamatae, causes to host erythrocytes, manifested by nuclear fragmentation, was used historically to distinguish this parasite from Hepatozoon catesbianae. Molecular characterization of these 2 Hepatozoon species has been equivocal in correlating genotype with gamont morphotype. Amplification and sequencing of multiple potential genotyping loci within the nuclear (18S ribosomal deoxyribonucleic acid [rDNA]; internal transcribed spacer 1), apicoplast (23S rDNA), and mitochondrial genomes (complete genomes, cytochrome c oxidase subunits I and III [COI and COIII], and cytochrome b) were conducted on Hepatozoon species that infect ranid frogs in Ontario. Sequence data were then used to evaluate the diversity of parasites present in these amphibian hosts and to assign genotypes to gamont morphotypes, if possible. Three distinct genotypes were identified at all loci; the data permitted the discovery of a third, formerly unrecognized Hepatozoon species in ranid frogs from Ontario. Although all genetic loci demonstrated differences between Hepatozoon species, mitochondrial COIII sequences were most suitable for genotypic differentiation of these parasites of frogs. Linking genotypes to gamont morphotypes proved impossible; genotypes identified as H. catesbianae and H. clamatae were found in infections with or without nuclear fragmentation of their host erythrocytes. This suggests that differentiating these species must rely on suitable genotyping methods for identification in the blood of their amphibian intermediate hosts.

Effects of 2 Neonicotinoid Insecticides on Blood Cell Profiles and Corticosterone Concentrations of Wood Frogs (Lithobates sylvaticus).

Neonicotinoids are widely used insecticides that are detectable in agricultural waterways. These insecticides are of concern due to their potential impacts on nontarget organisms. Pesticides can affect development of amphibians and suppress the immune system, which could impact disease susceptibility and tolerance. No previous studies on amphibians have examined the effects of these insecticides on differential blood cell proportions or concentrations of corticosterone (a general stress hormone). We investigated the effects of chronic exposure to 2 neonicotinoids, thiamethoxam and clothianidin, on immunometrics of wood frogs (Lithobates sylvaticus). Frogs were exposed to single, chronic treatments of 2.5 or 250 µg/L of clothianidin or thiamethoxam for 7 wk from Gosner stages 25 to 46. The juvenile frogs were then maintained for 3 wk post metamorphosis without exposure to neonicotinoids. We measured water-borne corticosterone twice: at 6 d and 8 wk after exposure in larval and juvenile frogs, respectively. We assessed differential blood cell profiles from juvenile frogs. Corticosterone was significantly lower in tadpoles exposed to 250 µg/L of thiamethoxam compared with other tadpole treatments, but no significant differences in corticosterone concentrations were found in treatments using juvenile frogs. Anemia was detected in all treatments compared with controls with the exception of tadpoles exposed to 2.5 µg/L of clothianidin. Neutrophil-to-leukocyte and neutrophil-to-lymphocyte ratios were elevated in frogs exposed to 250 µg/L of thiamethoxam. Collectively, these results indicate that chronic exposure to neonicotinoids has varied impacts on blood cell profiles and corticosterone concentrations of developing wood frogs, which are indicative of stress. Future studies should investigate whether exposure to neonicotinoids increases susceptibility to infection by parasites in both larval and adult wood frogs. Environ Toxicol Chem 2019;38:1273-1284. © 2019 Crown in the right of Canada. Published by Wiley Periodicals Inc. on behalf of SETAC.

Agrochemical Mixtures and Amphibians: The Combined Effects of Pesticides and Fertilizer on Stress, Acetylcholinesterase Activity, and Bioaccumulation in a Terrestrial Environment.

