Epizootic to enzootic transition of a fungal disease in tropical Andean frogs: Are surviving species still susceptible?

The fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis, has been linked to catastrophic amphibian declines throughout the world. Amphibians differ in their vulnerability to chytridiomycosis; some species experience epizootics followed by collapse while others exhibit stable host/pathogen dynamics where most amphibian hosts survive in the presence of Bd (e.g., in the enzootic state). Little is known about the factors that drive the transition between the two disease states within a community, or whether populations of species that survived the initial epizootic are stable, yet this information is essential for conservation and theory. Our study focuses on a diverse Peruvian amphibian community that experienced a Bd-caused collapse. We explore host/Bd dynamics of eight surviving species a decade after the mass extinction by using population level disease metrics and Bd-susceptibility trials. We found that three of the eight species continue to be susceptible to Bd, and that their populations are declining. Only one species is growing in numbers and it was non-susceptible in our trials. Our study suggests that some species remain vulnerable to Bd and exhibit ongoing population declines in enzootic systems where Bd-host dynamics are assumed to be stable.

Seasonal Patterns of Variation in Steroid Plasma Levels and Immune Parameters in Anurans from Brazilian Semiarid Area.

Elevated androgens and glucocorticoids displayed by males during the reproductive season have been proposed to mediate a possible trade-off between reproduction and immunocompetence. Anurans living in arid and semiarid environments display a strong seasonal reproduction, which could accentuate the variation in physiological, immunological, and behavioral parameters. We studied covariation between steroid plasma levels, morphometric variables associated with body condition and immunity, leukocyte profile, parasite load, and response to an immunological challenge across different phases of the annual life-history cycle of three anuran species from a Brazilian semiarid area. Our results showed a seasonal pattern of covariation among leukocyte parameters, kidney mass, and steroid plasma levels, with higher values measured during the reproductive season, particularly when males were sampled during calling activity. Moreover, these anurans showed a stronger response to an immunological challenge during the reproductive period. The immunosuppression during the dry period was particularly evident for the species that aestivate, indicating that the availability of energetic resources might be an important factor determining seasonal variation in inflammatory response. Intensity of the helminth infection was associated with eosinophil count but showed a more complex pattern with regard to androgens levels. These data emphasize that variations in the intensity of helminth infection might be more closely related to specific aspects of the immune response than to the general seasonal patterns of variation in steroid plasma levels, total circulating leukocytes, and inflammatory response.

Reduced innate immunity of Cuban Treefrogs at leading edge of range expansion.

During geographic range expansion, populations of non-indigenous species at the invasion front may benefit from directing resources away from immune defense. To test this hypothesis, we investigated the strength of two innate immune components in populations of invasive Cuban Treefrogs (Osteopilus septentrionalis) in a long-colonized area (core region) and at the invasion front (leading-edge region). First, we compared the region-specific metabolic response of frogs injected with an endotoxin that induces systemic inflammation (lipopolysaccharide, LPS) to sham-injected control frogs pooled from both regions. Males and females were analyzed independently because we detected a sex-related difference in mass-independent metabolism of control frogs, with males exhibiting a significantly higher metabolic rate (F1, 21  = 29.02, P < 0.001) than females. We observed a significantly higher metabolic rate in LPS-injected core frogs compared with control frogs for both males (P = 0.041) and females (P = 0.007). Conversely, in leading-edge populations, there was no significant difference in the metabolic rate of LPS-injected and control frogs (males, P  = 0.195; females, P  = 0.132). Second, we directly compared bacterial killing ability of frog blood plasma between regions. Bactericidal ability of plasma was significantly greater in frogs from the core region in comparison with those at the leading edge (F1, 26   = 28.67, P < 0.001). For both immune components that we examined, populations from the core exhibited stronger immune responses. Our findings support hypotheses predicting an inverse relationship between immunity and range expansion.

The lethal fungus Batrachochytrium dendrobatidis is present in lowland tropical forests of far eastern Panamá.

The fungal disease chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), is one of the main causes of amphibian population declines and extinctions all over the world. In the Neotropics, this fungal disease has caused catastrophic declines in the highlands as it has spread throughout Central America down to Panamá. In this study, we determined the prevalence and intensity of Bd infection in three species of frogs in one highland and four lowland tropical forests, including two lowland regions in eastern Panamá in which the pathogen had not been detected previously. Bd was present in all the sites sampled with a prevalence ranging from 15-34%, similar to other Neotropical lowland sites. The intensity of Bd infection on individual frogs was low, ranging from average values of 0.11-24 zoospore equivalents per site. Our work indicates that Bd is present in anuran communities in lowland Panamá, including the Darién province, and that the intensity of the infection may vary among species from different habitats and with different life histories. The population-level consequences of Bd infection in amphibian communities from the lowlands remain to be determined. Detailed studies of amphibian species from the lowlands will be essential to determine the reason why these species are persisting despite the presence of the pathogen.

