DGGE analysis of the structure of fungal communities associated to leaf litter and the gut of larvae of Triplectides (Trichoptera: Leptoceridae) in Atlantic Forest streams

Study Extent

Santa Clara and Banana stream, Santa Leopoldina, Espírito Santo, Brazil.

Sampling

At each stream, 10 larvae of Triplectides were manually collected on the streambed of a 200 m reach and stored in flasks containing stream water. The leaf patches on which the larvae were found were also collected and stored separately in plastic bags. The samples were then transported on the same day in thermal boxes to the laboratory. Each larva was submitted to surface disinfection by immersion in 1 mL of 70% alcohol (30 s), followed by immersion in 2% sodium hypochlorite solution (30 s) and washed in sterile distilled water. The dissections for removal of the gut of each larva were performed on individual slides superficially disinfected with 70% alcohol in a laminar flow hood, using scalpel blades and tweezers, also disinfected with 70% alcohol. Each gut was then transferred to eppendorf tubes containing 1 mL of sterile distilled water and homogenized. Discs (14 mm diameter) were cut from leaves of each leaf patch with a cork borer and stored separately in aluminum envelopes. The samples were kept at -20° C until molecular analyses. DGGE analyses were conduced by protocols below: Medeiros, A.; Duarte, S.; Pascoal, C.; Cássio, F.; Graça, M. Effects of Zn, Fe and Mn on leaf litter breakdown by aquatic fungi: A microcosm study. Int. Rev. Hydrobiol. 2010, 95, 12–26, doi:10.1002/iroh.200911182.

Method steps

1. Collect Dissection DNA extraction PCR DGGE analysis Analysis of data by PyElph program

1. Triplectides
   rank: genus

Santa Clara (20°00'37.68" S - 40°32'41.28" O; “SC”) and Banana (20°02'08.65" S - 40°31'58.01" O; “BA”) streams are located inside well-preserved Atlantic Forest fragments, in the municipality of Santa Leopoldina, Espírito Santo, Brazil.