Tank mixtures are popular within the agricultural community because they are time- and cost-effective, but field applications leave nontarget organisms at risk of exposure. We explored the effects of a common herbicide (atrazine and alachlor) and fertilizer (urea) tank mixture on juvenile frog corticosterone stress levels, acetylcholinesterase (AChE) activity, and pesticide bioaccumulation. Single agrochemical or tank mixtures were applied to terrestrial microcosms, and then individual Southern leopard frog (Lithobates sphenocephala) juveniles were added to microcosms for an 8-h exposure. Afterward, frogs were transferred to aquatic microcosms for 1 h to monitor corticosterone prior to euthanasia, brain tissues were excised to evaluate AChE, and tissue homogenates were analyzed for pesticide bioconcentation with gas chromatography-mass spectrometry. Atrazine significantly increased corticosterone in frogs, particularly when combined with alachlor and urea. Atrazine increased AChE and urea decreased AChE, although no interactive effects of chemical combinations were discernible. Relative to their individual treatments, the complete tank mixture with all 3 agrochemicals resulted in 64% greater bioconcentration of atrazine and 54% greater bioconcentration of alachlor in frog tissues. Our results suggest that agrochemical mixtures as well as their active ingredients can lead to altered stress levels and impaired physiological responses in amphibians. An improved understanding of the effects of co-exposure to environmental contaminants in amphibians is important in assessing the ecological risks these compounds pose. Environ Toxicol Chem 2019;9999:1-10. © 2019 SETAC.

Health status of Pelophylax ridibundus (Amphibia: Ranidae) in a rice paddy ecosystem in Southern Bulgaria and its importance in assessing environmental state: haematological parameters.

Pollution effects on haematological parameters in Pelophylax ridibundus individuals were investigated; animals were collected from two sites in Southern Bulgaria: the Tsalapitsa rice fields (RF) and the Vacha river (reference site, RS). Blood analysis showed significant differences between the haematological parameters of RBC, WBC, Hb, packed cell volume (PCV) and frogs' leucogram from RF and those from RS. These findings provide information on long-term background pollution of the habitat (RF) under investigation. In our view, the erythropenia, leucopenia, hypоchromia, lower values of PCV, St-neutrophilia, Sg-neutropenia, basopenia, eosinophilia, monocytosis and lymphopenia that were found in Pelophylax ridibundus individuals inhabiting the Tsalapitsa rice fields were probably caused by the pesticides and fertilizers that enter the paddy cages during the rice production process. The present study proves the practical usefulness of haematological parameters of Pelophylax ridibundus individuals in bioindication analyses for environmental assessment of agroecosystems.

The Influence of Six Pesticides on Physiological Indices of Pelophylax Ridibundus (Pallas, 1771).

The objective of the study is to screen for morphological, biochemical and histological changes induced by six widely used pesticides (Reldan 40EC, Actara 25WG, Tilt 250EC, Champion 50WG, Fusilade Forte, Dual Gold 960EC) in the amphibian species Pelophylax ridibundus (Pallas, 1771). Highly degenerative changes were observed in animals cultured at 22-24°C, compared to those cultured at 4-6°C. The hepatosomatic index increased upon exposure to almost all of the pesticides, the erythrocyte number decreased upon exposure to all pesticides except Reldan 40EC, while leucopenia was observed only for Reldan 40EC and Actara 25WG. Hyperglycemia was observed upon administration of pesticides (except Champion 50WG and Fusilade Forte, for which hypoglycemia is registered), while a decrease in cholesterol levels was induced by nearly all pesticides. Triglycerides varied only slightly. The results suggest that chronic pesticides exposure can lead to alteration of various indices, as well as to hepatic lesions in amphibians.

THE PHARMACOKINETICS OF TOPICAL ITRACONAZOLE IN PANAMANIAN GOLDEN FROGS (ATELOPUS ZETEKI).

