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Rationale of the study
A study carried out on a tree growing in Milano (Italy) revealed how cryptococcal yeasts interact with a number of arthropods living on the tree and how they can act as vectors for spreading in the environment (Fungal Ecology in press). In particular a strong interaction exists with ants that usually are the main hosts living on a tree.
Aim of the study
To understand which is the relationship between Cryptococcus and arthropods (in particular ants). Are they living in symbiosis? Are arthropods only vectors? Or they meet Cryptococcus only occasionally?
How to verify the frequency of interaction between Cryptococcus and arthropods?
This could be verified through an extensive environmental survey collecting arboreal ants and other arthropods as well as some samples from the tree. The large number of arthropods collected will allow to estimate: how frequent is the interaction with Cryptococcus, if there are arthropod species with a higher frequency, if there are different genotypes of Cryptococcus in the same community of arthropods, if there are hotspots area colonized by the same strain of Cryptococcus.
How to verify symbiosis?
Sampling of single ants or arthropods living in a tree colonized by Cryptococcus and dissecting single internal organs such as stomach. Culture of the organs will prove the presence of the fungus inside the arthropod.
Once verified this hypothesis section of a number of arthropods could be performed and observed under an electron microscope in order to understand where Cryptococcus is located in the arthropod body.
Further investigation could be done verifying the internal temperature of the ant nests, culturing ants present in the nest, the soil of the nest, and the ability of Cryptococcus to sexually reproduce on the soil of the nest.
Finally we could verify the presence of cryptococcal laccase in the arthropod body.
How to verify the role of arthropods as vectors?
Verifying the presence of different Cryptococcus genotypes in the same arthropod community.
Verifying the presence of genotypes different of those colonizing the tree on flying arthropods (winged males of ants, wasps, bees, flies, bugs, and others).
Verifying if among yeast-colonized arthropods are present arthropods coming from other colonies living on other trees.
How to verify interaction with other fungi?
Identifying the species or genus of fungi present in the micro habitat together with Cryptococcus.
Quantifying the number of growing colonies for each species.
Collecting samples in the different periods of the year.
Methods
How to catch arthropods
1) Ants and other arthropods moving rapidly on the tree: use a piece of adhesive tape to immobilize the arthropod and then transfer it into a vial by mean of tweezers.
2) Arthropods moving slowly on the tree: use tweezers.
3) Flying arthropods such as flies, bees, hornets and others: use a bottle containing water and sugar.
4) Winged males of ants: usually they move on the tree and can be easily caught with a piece of adhesive tape.
Cultures
Arthropods will be transferred in a vial with 1-3 ml (depending of the number of arthropods) of sterile distilled water plus CAF and then vortexed for at least 1 min. 100 µl of the supernatant will be cultured on two 90-mm Petri dish containing Niger seed agar (NSA, see Technical Information).
Then the samples will be fragmented with tweezers and again vortexed for 1 min. 100 µl of the suspension will be cultured on two 90-mm Petri dish containing NSA.
Incubate one plates at 25°C and one at 37°C for at least 2 weeks checking periodically the presence of brown colonies.
Identification and molecular typing
All brown colonies grown on NSA plates will be collected (at least 10 if more than 15 colonies are present) and identified by classical methods (see Technical Information). Cryptococcus species complexes will be identified by culturing the isolate on CGB agar medium (see Technical Information). Then DNA will be extracted and amplified for molecular type and mating type determination (see Technical Information).
Conservation of the isolates
All isolates can be easily conserved in sterile distilled water as describe in the Technical Information.
Survey data
Data of each survey must me entered in the Environmental survey e-form of the ScreenProject website.
It is important to indicate the date and the site of the survey with geographical coordinates as well as to recognize the species or the genus of the tree and of all arthropods collected. Alternatively it is important to take some photos for later identification.
For ant species identification you can refer to the following website http://formiche.chiave.free.fr/castes-formice.html
Strain collection
All isolates will be sent to Medical Mycology Lab of Università degli Studi of Milano (Italy) that will perform coding, storage, and molecular typing.
Shipping address:
Massimo Cogliati
Lab. Micologia Medica, 3° piano
Dip. Scienze Biomediche per la Salute
Università degli Studi di Milano
Via Pascal 36, 20133 Milano, Italy
Tel. +39 0250315144
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