presentation for symbios class

8/13/2019 Presentation for Symbios class.

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Leaf Cutter Ants

Part of the family Formicidae: leaf cutter antscomprise two genera, Acromyrmex and Atta, within the

tribe Attini.

Found in South America, Central America, Mexico, and

parts of southern United States.

Have an obligate mutualistic symbiosis with fungi.

Process fresh vegetation to serve as nutritional

substrate for their basidiomycetefungal cultivar.


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Fungus Gardens

Monocultures of a single cultivar strain.

The cultivated fungus together with the plant

substrate compose the fungal garden.

Reproduction is clonal.

Sometimes during cultivation the wrong type

of fungus can grow. Can kill and live off the fungus

garden


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Mutualistic Symbiosis

Fungi develop gongylidea, nutrient rich ends ofhyphae, that the ants use to nourish their brood.

Fungus is used to feed the ant larvae and principal foodof queen and adults.

Adult ants feed off the leaf sap.

The fungus in turn receives nutritional substrate, andis provided with an environment for suitable growth.

The ants also disperse the fungus when a queen startsa new colony.

Queen carries a small fungal inoculum from natal colony.


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Fungus gardens, being monocultures, lack geneticdiversity and are susceptible to diseases andparasites.

A specialized fungal parasite Escovopsis infectsnests of attine ant species. Escovopsis most frequently encountered non-

mutualistic fungus found in attine gardens. Escovopsis act as necrotrophic parasite.

Destroys the cultivars hyphae

Also exhibits a complex pattern of co-evolutionwith the cultivar.


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Attine ants harbor a community of other microbes intheir gardens, including micro-fungi.

Leafcutter ants can regulate the microbiota in gardens.

The function of the associated microbiota in the fungigarden is unknown.

Harmful invaders, weeds when found in high frequency.

Neutral and transient commensals, with negligible effectson garden homeostasis.

Beneficial ancillary components, serving for production ofenzymes or antibiotics.


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Hypothesis

Further understand the distribution andprevalence of micro-fungal species in gardens ofleaf-cutting ants.

Gardens of leaf-cutting ants harbor several soiland plant-borne fungi. Also shows comparatively low infection rate by

Escovopsis.

Diversity of soil and plant-borne fungi may

function under certain conditions asopportunistic pathogens in fungal garden. Stresses the symbiosis by competing with the fungal

cultivar for nutrient resources.


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Fungus Garden Sampling

4-17 September 2004

Rio Grande do Sul, Brazil

37 mature nests 10Acromyrmexspecies

Recorded type of substrate carried by foragers

and nesting habitat Nests were carefully excavated or opened to

prevent contamination


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Microfungi Isolation

2 different techniques

Ten fragments from each garden collection were removed and inoculated

in potato-dextrose agar (PDA) plates supplemented with chloramphenicol

Six fragments were freed of workers and brood and placed into a sterile,

humidified petri dish All plates were incubated at 25C for 7-14 days in the dark

Plates and chambers were checked daily for signs of filamentous fungal

growth

Once fungal growth emerged, an inoculum was transferred to malt agar

2% plates to obtain pure cultures

17 isolations were PDA only, 4 isolations were wet chambers only, and 16

isolations with both methods


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Fungal Identification

Morphological Methods

Molecular Methods

Isolates grown in aerated liquid cultures for 7 days at 25C

Mycelia harvested and lyophilized (freeze dried)

Genomic DNA extracted with cetyl trimethylammonium bromide

PCR was performed

ITS4 and ITS5 primers used to amplify the internal transcribed

spacer regions of the ribosomal DNA

For Escovopsis, modified eafF and eafR were used to amplify a

fragment spanning the exon 6 of the elongation factor 1-alpha

Amplicons sequenced on an ABI Prism 377 DNA sequencer


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Sequence Analysis

The consensus sequence was used in BLASTN

searches and sequences presenting 99%

similarity were considered as identified.


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Microfungal Distribution inAcromyrmexNests

Cunninghamella, Escovopsis, Fusarium, Mucor,

Penicillium, andTrichodermawere the most

prevalent, occuring in at least 18% of the gardens

Ten of the 16 genera were observed in monocot-cutting ants

14 of the 16 were found in dicot-cutting ant gardens

8 were found with both monocot and dicot-cuttingants


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Phylogenetic Analyses

Evaluated the phylogenetic relationshipbetween ITS haplotypes from their F.oxysporum isolates compared with published

ITS haplotypes from F. oxysporum strainscommonly found in soil or plant substrates.

Confirmed that the major ITS2 type present inthe isolates was the ITS2-type I.

Low polymorphism of the ITS1 and ITS2 regions.

One nucleotide difference on average.


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Phylogenetic Analyses cntd.

No species-specificity was detected between

Acromyrmex ants and Escovopsis strains from

Southern Brazil.

Closely related Escovopsis strains were associated

with different ant species

Gardens of the same ant species were associated

with more distantly related Escovopsis strains.


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Take Home Messages

Gardens of monocot of dicot-cutting and mound-builder orsoil-dweller leafcutter ants harbored slightly distinctmicrofungal communities. Garden substrate and nest-type may influence microfungal

contamination.

No microfungal lineage was clearly specialized on eithergarden substrate or nest type.

There are many factors that can influence the gardenmicrobiota.

Specific plant species harvested by the ants, age of the colony,garden contaminants.

Presence of microbiota can have drastic effects onleafcutter gardens.


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References

Rodrigues A, Bacci Jr M, Mueller UG, Ortiz A,

Pagnocca FC (2008) Microfungal Weeds in

the Leafcutter Ant Symbiosis. Microb Ecol

56:604-614

Currie CR, Mueller UG, Malloch D (1999) The

Agricultural pathology of ant fungus gardens.

Proc Natl Acad Sci USA 96:7998-8002

presentation for symbios class

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