Chytridiomycosis is caused by the fungus Batrachochytrium dendrobatidis and is one of the primary causes of the global decline in amphibian populations and specifically of the Panamanian golden frog ( Atelopus zeteki ). Itraconazole has been demonstrated to be an effective treatment for chytridiomycosis by inhibiting cytochrome P450, a major enzyme important for the structure of B. dendrobatidis zoospores' plasma membranes. However, anecdotal reports of toxicity in this and other amphibian species have been reported at the 0.01% concentration. This study is the first to determine pharmacokinetics of 0.01% and 0.001% itraconazole in the Panamanian golden frog. Frogs were bathed 10 min, euthanized, and skin, liver, and heart were collected at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, and 36 hr. Itraconazole concentrations were measured using high performance liquid chromatography, and the minimum inhibitory concentration (MIC) of itraconazole (0.032 µg/ml) for B. dendrobatidis was used to determine whether therapeutic concentrations were attained. Itraconazole was detected in all tissues at both concentrations, indicating systemic absorption. At the 0.01% itraconazole bath, itraconazole concentrations in all tissues exceeded the MIC at all time points, and the lack of decline until the end of the study at 36 hr precluded determining a disappearance half-life. With the 0.001% bath, itraconazole exceeded the MIC and declined with a disappearance half-life that markedly varied (14.1-1,244 min). This study augments the growing literature base on chytridiomycosis and seeks to aid in further experimental attempts to find the most-optimal treatment protocol for this disease.

A Threshold Dosage of Testosterone for Female-to-Male Sex Reversal in Rana rugosa Frogs.

Androgens play a critical role in testicular differentiation in many species of vertebrates. While female-to-male sex reversal can be induced by testosterone (T) in some species of amphibians, the mechanism still remains largely unknown even at the histological level. In this study, we determined a threshold dosage of T to induce female-to-male sex reversal in the Japanese frog Rana (R.) rugosa. Tadpoles were allowed to metamorphose into frogs with T present in the rearing water. At 0.2 ng/mL T, female frogs formed tissue comprising a mixture of ovary and testis, the so-called ovotestis, the size of which was significantly smaller than the wild-type ovary. Histological changes occurring in the oocytes of T-treated ovaries induced oocyte degeneration in the masculinizing ovaries leading to their final disappearance. In parallel, many germ cells emerged in the cortex of the ovotestis and, later, in the medulla as well. RT-PCR analysis revealed upregulated expression of CYP17 and Dmrt1 but not 17ßHSD in the ovotestis, and downregulation of Pat1a expression. Furthermore, immunohistology revealed CYP17-positive signals in the cortex of the masculinizing ovary, spreading throughout the whole area as the testis developed. These results indicate that oocytes are sensitive to T in the ovary of R. rugosa and that male-type germ cells expand in the masculinizing gonad (testis) contemporaneous with oocyte disappearance.

Hematologic reference intervals for Rana sylvatica (Lithobates sylvaticus) and effect of infection with Frog Virus 3 (Ranavirus sp., Iridoviridae).

BACKGROUND: Although the Wood Frog, Rana sylvatica, is used in research on infectious diseases of amphibians, hematologic RIs or response to infection have not been established. OBJECTIVES: The purpose of the study was to determine hematologic RIs for adult Wood Frogs and alterations associated with infection with Frog Virus 3 (FV3, Ranavirus sp.). METHODS: Blood was collected from 40 wild-caught adult Wood Frogs that had been in captivity for 6 months. Complete (Natt-Herrick solution hemocytometry) and differential (Wright-Giemsa-stained smears) WBC, RBC, and thrombocyte cell counts, PCV, and automated total cell counts (WBC+RBC+thrombocytes, Sysmex particle counting) were determined. Concordance correlation coefficients determined agreement between hemocytometric and automated total cell counts. Thirteen frogs were orally infected with a lethal dose of 10(4.43) plaque-forming units of FV3 and terminally sampled 4, 9, or 14 days postinfection (dpi). Pre- and postinfection variables for each frog were compared. RESULTS: Leukocyte morphology was similar to that of other amphibians and mammals. Lymphocytes were the most numerous WBC. PCV and RBC counts were similar to other frogs in the same family. Agreement was good between hemocytometry and automated total cell counts. Infection with FV3 caused neutrophilia, increase in undifferentiated blast-like cells, and reduction in the percentage of basophils. Lymphocytes decreased at 4 and 9 dpi but increased at 14 dpi. From 9 dpi onwards, nuclear deterioration and mild toxic change were present in neutrophils; viral cytoplasmic inclusion bodies were observed in lymphocytes, monocytes, neutrophils, and eosinophils. CONCLUSION: We provide hematology RIs for Rana sylvatica, and report hematologic changes associated with a lethal FV3 infection.

