II. Studies on In Vivo
Development of Helicosporidium
Recently, Helicosporidium has been isolated from
larvae of the black fly Simulium jonesi
Stone & Snoddy (Diptera, Simuliidae) collected in 1998 from Hatchet Creek,
Alachua Co, Fl. and from the weevil Cyrtobagous
salviniae Calder & Sands (Coleoptera: Curculionidae) also found in
Gainesville, Florida. This insect is an introduced biological control agent for
the aquatic weed Salvinia molesta (Salviniaceae). Phase contrast microscopy of tissue smears
revealed discoid cysts that measured 6.5 ± 0.2 x 5.9 ± 0.3 µm. Light and
electron microscope studies demonstrated that the cysts contained a core of
three ovoid cells and a single filamentous cell. 
The filamentous cell
makes 3-4 coils, forming a helix around the ovoid cells within the mature
cysts. The outer wall or pellicle is a multilaminate structure, enclosing the
peripheral filamentous cell within its innermost wall layer. Within intact
cysts, three centrally located ovoid cells are compressed in an accordion-like
fashion. Each of these cells possesses a peripheral nucleus that encloses a
cytoplasmic region that contain a variety of vacuoles and granules.
When stimulated by
pressure, the outer pellicle layer of the cyst splits open or dehisces releasing
the filamentous cell-ovoid cell complex from the pellicle. 
Release
from the cyst stage produces an expanded ovoid cell aggregate and results in
the uncoiling of filamentous cells. The filamentous cells, measuring 37 ± 4.3 mm in
length by 0.9 ±
0.13 mm
in diameter, are coated with short projections (340 ± 60 nm) orientated in the
same direction providing polarity to the filamentous cells. 
Cyst
dehiscence was triggered readily by the application of gentle pressure to the
coverslip covering a cyst suspension. Alternatively, the incubation of purified
cysts in midgut fluid extracted from Heliocoverpa
zea larvae stimulated the release of the filamentous cells from both cyst
suspensions. A twenty minute exposure to midgut fluids resulted in more than
50% of the cysts releasing their filamentous cells. Upon activation these cysts
increased in volume resulting in pellicle rupture and release of the
filamentous cell. Incubated in the midgut luminal fluid the ovoid cells lysed,
whereas the released filamentous cell became uncoiled and remained intact.
Presently, the component(s) in the midgut fluid that signals dehiscence is not
known. Uncoiled filamentous cells of the blackfly isolate readily clustered
with other filamentous cells producing rosettes. Whether this clumping was due
to a specific surface adhesion or was simply a result of entanglement of the
surface projections is unknown. The filamentous cells of the weevil isolate did
not aggregate when released from the cysts.
The two Helicosporidia isolates are capable of infecting and
replicating in a variety of dipteran and lepidopteran species. Oral challenge
of H. zea and the tobacco hornworm Manduca sexta larvae with cyst
preparations was lethal to tested insects. Examination of dissected alimentary
tracts revealed that ingested cysts bound initially to the peritrophic matrix
in challenged larvae. Within 2 h after ingestion, cysts dehisced releasing
filamentous cells from the ovoid cell-pellicle complex. SEM of the midguts
dissected from M. sexta larvae at 4 h
post-ingestion revealed that the released filamentous cells penetrated the
peritrophic matrix and attached to the midgut columnar epithelium 
. Filamentous cells penetrated the midgut with the projections oriented away from
the penetration point, suggesting that these spines may play a role in
anchoring the filamentous cell to the gut epithelium. In the case of M. sexta, vegetative cells were observed
in the hemolymph within 2 days after ingestion. Vegetative cells, containing
variable numbers of cells within the pellicles, were observed to be both
associated with circulating hemocytes and present as freely circulating cells. 
By 6 days post ingestion plasmodial-like hemocytes displayed marked
cytopathic effects (CPE). It is unclear whether the Helicosporidium sp. induced a haemocyte fusion, blocked
cytokinesis, or stimulated hemocyte nuclear division. Within 10-14 days,
treated larvae contained massive numbers of mature cysts in the cream-colored
hemolymph. 
At this point large numbers of cysts could be extracted
easily using several cycles of centrifugation followed by high-speed
centrifugation through a Ludox gradient.