W.W. Pong, Y. Xu, & J.G. Kunkel
Department of Biology, University of Massachusetts
Figures and Tables
In urban domiciles, Blattella germanica allergens have been implicated in human asthma and allergies and are noted triggers in the increasing cases of childhood asthma. These allergens found in the dust and air of homes have been identified in fecal and whole body extracts of cockroaches. The gregarine (Apicomplexa: Gregarinasina) parasite, Gregarina blattarum, is found infesting the midgut of B. germanica and their cysts and spores are found in the cockroach feces. It is not known whether gregarine spores are related to childhood asthma. In order to study the correlation between human asthma and Gregarina blattarum, it is necessary to investigate both the sporadin and the spore stages.
Gregarines were removed from the host, Blattella germanica, and cultivated in a modified culture media (Abe and Aoyama, 1979). The sporadin were found to live and move for at least five days in this solution. Isoelectric point focusing was employed with glutamic acid, serine, lysine, and a simplified culture solution to produce a pH gradient from four to twelve, which was monitored using the non-invasive vibrating probe. Gregarines were introduced into the solution and their movement was observed as an indicator for the effect of the pH. Results indicated that the sporadin can survive in a wide pH range and that their rate of movement may be affected by differences in pH.
Cysts were obtained from cockroach feces, cleaned, and placed in a moist chamber to facilitate sporulation. Extruded spores in long strings were collected, but purification has not yet been successful.
Further work includes using the vibrating probe on the sporadin to detect ion fluxes associated with its gliding mode of locomotion, purification of the spores, and immunohistochemistry on the spores using human allergic serum.
Childhood asthma is recently becoming a more serious problem, and much
research has pointed to allergens from dust mites and cockroaches.
The main cockroach allergens identified were from Blattella germanica
whole body and fecal extracts. Since most cockroaches are parasitized
by gregarines (Apicomplexa: Gregarinasina), it is possible that some gregarine
allergens have been misidentified as cockroach antigens. Most notably,
blattarum spores may be causally related to human asthma in cities,
where infestations of the host cockroach, B. germanica, are common,
1. Elimination or control of spores may be accomplished by killing
the spores directly or indirectly by targeting the sporadin stage within
the cockroach host. This parasite may also be a method of controlling
populations of the cockroaches. Therefore, it may be useful to study
the sporadin as well as the spore stages of the gregarine.
Blattella germanica were decapitated and dissected. The midguts were removed and the gregarines were gently cleaned from the intestinal material and placed into 0.9% saline. Then, they were put into a modified culture media (Abe and Aoyama, 1979) in petri dishes and observed over the course of 5 days.
A ten centimeter quartz trough was half filled with 2% agar made with a simplified culture media (Table 1) and isoelectric point focusing was applied. Then a layer of solution was added to allow use of the non-invasive vibrating probe to measure the pH gradient (Fig. 2).
Gregarines were introduced, and ASET v.2.00 (Eric Karplus; Science Wares) was used with the non-invasive vibrating probe (Alan M. Shipley; Applicable Electronics) to detect the pH gradient, while MGI VideoWave v.1.0c (MGI Software Corp.) was used to record the movement at every centimeter. Frames were extracted from the movies with AVIQuick v.1.0 (Paul A. Roberts), and ImageJ v1.16f (Wayne Rasband; NIH) was used to find edges and overlap frames. The resulting frames were then analyzed with UmassJJa v1.0, developed by one of the authors (Y.X.).
Cysts and spores were collected from the B. germanica feces (Hoshide
et al., 1993) with some slight modifications. Cysts were placed on
coverslips and incubated at 27ºC in a Petri dish moist chamber.
Extruded spores, Fig. 3, were removed from the
coverslip by suspending them in saline and using a pipet to transfer them
into Eppendorf tubes. Pictures in Fig. 4
were taken using Kodak Digital Science Microscopy Documentation System
120 (Eastman Kodak Company) with Paint Shop Pro v. 5.01 (Jasc Software,
The modified culture media allowed the gregarine sporadins to live for at least five days. By five days, however, bacteria and fungus were overgrown in the Petri dishes, and this impeded the gregarine movement. We designed our experiments to conclude within 24 hours.
Isoelectric point focusing produced a pH gradient from 4-12 over a distance
of six centimeters in the trough. Motion of the gregarines was recorded
to AVI files and velocities analyzed, Fig. 5.
The modified culture media was adequate for keeping gregarines in vitro for the duration of most experiments. Typically, experiments were designed for one day.
Although there is no obvious optimum pH, gregarines could live in a wide pH range, and suggests pH association with motility (Fig. 5). It is uncertain whether the sporadin are moving away or towards a certain pH, or whether there are optimal proton levels for movement at pH 8. The gregarines were motionless at the extreme ends of the gradient - dying and bursting at the highest pH end, but retaining their shape at the lowest pH.
The integration of both hardware and software in the MRMS was successfully
applied in this experiment, and can be used as a foundation for a broad
range of applications, such as immunology, oncology, and embryology.
This work was supported by the Research Experience for Undergraduates,
Howard Hughes Medical Institute’s Undergraduate Biological Science Education
Program and was done in cooperation with Alan M. Shipley of Applicable
Electronics and Eric Karplus of Science Wares. I would also like
to thank all the REU advisors and participants for their support and guidance.
Special thanks to Jessica Jeffrey, for her advice and sincere support throughout
Abe, H., Aoyama, M. 1979. In vitro culture of the Gregarina blattarum. Bulletin of the Faculty of Education, Yamaguchi University. 29 (2); 1-10.
Hoshide, K., Nakamura, H., & Todd, K. S.
1993. In vitro excystation of Gregarina blattarum oocysts.
Acta Protozoologica. 32; 67-70
Figure 1. Model
Figure 2. Motion Recording and Measurement System (MRMS)
1. Ion-selective probe
2. Reference probe
3. Quartz Trough
4. Microscope + Video Camera
5. Amplifiers + Motion Control + Computer + Software
Figure 3. Cyst surrounded by strings of spores
Figure 4. Magnified sporoduct and spore from cyst
Figure 5. pH versus
gregarine average speed (+/-95% CI).
|Components||Abe and Aoyama||Modified to|
|CaCl 2 •2aq||0.026%||0.026%|
|Yeast extract||0.1%||0.1% (nutrient broth)|
|Penicillin||5 unit/ml||5 unit/ml|
|Streptomycin||5 x 10-4 Gm/ml||5 x 10-4 Gm/ml|
|buffer- Adjusted to pH 7.4 with 2.5mM||phosphate buffer||none|