Seasonal changes of basic erythrocyte-metric parameters in Pelophylaxridibundus (Amphibia: Ranidae) from anthropogenically polluted biotopes in Southern Bulgaria and their role as bioindicators.

The purpose of this research work is to present data that show the seasonal changes (spring-summer-autumn) of basic erythrocyte-metric parameters (ЕL: Erythrocyte length, ЕW: Erythrocyte width, ЕL/ЕW, ES: Erythrocyte size; NL: Nucleus length, NW: Nucleus width, NL/NW; NS: Nucleus size, NS/ES: Nucleus-cytoplasmic ratio) in Pelophylax ridibundus populations from three biotopes located on two rivers in Southern Bulgaria (less disrupted biotope, with domestic sewage pollution and heavy metal pollution). Differences of high statistical significance were found among the different populations. Within the population living in conditions of domestic sewage pollution, for the entire period of the investigation the erythrocytes and their nuclei had an elliptical shape (a slight elongation of ellipses in autumn) and the biggest sizes (EL, EW, ES, NL and NS were constantly higher than the less disrupted biotope), NS/ES, became significantly smaller in autumn. Throughout the period of investigation, the values of all nine cellular and nuclear parameters were statistically-significantly the lowest in the population from the biotope with heavy metal pollution. The parameters: EL, ЕW, NL, NW and ES became significantly lower, progressively and statistically, during seasonal transitions. Cells and nuclei grew ovular in shape in comparison to the populations from the other two biotopes (this process was most pronounced in autumn) and NS/ES numbers were significantly decreased in summer and autumn.

Plasma retinoids concentration in Leptodactylus chaquensis (Amphibia: Leptodactylidae) from rice agroecosystems, Santa Fe province, Argentina.

Retinoids are known to regulate important processes such as differentiation, development, and embryogenesis of vertebrates: Alteration in endogenous retinoids concentration is linked with teratogenic effects. Retinol (ROH), retinoid acid (RA), and isoform 13-Cis-retinoic acid (13-Cis-RA), in plasma of a native adults frog, Leptodactylus chaquensis from a rice field (RF) and a forest (reference site; RS) were measured. ROH did not vary between treatment sites. RA and 13-Cis-RA activities were higher (93.7±8.6 µg mL(-1) and 131.7±11.4 µg mL(-1), respectively) in individuals collected from RF than in those from RS (65.5±8.6 µg mL(-1) and 92.2±10.2 µg mL(-1), respectively). The ratios retinoic acid-retinol (RA/ROH) and 13-Cis-RA/ROH revealed significantly higher values in RF than in RS. RA and 13-Cis-RA concentrations in plasma on wild amphibian's species such as L. chaquensis would be suitable biomarkers of pesticide exposure in field monitoring. Finally, the mechanism of alteration in retinoid metabolites alteration should be further explored both in larvae and adult, considering that the potential exposition and uptake contaminants vary between the double lives of these vertebrates.

Elevated stress hormone diminishes the strength of female preferences for acoustic signals in the green treefrog.

Mate selection can be stressful; time spent searching for mates can increase predation risk and/or decrease food consumption, resulting in elevated stress hormone levels. Both high predation risk and low food availability are often associated with increased variation in mate choice by females, but it is not clear whether stress hormone levels contribute to such variation in female behavior. We examined how the stress hormone corticosterone (CORT) affects female preferences for acoustic signals in the green treefrog, Hyla cinerea. Specifically, we assessed whether CORT administration affects female preferences for call rate - an acoustic feature that is typically under directional selection via mate choice by females in most anurans and other species that communicate using acoustic signals. Using a dual speaker playback paradigm, we show that females that were administered higher doses of CORT were less likely to choose male advertisement calls broadcast at high rates. Neither CORT dose nor level was related to the latency of female phonotactic responses, suggesting that elevated CORT does not influence the motivation to mate. Results were also not related to circulating sex steroids (i.e., progesterone, androgens or estradiol) that have traditionally been the focus of studies examining the hormonal basis for variation in female mate choice. Our results thus indicate that elevated CORT levels decrease the strength of female preferences for acoustic signals.

