Utility of Calcofluor M2R and Sytox Green Dual Staining for Assessment of Microsporidia

Thelohania solenopsae Invading Solenopsis invicta

M. Walker Hale and S.B. VinsonDepartment of Entomology, Entomology Research Laboratory,

Texas A&M University, College Station, Texas 77843Introduction

Currently, there are no published reports on spore viability of T. solenopsae when removed from the host S. invicta, and no published reports on spore viability when spores are infused into oils, protein, or carbohydrates, ingested by RIFA. Likewise, there are no published reports on the viability of those spores when stored in the oils, protein, or carbohydrates. This study sought to answer questions concerning T. solenopsae viability after centrifugation, and infusion into a bait via a dual staining method developed by Green et al (2000),using Calcoflouor White M2R, a chitin binding stain (a component of microsporidian spores), followed by Sytox Green a nuclear binding stain which fluoresces spores that have everted their polar filament.

Methods and Materials

RIFA workers and dealate reproductives were taken from colonies assessed positive for T. solenopsae infection. The workers were homogenized using 0.5 mm beads in a Mini Bead Beater® and 500µl of water. This procedure was repeated 25, and the resulting homogenate was strained into a 500ml Falcon tube using a Falcon 40 micron screen to remove larger ant debris. 200µl of this homogenate was used to fill 100 1.5ml centrifuge tubes. Modified centrifuge techniques derived from Shapiro et al. (2003) were used to pellet the T. solenopsae spores. The centrifuge spun at 6000 rpm for 5 min., re-suspension in 100µl and then centrifuged at 6000 rpm for 5 min. Resultant pellets were re-suspended in 99µl water, to check for viability ratios after centrifugation, or re-suspended with 99µl egg albumin, 99µl 10% glucose solution, 99µl 20% glucose solution, or 99µl of high oleic safflower oil. Spores from centrifuge tubes were checked weekly for 10 weeks using Green et al (2000) methodology for discrimination between viable and dead microsporidian spores using dual staining Sytox Green and Calcofluor White M2R. This method was repeated utilizing binucleate free spores, the only difference in methodology was dealated reproductives replaced the workers at the first step of homogenization.

Methodology for Dual Staining with Sytox Green and Calcofluor White M2R:

1µl of Sytox Green was added to centrifuge tubes with 99µl of solution with a spore pellet. This solution was vortexed and then incubated for 6 min. A 20µl aliquot of the resultant solution was added to a haemocytometer and counter stained with 20µl of Calcofluor White M2R, a chitin binding stain, and assesses at 400 under a fluorescing microscope. Yellow-green ovals through the 470-490 nm excitation wavelength used for Sytox Green staining were counted as dead spores. Turquoise or white ovals at 395nm to 415 nm excitation wavelength filter used for Calcofluor White M2R staining were counted as total spores.

Results and DiscussionThe within subject factor of time in had an effect on queen and worker derived spores at the (α=.05) level, significance (P<0.000) Repeated Measures. The only food class that differed significantly between the treatments was a greater decrease of spore viability at 20% glucose concentration (P=0.02) Fisher’s LSD. Since there is a general increase in spores detected by Sytox Green this indicates an increase of polar filament extrusion over the time course. This evidence supports that a ratio of spores survive the extraction and centrifugation process, and are viable. The difference at 20% glucose may relate to the osmotic pressure on the spores at this carbohydrate concentration.

Fig. 1 Calcofluor White M2R a chitin stain Fig. 2 Sytox Green a nuclear stain fluorescing at 415 nm fluorescing at 480 nm

Fig. 3 Queen derived spores showing decrease in viability over time

REFERENCES CITED

Green, L.C., P.J. LeBlanc, E.S. Didier. 2000. Discrimination between Viable and Dead Encephalitozoon cuniculi (Microsporidian) Spores by Dual Staining with Sytox Green and Calcofluor White M2R.

Shapiro, A.M, J.J. Becnel, D.H. Oi and D.F. Williams. 2003. Ultrastructural characterization and further transmission studies of Thelohania solenopsae from Solenopsis invicta pupae. J. Inverteb. Pathol. 18(2):177-180.

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