Bd in Bahia and Espirito Santo

Jan. 22, 2014.

The Bd in Brazil crew is back again this year, this time moving the transect north into the states Espirito Santo and Bahia of the Atlantic Forest. We are a team of mycologists and herpetologists interested in understanding the biology of a fungus disease that globally is decimating amphibian populations (Wake and Vredenburg 2008). The ultimate goal of the Brazilian research is to sample across the remaining fragments of the Brazilian Atlantic Forest from Rio Grande do Norte to Rio Grande do Sul for molecular epidemiological studies of the fungus.

This year’s crew is again a combination of researchers at UNICAMP (Felipe Toledo Lab, Campinas, Brasil) and University of Michigan (Tim James Lab) and Cornell University (Kelly Zamudio Lab). We also had the pleasure of adding a member from UFAL (Universidade Federal de Alagoas), Anyelet Valencia from Tamí Mott’s lab.

In brief, we found Bd at 4/5 sites in tadpoles, with prevalence ranging from very high (50%) to very low (1%) on the basis of morphological examination of tadpoles. The three sites with high levels of Bd were all high elevation (>600 m) localities, whereas the lowland rainforest showed a near absence of Bd in tadpoles. It will be of great interest to compare the results we have on Bd prevalence from the larval data to that of the adults. At each of the 5 localities, over 100 adults were swabbed and analysis of Bd infection by qPCR is in process at UNICAMP. At one locality near Pedra Azul, we collected a second year North American bullfrog tadpole, infected with Bd, in among a diverse pool of native tadpoles, some also infected.

girinos
A couple tadpoles from the pond near Pedra Azul, Espirito Santo.

What is becoming abundantly clear is that there is a strong correlation with detectable Bd infection of larvae and temperature of the water from which they are collected. Cool streams running down from high elevation sites seem to invariably yield infected tadpoles, whereas warm unshaded ponds as found in deforested sites with daily maximum temperature above 25 have all uninfected larvae. Of course this fits in very well with what we know about the tolerance of Bd in the laboratory. That is, growth is essentially halted when Bd experiences temperatures at or above 28 C (Piotrowski et al. 2004). On the other hand, adults collected from the same warm ponds can be quite infected with Bd. These observations of differential infection of adults and larvae prompt at least a couple questions regarding the disease dynamics. Firstly, how do the adults become infected in the first place? Where is Bd lurking among the places to which adults might journey? Are adults acquiring their infections by crossing these Bd-infested streams or is there another refuge for the fungus in these warmer sites? Secondly, what effect does this high level of Bd infection in tadpoles have on the adults of riparian species of frogs? Are there sub-lethal effects or mortalities that have gone undetected.


dendropsophus
Dendropsophus branneri from southern Bahia. These frogs breed in permanent and vernal pools and live in a variety of habitats except closed canopy forest. Bd infection status is unknown at present.

The answer to this second question is critical to our understanding of the impacts of Bd on Brazilian amphibians. While the consensus, or absence of evidence, points to no known Bd-related declines in the Atlantic Forest of Brazil, it is a statement which largely speaks to poor data. Also, Bd could also have large impacts on population density without obvious extinctions and observed die offs. As it stands, it is an interesting correlation that the 31 species suspected of undergoing declines are enriched in those who inhabit high elevation riparian sites (Eterovick et al. 2005). Our crew has now visited a couple sites where declines are reported (PARNASO, Teresopolis, RJ and Santa Teresa, ES) and these two localities are sites where Bd occurs at high prevalence.

Our sampling has now expanded over a large portion of the Atlantic Forest and has sadly extended the known distribution of Bd into additional states, Bahia and Espirto Santo. The once continuous Atlantic Coastal Forest of Brazil appears to have Bd wherever conditions are favorable for the fungus. Interestingly, there appears to be a negative correlation between Bd prevalence and degree of forest fragmentation (Becker and Zamudio, 2011). This paradox has been explained in a couple ways, either the degraded habitat selects for weedy species of frogs that thrive in disturbed areas, and there may be a correlation between aggressive weedy species and resistance to Bd, or the reduction in diversity may reduce the equilibrium infection frequency of Bd in a community. Alternatively the increased fragmentation leads to increased solar radiation and thus higher temperatures, making water and terrestrial habitats perhaps less favorable to Bd.

Figure 7
Map of the distribution of Bd in Brazil as known in 2012 (Fig 7 of Schloegel et al. 2012).

