I. Introduction. - The class Trichomycetes comprises a diverse assemblage of fungi that live almost exclusively in the digestive tracts of arthropods (Lichtwardt, 1986). These fungi do not invade the body cavity of their hosts, but instead attach to the gut lining by means of an adhesive holdfast and absorb nutrients from the gut contents. Most species exhibit considerable host specificity and are generally restricted to specific regions of the digestive tract; for example, the midgut and hindgut regions. Spores are eventually produced inside the gut, which often must occur before the gut lining with its attached thalli are shed during molting. Once defecated into the environment, these spores are ingested by new host individuals and thus serve to maintain infestations within a host population. Spores of Trichomycetes are confronted with several problems once they are ingested by a suitable host: (1) They must "germinate" and attach to the gut lining very quickly before passing out of the host; (2) They must attach to the specific region of the gut where the fungus lives.

Smittium culisetae is frequently found in the hindguts of freshwater Diptera larvae, including mosquitoes (Culicidae), black flies (Simuliidae), and midges (Chironomidae). Hyphae of this fungus produce a unique propagule called a trichospore. The complexity of the trichospore emphasizes how marvelously adapted this fungus is to its unusual habitat. The trichospore (Fig. 1a) consists of an elongate sporangium that contains a single, closely appressed sporangiospore. A single filamentous appendage extends from the base of the trichospore and presumably allows the trichospore to become entangled in submerged debris in flowing water. When trichospores of S. culisetae are fed to mosquito larvae in the laboratory, they appear unchanged during passage through the midgut (see Fig. 2), the main digestive organ of the insect (Horn, 1989a). However, once encountering the posterior hindgut where the fungus lives, trichospores explosively extrude their sporangiospores, which then immediately form terminal holdfasts (< 30 sec) (Fig. 1b). The attached sporangiospore eventually grows into a branched septate thallus.

The specific stimuli in the mosquito gut responsible for sporangiospore extrusion and holdfast formation occur in two stages (Horn, 1989a,b; 1990). Trichospores are initially preconditioned in the midgut by exposure to potassium largely supplied by the insect's excretory organs, the Malpighian tubules (Fig. 2), under conditions of high alkalinity (pH 10). Once the trichospore passes into the hindgut (pH 7), the rapid drop in pH stimulates both sporangiospore extrusion and holdfast formation. This specific succession of stimuli--potassium at pH 10 followed by a drop in pH to 7--ensures that S. culisetae will become established only in an appropriate host (most insect midguts are not highly alkaline) and only in the hindgut of that host.

S. culisetae is easily cultured in the laboratory and readily produces trichospores. In this experiment, trichospores will be exposed to a solution that represents conditions in the midgut and hindgut of mosquito larvae. Trichospores will also be treated with solutions that lack either potassium or a shift in the pH to demonstrate the requirement for these stimuli. The response of trichospores to these different treatments will be observed under the microscope.

II. Materials:

• Culturing
  1. Culture of Smittium culisetae (isolate NE-27-1; ATCC 46885)
  2. Brain heart infusion (Difco)
  3. Agar
  4. Sterile distilled water
  5. Petri dishes
• Treatment of Trichospores
  1. Potassium chloride (KCl)
  2. Ethylenediamine (1,2-Diaminoethane)
  3. Citric acid (free acid)
  4. Microscope slides and coverslips
  5. pH meter
• Light microscope with high dry objective (e.g. 40x); an oil immersion objective (e.g. 100x) is also helpful. Certain features of trichospores are best observed with phase contrast microscopy.