Immmunological clearance of Batrachochytrium dendrobatidis infection at a pathogen-optimal temperature in the hylid frog Hypsiboas crepitans.

Amphibian declines worldwide have been linked to the fungal disease chytridiomycosis. Its causative agent (Batrachochytrium dendrobatidis, hereafter Bd), however, also infects many nondeclining species. Experimental infections have shown species-specific and temperature-dependent frog responses to Bd infection. Although Bd infection may be eliminated by housing amphibians at temperatures above those tolerated by the fungus, the question of whether frogs can eliminate infection under more favorable conditions remains unanswered. Repeated diagnostics using real-time polymerase chain reaction (rt-PCR) assays of postmetamorphic individuals at 28, 38, 45, 53, and 62 days after exposure demonstrated that Hypsiboas crepitans is able to clear infection within a few weeks at 23°C. Thus, we demonstrate a temperature-independent and likely immunological mechanism for the clearance of Bd in a resistant amphibian species. Future studies are needed to determine the generality of this mechanism among amphibians and to describe the immune factors affecting different outcomes of Bd exposure including resistance to infection, tolerance of infection, and clearance of infection.

Post-secretory events alter the peptide content of the skin secretion of Hypsiboas raniceps.

A novel family of antimicrobial peptides, named raniseptins, has been characterized from the skin secretion of the anuran Hypsiboas raniceps. Nine cDNA molecules have been successfully cloned, sequenced, and their respective polypeptides were characterized by mass spectrometry and Edman degradation. The encoded precursors share structural similarities with the dermaseptin prepropeptides from the Phyllomedusinae subfamily and the mature 28-29 residue long peptides undergo further proteolytic cleavage in the crude secretion yielding consistent fragments of 14-15 residues. The biological assays performed demonstrated that the Rsp-1 peptide has antimicrobial activity against different bacterial strains without significant lytic effect against human erythrocytes, whereas the peptide fragments generated by endoproteolysis show limited antibiotic potency. MALDI imaging mass spectrometry in situ studies have demonstrated that the mature raniseptin peptides are in fact secreted as intact molecules within a defined glandular domain of the dorsal skin, challenging the physiological role of the observed raniseptin fragments, identified only as part of the crude secretion. In this sense, stored and secreted antimicrobial peptides may confer distinct protective roles to the frog.

Antimicrobial peptide from the skin secretion of the frog Leptodactylus syphax.

Antimicrobial peptides are considered part of the innate immune system of the majority of living organisms. Most of these molecules are small, cationic and show amphiphilic nature. The skin secretions of Leptodactylus syphax were extracted by mild electrical stimulation and its semipreparative reverse-phase chromatography was resolved in more than 40 fractions. Among these fractions, an antimicrobial peptide was isolated and its amino acid sequence determined by de novo sequencing. Six other truncated forms were characterized in skin secretion. The longest one (25 amino acid residues), named syphaxin (SPX), is amidated at the C-terminal, and shares strong sequence similarity with antimicrobial peptides found in the skin secretion of leptodactylid frogs. Two of the truncated peptides (SPX(1-22) and SPX(1-16)) were tested against Escherichia coli and Staphylococcus aureus, showing low minimal inhibitory concentration (MIC) and no significant toxicity towards blood cells, including both leukocytes and erythrocytes, based on their direct incubation in whole blood at the highest MIC concentration (64 microg/mL).

Leptospires in the whistling frog (Eleutherodactylus johnstonei) on Barbados.

Two groups of whistling frogs (Eleutherodactylus johnstonei) comprising 99 and 117 animals were examined for leptospiral infection. Group I animals were caught in 14 areas of Barbados, and Group II animals in seven areas of suburban Bridgetown. Leptospires were isolated from the kidneys or body fluid of six frogs in Group I and the kidneys of 3 frogs in Group II. Two of the Group I isolates died out; the others were identified as bajan (a new serovar in the Australis serogroup) (6) and bim (Autumnalis) (1). The macerated body tissues and fluid of Group I frogs were put into phosphate buffered saline and examined by the microscopic agglutination test using 22 antigens. The results were all negative. For the Group II frogs the methodology was altered; blood was collected onto filter paper discs and allowed to dry out before being agitated in PBS and examined by the MAT. 15/117 (12.8%) animals were positive at greater than or equal to 1:100 and 19 (16.2%) at greater than or equal to 1:50. The geometric mean titre was 179. Seventeen of the sera reacted predominantly to antigens in the Australis serogroup, and two to Pyrogenes on its own. The serological results reflected the identity of the isolates. Serovars of Australis are not known to cause illness on Barbados, but bim is the commonest cause of severe leptospirosis on the island.