Despite the observation that Bd may occur in lower frequencies in deforested sites, stopping or reversing deforestation in Brazil should be a central priority of conservation biologists working here. Though not based on any specific data or experiments, my impression is that the the Atlantic Forest and its biodiversity could rebound if the deforestated areas are allowed to recover, a change in land use policies is implemented, and enough time elapses. While settlement contributes a large proportion to the loss of forest, obvious are also the hectares infinitos devoted to raising cattle, growing eucalyptus, sugarcane, coffee, soy, bananas, and etc. With a Brazilian economy emerging rapidly, including a burgeoning middle class with US sized consumerism, it will require progressive leadership to value future resources over money in pockets. Nevertheless, the people are concentrated into urban environments, with a rural landscape that is deforested primarily for agriculture or logging. When I look at this fragmented landscape, I see potential for regrowth of the forest, for the salvation of the wildlife and plant species and their diversity. What is left must be protected at all costs because extinctions are much more likely given small fragment sizes and invasive species. What has been razed for cattle and sugarcane must be allowed to grow back by a few centuries of abandon. I remain hopeful.

Back to our research. Brazil is known to harbor three genotypes of Bd: Bd-GPL (a global genotype associated with all known Bd related declines), Bd-Brazil (a genotype known only from Brazil and possibly Japan with unknown virulence), and Bd-Hybrid (sexual genotypes produced between Bd-GPL and Bd-Brazil). We are attempting to use our cultures of Bd to understand how temporal and spatial patterns of genotypes can answer the question of what and when Bd came to Brazil. We predicted last year that Teresopolis, where declines are recorded, would have the hyper virulent Bd-GPL genotype, and that held true (in prep). We predict the same for Santa Teresa, ES, where species like Colostethus olfersioides are in decline (Weygoldt 1989; Stud. Neotrop. Fauna Environ. 243: 249). As part of the same hypothesis we predicted that Bd-Brazil was endemic, perhaps extending deep into the time when there were multiple, isolated refugia for fauna during Pleistocene global cooling and drying events. Maybe there are more genotypes of Bd in the Atlantic Forest that have yet to be recovered centered around these refugia. In frogs at least, there are known to be three refugia in the AF relating to concentrated forest fragments (Carnaval et al. 2009). Maybe the presence of these endemic genotypes has provided some sort of defense against Bd-GPL, some sort of immunization or competition among the global hyper virulent lineage? Currently, we have found Bd-GPL at every site we have sampled, but Bd-Brazil only at sites in Paraná, Santa Catarina, and São Paulo. This would seem to be consistent with a model of Bd-GPL recently replacing Bd-Brazil, perhaps through their introduction on bullfrogs, which arrived in Brazil in the 1930’s. However, this hypothesis is challenged by the recent observation that both Bd-Brazil and Bd-GPL genotypes have been detected from museum specimens dating back to at least 1916 (Rodriguez et al. 2014). Bd has apparently been in the Atlantic Forest since 1894.

proceratophyrisproceratophyris_inflatus

Proceratophrys schirchi. These Leptodactylids are able to defend themselves from predation by inflation. Photos: T. James (Top), K. R. Zamudio (bottom).

Getting to the bottom of the genotype mystery will require integrated studies of museum collections, population genetics, and infection experiments. Detecting population expansion could use coalescent methods in population genetics, but a general lack of variation and polyploidy has been a big hindrance. We are currently developing new markers to ask the question of whether the Bd-Brazil or the Bd-GPL strains have greater heterozygosity in the AF. That seems to have been the case based on genome resequencing (Rosenblum et al. 2013), but many more isolates need to be tested. I dream of establishing long term monitoring plots in the area we know that all three genotypes occur so that the dynamics of the genotypes in time and space can be studied properly.

Até mais!

Chytridiomycosis in Rio: Serra dos Órgãos

Feb. 3, 2013. We've recently returned from a trip east to a national park called Serra dos Órgãos (PARNASO) in the state of Rio de Janeiro. This region of Brazil is particularly interesting because it is a place where a few endemic species have declined in recent times (Silvano and Segalla 2005), including the species Thoropa petropolitana. Whether or not chytridiomycosis is involved in the declines is something we hope to address with our research. Many things about PARNASO seem like a good place for Bd: it is a cloud forest, extremely wet with many streams, pools and rivers, cool temperatures, and proximity to a major metro area, the town of Teresópolis. Amphibian diversity is high here, including about 100 species, with some species like T. petropolitana and Aplastodiscus musicus only known from this location.


Bd group at PARNASO: L to R: Joice Ruggeri Gomes, Carlos Henrique Nunes-de-Almeida, Clarisse Betancourt-Roman, Thomas Jenkinson, Tim James

If there are chytridiomycosis related declines in Brazilian Atlantic Forest then this might be the place. Could all the environmental conditions be met such that chytrid can actually lead to species decline and even extinctions here unlike other parts of the AF where no disease related declines are known? And is this region a bubbling breeding ground for the formation of virulent strains of Bd that have been dispersed to parts far and wide and have caused havoc in other parts of the world. Is this the heart of the global chytridiomycosis epidemic?


Aplastodiscus is found in PARNASO in epiphytic bromeliads.

