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.

Introduction

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, Gregarina blattarum spores may be causally related to human asthma in cities, where infestations of the host cockroach, B. germanica, are common, Fig. 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.
 

Methods

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, Inc.).
 

Results

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.
 

Conclusions

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.
 

Future work

• How can the gregarines detect the pH gradient?
• How do they achieve their directed movement?
• Investigate a possible role of proton fluxes associated with the gliding motion of sporadin using the non-invasive ion probe.
• Measure the velocity in various pH over several time intervals to see if velocities are constant.
• If constant velocity conditions can be found, use various channel inhibitors to determine possible roles of ions in maintaining velocity.
• Purification of the spores for possible use in skin tests with asthmatic/allergic patients.
• Immunohistochemistry on the spores using human allergic serum.

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 the project.
 

References
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).
 
 
 

Table 1. Simplified culture media 

Components	Amount
Glutamic acid	5mM
Lysine	5mM
Serine	5mM
K2HPO4	50µM
NaCl	0.90%
KCl	10mM

 
 
 

Table 2.  Culture media for G. blattarum sporadin.

Components	Abe and Aoyama	Modified to
NaCl	0.85%	0.90%
KCl	0.035%	0.035%
CaCl 2 •2aq	0.026%	0.026%
Intestine extract	10%	10%
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