[Early stages of development of Trypanosoma rotatorium (Mayer, 1843) from peripheral blood and internal organs of Anurans Bufo bufo (Linnaeus) and Rana sp. (Anura)].

The data on the fauna of trypanosomes of Anura of the Leningrad Province are given. The initial development stages of Trypanosoma rotatorium in peripheral blood and internal organs of the frog are described for the first time.

Vitamin A values of wild-caught Cuban tree frogs (Osteopilus septentrionalis) and marine toads (Rhinella marina) in whole body, liver, and serum.

Recent issues surrounding captive amphibians are often nutritionally related problems, such as hypovitaminosis A. Although supplementation of frogs with vitamin A is a topic of investigation, the underlying issue is understanding vitamin A metabolism in amphibian species. To develop a range of "normal" vitamin A concentrations for captive amphibians, baseline vitamin A concentrations must be established in wild amphibian species. In this study, two species, Cuban tree frogs (Osteopilus septentrionalis; n = 59) and marine toads (Rhinella marina; n = 20) were collected from the wild as part of an invasive species control program at Zoo Miami, Miami, Florida. Serum, liver, and whole body samples were analyzed for vitamin A content. The Cuban tree frogs showed higher concentrations on average of vitamin A in serum (82.8 ppb), liver (248.3 IU/g), and whole body (5474.7 IU/kg) samples compared with marine toads (60.1 ppb; 105.3 IU/g; 940.7 IU/kg, respectively), but differences were not significant (P = 0.22). What can be considered "normal" values of vitamin A concentrations across different amphibian species requires further investigation. Although all amphibians collected in this study appeared healthy, a larger sample size of animals, with known health histories and diets, may provide stronger evidence of normal expectations.

Hibernation physiology, freezing adaptation and extreme freeze tolerance in a northern population of the wood frog.

We investigated hibernation physiology and freeze tolerance in a population of the wood frog, Rana sylvatica, indigenous to Interior Alaska, USA, near the northernmost limit of the species' range. Winter acclimatization responses included a 233% increase in the hepatic glycogen depot that was subsidized by fat body and skeletal muscle catabolism, and a rise in plasma osmolality that reflected accrual of urea (to 106±10 µmol ml(-1)) and an unidentified solute (to ~73 µmol ml(-1)). In contrast, frogs from a cool-temperate population (southern Ohio, USA) amassed much less glycogen, had a lower uremia (28±5 µmol ml(-1)) and apparently lacked the unidentified solute. Alaskan frogs survived freezing at temperatures as low as -16°C, some 10-13°C below those tolerated by southern conspecifics, and endured a 2-month bout of freezing at -4°C. The profound freeze tolerance is presumably due to their high levels of organic osmolytes and bound water, which limits ice formation. Adaptive responses to freezing (-2.5°C for 48 h) and subsequent thawing (4°C) included synthesis of the cryoprotectants urea and glucose, and dehydration of certain tissues. Alaskan frogs differed from Ohioan frogs in retaining a substantial reserve capacity for glucose synthesis, accumulating high levels of cryoprotectants in brain tissue, and remaining hyperglycemic long after thawing. The northern phenotype also incurred less stress during freezing/thawing, as indicated by limited cryohemolysis and lactate accumulation. Post-glacial colonization of high latitudes by R. sylvatica required a substantial increase in freeze tolerance that was at least partly achieved by enhancing their cryoprotectant system.

Sexually dimorphic effects of melatonin on brain arginine vasotocin immunoreactivity in green treefrogs (Hyla cinerea).