This leads me back to the discussion of whole reason why the Brazilian Atlantic Forest is so important for chytridiomycosis research. First, the biodiversity and endemism of the region is phenomenal, yet horribly threatened by anthropogenic disturbance. Second, the region has many threatened species and many species for which data are inadequate (see note by Pimenta et al. 2005), but those declines and disappearances are suspected of being due to habit destruction with no known role for chytrid. Yet, when you look at the models that attempt to predict the distribution of chytrid based on existing data and climate/species variables, they strongly suggest that chytridiomycosis should be very prevalent throughout the AF, with some non-negligible risk of decline or distinction centered around Rio de Janeiro (see Figure C below from Rödder et al.2009, Diversity 1:52). These models are entirely consistent with the existing publications showing chytrid to be widespread in the AF (Carnaval et al. 2006, EcoHealth 3:41; Toledo et al. 2006, South American Journal of Herpetology 1:185) and our own observations from this trip. Yet based on the analysis of Bielby et al. (2008, Con. Letters 1: 82), many of the life history characteristics of species declining due to Bd in other geographic regions are not observed in the AF. The details of this analysis regarding amphibians of the AF are a little curious to me, as Bielby et al. identified high altitude, low fecundity, and aquatic life history traits as factors associated with risk of species displaying Bd-associated declines, and all of these characterize at least parts of the AF herpetofauna. Something also is a bit imperfect in the models as Panama doesn't show a smoking signal in the models, yet it is the most notorious region of Bd declines.


Figure 2 of Rödder et al. 2009. A). Species Distribution Model showing the potential distribution of Bd based on existing occurrence data and climate variables. B). Overlap of the potential Bd distribution and amphibian diversity shows the Brazilian Atlantic Forest to have the huge potential for species declines and possibly Bd diversity. C). However, Risk Factor analysis suggests that by overlaying potential Bd distribution and life history traits of the animals, the Atlantic Forest amphibians are not nearly in as much jeopardy as Andean frogs.

Just as expected, we found evidence for Bd in PARNASO on from infected tadpoles collected from streams. We also swabbed exactly 100 adults for later molecular diagnostic tests for Bd that will be done at UNICAMP. Adults were a bit hard to find, which our local expert, Joice Ruggeri, suggested was due to the incessant rain rather than chytridiomycosis, based on her experiences at PARNASO. We are currently working on isolation of the pathogen from the tadpoles and are ultimately very interested in determining the genotypes of the Bd strains. Our hypothesis is that PARNASO may show an epidemic population structure with a single genotype of the hyper-virulent Bd-GPL group.

I wanted to share some excellent photos that Clarisse Betancourt-Roman took of the infected tadpole mouthparts. These poor tads can get so infected by Bd, that their foraging behavior is significantly reduced (Venesky et al. 2009).

Figure A) the upper jawsheath is clear and black across, whereas in B) most of the pigment of the upper jawsheath (red arrow) is missing. C) another jawsheath shows a large region of depigmentation in the center (arrow). D). Under higher magnification, numerous spheroid Bd sporangia can be seen. credit (Clarisse Betancourt-Roman).

PARNASO also has tons of amazing wildlife, plants, and fungi.




Brachycephalus ephippium (sapinho-pingo-de-ouro or pumpkin toadlet). B. ephippium moves very slowly but it can afford to because its color is a warning that it is highly poisonous. However, it can also leap when needed and does an odd karate kid style move when threatened by other males.



Marasmius


Favolaschia




fruit flies aggregating on the surface of a Ganoderma mushroom


rufous-crowned greenlet (credit Carlos Henrique Nunes-de-Almeida)


pin-tailed manakin (credit Carlos Henrique Nunes-de-Almeida)

These last two pictures were taken by Carlos Henrique who is a graduate student with Felipe Toledo and also an amazing birder who leads tours in southern Brazil (http://www.carduelis.bio.br/).

Falamos depois!

Samba School!

Jan 18, 2013. Samba School in Bragança Paulista. It’s Carnaval season.



First Bday!

Jan 16, 2013. Today we had the first ever meeting of scientists interested in chytridiomycosis in Brazil at UNICAMP. Approximately 30 scientists were in attendance. Tommy gave an impromptu talk about his thesis project to fill in for Ananda who couldn’t make it.





The Road South

Jan 13, 2013. We return from our trip south searching for Bd-Brazil and hybrid genotypes. We have spent a week swabbing frogs and collecting tadpoles for isolation of the chytrid fungus. We visited localities in these three states: São Paulo, Paraná, and Santa Catarina. The final point of our transect was near Rancho Queimado in Santa Catarina. We found chytrid infected tadpoles at each of the localities. The rate of chytrid infection was really high, roughly the 50% previously reported for Brazilian Atlantic Forest tadpoles (Toledo et al. 2006, South American Journal of Herpetology 1:185). This blew me away as we find almost zero percent tadpoles with dekeratinized mouthparts back in Michigan. What did make me feel more at home was the presence of the North American bullfrog Lithobates catesbeianus at one of the localities. Multiple attempts were made to shoot and kill this invasive pest.



We swabbed over 500 frogs including this Phyllomedusa.


The most common frog was Hypsiboas faber.