III. Procedures:

• A.Culturing
  1. Make up 5-10 petri dishes containing 1/10th strength brain heart infusion agar (1/10 BHI) (3.7 g brain heart infusion; 20 g agar; 1 L distilled water). After medium has solidified, aseptically pour a thin overlayer of sterile distilled water.
  2. Petri dishes are inoculated with S. culisetae by transferring a loopful of mycelium to the water overlayer and vigorously breaking up the mycelium with the transfer loop.
  3. Incubate cultures at 25^oC for 7 d. S. culisetae is best stored at 5^oC on slants of 1/10 BHI containing approximately 1 ml of sterile water as an overlayer.
• B.Solutions
  1. Although other buffers at appropriate pH levels will stimulate sporangiospore extrusion from trichospores, ethylenediamine/citric acid buffer is used in this experiment because it covers a pH range of 7-10 and does not contain any monovalent cations. Make up stock solutions of 1 M ethylenediamine (9 ml ethylenediamine + 124 ml distilled water) and 1 M citric acid.
  2. Add 2 ml of 1 M ethylenediamine to 100 ml of distilled water or 0.05 M KCl for the following treatments:

     Treatment	Solution	pH
     A	0.05 M KCl	10
     B	0.05 M KCl	7
     C	Distilled water	10

     Adjust the pH with 1 M citric acid to pH values given above.
• C.Trichospore treatments
  1. Before starting the experiment, examine the trichospores under the microscope by mounting a small piece of mycelium from a culture of S. culisetae.
  2. Next agitate the water overlayers of the cultures with a transfer loop, then remove the slurry of mycelium/trichospores with a pipet and transfer to a test tube.
  3. Further dislodge the trichospores from the mycelium by vortexing or shaking the test tube.
  4. Add 2 ml of the slurry to each of three small beakers containing 20 ml of pH-adjusted solutions for treatments A, B, and C (midgut conditions). Initially stir the solutions for several seconds, then incubate 10 min. at room temperature.
  5. Just before the 10 min. incubation period is over, remove a drop of the solution from Treatment A and examine the trichospores under the microscope.
  6. Following incubation, quickly drop the pH from 10 to 7 for solutions in Treatments A and C using 1 M citric acid (hindgut conditions).
  7. Immediately examine trichospores from all treatments. In Treatment A, observe the sporangial case (empty sporangium) and the extruded sporangiospore (Fig. 1b) which often has a holdfast at its distal end (Horn 1989b). The membrane-like "sleeve" that extends from the empty sporangial case at the exit point of the sporangiospore is derived from a wall layer of the trichospore. Note that the sporangiospore is much larger than the original sporangium, indicating that pressure within the sporangiospore is involved in its extrusion. The lack of sporangiospore extrusion from trichospores in treatments B and C emphasizes the importance of potassium and a pH shift.

IV. References

Horn, B.W., 1989a. Requirement for potassium and pH shift in host-mediated sporangiospore extrusion from trichospores of Smittium culisetae and other Smittium species. Mycol. Res. 93:303-313.

Horn, B.W., 1989b. Ultrastructural changes in trichospores of Smittium culisetae and S. culicis during in vitro sporangiospore extrusion and holdfast formation. Mycologia 81:742-753.

Horn, B.W., 1990. Physiological changes associated with sporangiospore extrusion from trichospores of Smittium culisetae. Exp. Mycol. 14:113-123.

Lichtwardt, R.W., 1986. The Trichomycetes, Fungal Associates of Arthropods. Springer-Verlag, New York.

Fig. 1 Sporangiospore extrusion from trichospores of S. culisetae.

• A. Trichospores before treatment. Note the filamentous appendage at the base of the trichospores.
• B. Extruded sporangiospores from trichospores treated with 50 mM KCl at pH 10 (10 min) followed by a drop in the pH to 7. Note the empty sporangia and the membrane-like sleeve extending from the exit point of the sporangiospore; terminal holdfasts are present on many of the sporangiospores. Phase contrast microscopy; bar = 20 µm; s, sporangium; ss, sporangiospore. Reprinted from Horn (1989a).

Fig. 2 Digestive tract of a mosquito larva. The head is not shown; abdominal segments are designated 1-8. The midgut extends from the caeca to the pyloric chamber and the hindgut consists of the intestine and rectum. C=midgut caecum; Int=intestine; Mg=midgut; Mt=Malpighian tubule; Pc= pyloric chamber; R=rectum. Reprinted from Horn (1989a).