Arginine vasotocin (AVT) and its mammalian homologue, arginine vasopressin (AVP), regulate a variety of social and reproductive behaviors, often with complex species-, sex- and context-dependent effects. Despite extensive evidence documenting seasonal variation in brain AVT/AVP, relatively few studies have investigated the environmental and/or hormonal factors mediating these seasonal changes. In the present study, we investigated whether the pineal hormone melatonin alters brain AVT immunoreactivity in green treefrogs (Hyla cinerea). Reproductively active male and female frogs were collected during the summer breeding season and a melatonin-filled or blank silastic capsule was surgically implanted subcutaneously. The duration of hormone treatment was 4 weeks, at which time frogs were eutha-nized and the brains and blood collected and processed for AVT immunohistochemistry and steroid hormone assay. We quantified AVT-immunoreactive (AVT-ir) cell bodies in the nucleus accumbens (NAcc), caudal striatum and amygda- la (AMG), anterior preoptic area, suprachiasmatic nucleus (SCN) and infundibular region of the ventral hypothalamus. Sex differences in AVT-ir cell number were observed in all brain regions except in the anterior preoptic area and ventral hypothalamus, with males having more AVT-ir cells than females in the NAcc, amygdala and SCN. Brain AVT was sensitive to melatonin signaling during the breeding season, and the effects of melatonin varied significantly with both region and sex. Treatment with melatonin decreased AVT immunoreactivity in both the NAcc and SCN in male H. cinerea. In contrast, brain AVT was relatively insensitive to melatonin signaling in females, indicating that the regulation of the AVT/AVP neuropeptide system by melatonin may be sexually dimorphic. Finally, melatonin did not significantly influence testosterone or estradiol concentrations of male or female frogs, respectively, suggesting that the effects of melatonin on AVT immunoreactivity are independent of changes in gonadal sex steroid hormones. Collectively, our results indicate that the AVT/AVP neuronal system may be an important target for melatonin in facilitating seasonal changes in reproductive physiology and social behavior.

Blood cell profiles of the tadpoles of the Dubois's tree frog, Polypedates teraiensis Dubois, 1986 (Anura: Rhacophoridae).

The present paper describes a sequential study of the leukocyte profiles and the changes in morphometry and morphology of erythrocytes in the tadpoles of Polypedates teraiensis during their development and metamorphosis, that is, transfer from an aquatic mode to a terrestrial mode of life. Blood smears of 21 different stages (Gosner stage 26 to 46) of tadpoles were investigated. Population of erythrocytes was heterogeneous in population represented by various forms (oval, elliptical or rounded cells, comma shaped, teardrop shaped, schistocytes, senile erythrocytes, crenulated RBCs). Correlation between various morphometric values of erythrocytes was determined with different developing stages of tadpoles. Amongst the leucocytes, the lymphocytes were the most abundant cells followed by neutrophils. Neutrophils and monocytes showed varied morphologic forms. The percentage of lymphocytes and neutrophils showed a negative whereas percentage of eosinophil, basophil, and monocytes showed a positive correlation with the developmental stages of tadpoles. Blood platelets were also observed, which were rounded in shape and found in aggregates.

Seasonal variation and response to osmotic challenge in urea transporter expression in the dehydration- and freeze-tolerant wood frog, Rana sylvatica.

Urea accumulation is a universal response to osmotic challenge in anuran amphibians, and facilitative urea transporters (UTs) seem to play an important role in this process by acting in the osmoregulatory organs to mediate urea retention. Although UTs have been implicated in urea reabsorption in anurans, little is known about the physiological regulation of UT protein abundance. We examined seasonal variation in and effects of osmotic challenge on UT protein and mRNA levels in kidney and urinary bladder of the wood frog (Rana sylvatica), a terrestrial species that tolerates both dehydration and tissue freezing. Using immunoblotting techniques to measure relative UT abundance, we found that UT numbers varied seasonally, with a low abundance prevailing in the fall and winter, and higher levels occurring in the spring. Experimental dehydration of frogs increased UT protein abundance in the urinary bladder, whereas experimental urea loading decreased the abundance of UTs in kidney and bladder. Experimental freezing, whether or not followed by thawing, had no effect on UT numbers. UT mRNA levels, assessed using quantitative real-time polymerase chain reaction, did not change seasonally nor in response to any of our experimental treatments. These findings suggest that regulation of UTs depends on the nature and severity of the osmotic stress and apparently occurs posttranscriptionally in response to multiple physiological factors. Additionally, UTs seem to be regulated to meet the physiological need to accumulate urea, with UT numbers increasing to facilitate urea reabsorption and decreasing to prevent retention of excess urea.