Leptospires in the whistling frog (Eleutherodactylus johnstonei) on Barbados

Two groups of whistling frogs (Eleutherodactylus johnstonei) comprising 99 and 117 animals were examined for leptospiral infection. Group I animals were caught in 14 areas of Barbados, and group II animals in seven areas of suburban Bridgetown. Leptospires were isolated from the kidneys or body fluid of six frogs in Group I and the kidneys of 3 frogs in Group II. Two of the group I isolates died; the others were identified as bajan (a new serovar in the Australis serogroup) (6) and bim (Autumnalis) (1). The macerated body tissues and fluid of Group I frogs were put into phosphate buffered saline and examined by the microscopic agglutination test using 22 antigens. The results were all negative. For the Group II frogs the methodology was altered; blood was collected unto filter paper discs and allowed to dry out before being agitated in PBS and examined by the MAT. 15/117 (12.8 percent) animals were positive at o 1:50. The geometric mean titre was 179. Seventeen of the sera reacted predominantly to antigens in the Australis serogroup, and two to Pyrogens on its own. The serological results reflected the identity of the isolates. Serovars of Australis are not known to cause illness on Barbados, but bim is the commonest cause of severe leptospirosis on the island. (AU)

Estudios inmunológicos en dos especies afines de Ceratophrys (Anura Leptodactylidae): C. ornata (Bell, 1843) y C. cranwelli (Barrio, 1979) / Immunological studies in 2 species of Ceratophrys (Anura Leptodactylidae): C. ornata (Bell, 1843) and C. cranwelli (Barrio, 1979)

Se estudiaron dos especies afines del género Ceratophrys: C. ornata y C. cranwelli, mediante técnicas inmunológicas para hallar caracteres no morfológicos que contribuyan a su mejor clasificación. En las inmunodifusiones bidimensionales de las proteínas séricas de ambas especies se observó una gran reactividad cruzada, lo que halla en favor de un parentesco muy próximo. No obstante, empleando la técnica de inmunoelectroforesis se detectaron diferencias significativas. Los antisueros utilizados mostraron distinta capacidad para reaccionar con el suero de cada especie, dando arcos de precipitación más nítidos con el suero homólogo. Se caracterizaron, además, dos fracciones de naturaleza lipoproteica en el suero de ambas especies, que por su movilidad electroforética se las clasificó como alfa y beta lipoproteínas

Estudios inmunológicos en dos especies afines de Ceratophrys (Anura Leptodactylidae): C. ornata (Bell, 1843) y C. cranwelli (Barrio, 1979) / Immunological studies in 2 species of Ceratophrys (Anura Leptodactylidae): C. ornata (Bell, 1843) and C. cranwelli (Barrio, 1979)

Se estudiaron dos especies afines del género Ceratophrys: C. ornata y C. cranwelli, mediante técnicas inmunológicas para hallar caracteres no morfológicos que contribuyan a su mejor clasificación. En las inmunodifusiones bidimensionales de las proteínas séricas de ambas especies se observó una gran reactividad cruzada, lo que halla en favor de un parentesco muy próximo. No obstante, empleando la técnica de inmunoelectroforesis se detectaron diferencias significativas. Los antisueros utilizados mostraron distinta capacidad para reaccionar con el suero de cada especie, dando arcos de precipitación más nítidos con el suero homólogo. Se caracterizaron, además, dos fracciones de naturaleza lipoproteica en el suero de ambas especies, que por su movilidad electroforética se las clasificó como alfa y beta lipoproteínas (AU)

[Immunological studies in 2 species of Ceratophrys (Anura Leptodactylidae): C. ornata (Bell, 1843) and C. cranwelli (Barrio, 1979)]. / Estudios inmunológicos en dos especies afines de Ceratophrys (Anura Leptodactylidae): C. ornata (Bell, 1843) y C. cranwelli (Barrio, 1979).

Two related species of the genus Ceratophrys (C. ornata and C. cranwelli) were studied by immunological techniques in order to find non-morphological characters which would allow to classify them more precisely. Bidimensional immunodiffusion of serum proteins showed extensive crossreactivity between both species, thus revealing their close relatíonship. However, by immunoelectrophoretic techniques, differences between their serum fractions were detected. The antisera raised in rabbits against the serum proteins showed different potency in reacting with the sera of each species. Sharper precipitation bands were obtained with the homologous serum. Two lipoproteic fractions, in the serum of both species, were also characterized.