Below are descriptions and photos of some of the methods we commonly use in studying Plasmodium mexicanum.
Lizards (Sceloporus occidentalis) are captured using a noose on the end of a fishing pole. See the Schall lab website for instructions on how to tie a noose.
A small sample of blood is collected from a toe clip. A few drops are preserved dried (air dried, then put in ziplock bag with silica beads) and frozen on filter paper for genetic analysis. One or two drops of blood are used to make a thin blood smear, which is fixed with Methanol and stained with Giemsa. The Schall lab website includes a video on making thin blood smears and photos of stained and unstained smears.
DNA is extracted from dried, frozen blood dots using a Qiagen DNEasy or Omega EZNA kit. It is then amplified at microsatellite loci using the primers described by Schall and Vardo (2007; Molecular Ecology Notes 7:227-229). All blood stages of malaria parasites are haploid, so each microsatellite allele represents a separate clone. In the pherogram above, the first sample has at least 1 clone, and the second has at least 2. The heights of the peaks are also related to the relative abundance of the allele in the sample.
Infections of P. mexicanum can be initiated in uninfected lizards by intraperotineal injection of infected blood mixed with saline. We can then monitor infections over time by sampling blood (via toe clip, as above) at different time points. Experimentally infected lizards are housed in outdoor cages during the course of the experiment.
The insects that transmit Plasmodium mexicanum are sand flies in the genus Lytzomyia. They rest in ground squirrel burrows during the day and emerge at night to feed. The primary way we collect sand flies is using funnel traps placed over the openings of ground squirrel burrows. These traps consist of a plastic funnel with a fabric “skirt” which is placed over the burrow just before dusk (5-6pm during July/August in Hopland, Mendocino County, CA) and kept in place with dirt and rocks (to ensure a nice “seal” over the hole). A cup with mesh is placed over the funnel opening to collect sand flies as they emerge. We check these traps around 10-11pm.
We keep live sand flies in an incubator at 25.8 Celcius. Flies are kept in jars with a layer of moist plaster of paris in the bottom few cm to maintain a humid environment. The tops of the jars are covered with mesh, and a flattened cotton ball soaked in 10% glucose solution is placed on top of the mesh to allow the sand flies to feed. (Note: the vial pictured is one we use to transport sand flies from the field to the lab, not the jar used in the incubator.)
Sand flies can also be kept in fabric/mesh cages in the incubator. We usually keep these cages in plastic bags with a damp sponge, again to maintain high humidity. These cages can also be used for experiments that involve feeding female sand flies on lizards. The masked lizard (masked to prevent the lizard from seeing and eating sand flies) is usually placed in the cage first and allowed to acclimate for 15 min (this reduces the number of flies that are trampled the lizard). Sand flies are then added and can be removed after they have fed. We usually allow them up to 24 hours.
We commonly catch 2 species of Lutzomyia at HREC- L. vexator and L. stewardii. Females are distinguishable by the shape of their spermotheca (left photo), and males by the placement of hairs on their claspers (right photo). A paper by Young and Perkins provides very useful illustrations for differentiating species (Young, D. G. and P. V. Perkins. 1984. Phlebotomine sand flies of North America [Diptera: Psychodidae]. Mosquito News 44:263-304; not available in digital copy)
We only find one species of malaria parasite in the western fence lizards at HREC. See the life cycle page for pictures of different parasite stages. We do commonly encounter 2 other blood parasites, however. Haemogregarines (left photo) are similar in size and color to P. mexicanum gametocytes, but the nucleus stains much darker and is always centrally located. Schellackia (right photo, shown in a cell with a P. mexicanum male gametocyte) also looks a bit like a gametocyte, but is smaller, pear shaped, and the pink staining always forms a band across the entire cell.