Pathophysiology in mountain yellow-legged frogs (Rana muscosa) during a chytridiomycosis outbreak.

The disease chytridiomycosis is responsible for declines and extirpations of amphibians worldwide. Chytridiomycosis is caused by a fungal pathogen (Batrachochytrium dendrobatidis) that infects amphibian skin. Although we have a basic understanding of the pathophysiology from laboratory experiments, many mechanistic details remain unresolved and it is unknown if disease development is similar in wild amphibian populations. To gain a better understanding of chytridiomycosis pathophysiology in wild amphibian populations, we collected blood biochemistry measurements during an outbreak in mountain yellow-legged frogs (Rana muscosa) in the Sierra Nevada Mountains of California. We found that pathogen load is associated with disruptions in fluid and electrolyte balance, yet is not associated with fluctuations acid-base balance. These findings enhance our knowledge of the pathophysiology of this disease and indicate that disease development is consistent across multiple species and in both laboratory and natural conditions. We recommend integrating an understanding of chytridiomycosis pathophysiology with mitigation practices to improve amphibian conservation.

Blood collection from the facial (maxillary)/musculo-cutaneous vein in true frogs (family Ranidae).

Collection of blood from amphibians, as in other classes of vertebrate animals, is essential to evaluate parameters of health, diagnose hemoparasitism, identify viral and bacterial pathogens, and measure antibodies. Various methods of blood collection have been described for amphibians. Most can be cumbersome (venipucture of femoral vein, ventral abdominal vein or lingual venus plexus) or result in pain or deleterious health consequences (cardiac puncture and toe-clipping). We describe an easy and practical technique to collect blood from frogs and toads that can be used in multiple species and is minimally invasive. The technique consists of puncturing either the facial or, less commonly, the musculo-cutaneous vein and collecting the blood with a capillary tube. These veins run dorsal and parallel to the maxillary bone and can be accessed by quick insertion and withdrawal of a needle through the skin between the upper jawline and the rostral or caudal side of the tympanum. The needle should be of 27 or 30 gauge for anurans weighing more or less than 25 g, respectively. Although the technique has been used by some amphibian researchers for years, it is little known by others and has never been fully described in a peer-reviewed publication.

Relationships between leukocytes and Hepatozoon spp. In green frogs, Rana clamitans.

There are few published data on amphibian leukocyte profiles, and relationships between amphibian leukocytes and parasites are even less well known. Using counts from 35 pairs of blood smears taken 2 days apart, we tested for correlations between leukocyte proportions and infection intensities of Hepatozoon spp. (either Hepatozoon catesbianae or Hepatozoon clamatae) in green frogs (Rana clamitans). On average (SE), we counted 65.4 (1.7) lymphocytes, 14.0 (1.3) neutrophils, 19.3 (1.6) eosinophils, 0.9 (0.1) monocytes, and 0.4 (0.1) basophils per 100 leukocytes. All frogs harbored Hepatozoon spp. (median seven parasites per 100 leukocytes; range 1-250). Significant relationships were not observed between numbers of leukocytes and infection intensities of Hepatozoon spp. Among the possible explanations for these null results are that Hepatozoon spp. is benign, that Hepatozoon spp. is able to evade detection by the immune system, that Hepatozoon spp. is able to manipulate leukocyte investment, or that other unmeasured or undetected parasites were more important in affecting immune response.