Leong 1997
Leong 1997
Leong 1997
79 0022-2011/97 $25.00
Copyright r 1997 by Academic Press
All rights of reproduction in any form reserved.
80 LEONG ET AL.
infection by this neogregarine. We report herein the (1) milkweed leaves daily within these containers until the
effect of spore concentration fed to larvae on the fifth instar, at which time they were transferred one per
neogregarine parasite load in the resulting adult butter- cup to inverted 355-ml clear plastic tumblers with
flies, (2) viability of spores after passage through the plastic lids. Five holes were made in the bottom of the
larval intestine, (3) infectivity of 1-year-old spores, and plastic cups for aeration. The larvae were fed daily
(4) susceptibility of first, third, and fifth larval instars within these clear plastic cups until they pupated.
to the protozoan parasite. Upon adult eclosion, the butterflies were harvested and
their abdomens examined for spores, using the wash
MATERIALS AND METHODS
method described above.
Effect of larval spore concentration on adult parasite
The butterflies used for the study were laboratory load. In the first experiment, first instar larvae were
reared (19.4°C 6 0.4 SE and 44.9 R.H. 6 1.5 SE) and exposed to milkweek bouquet leaves sprayed with one
free of the neogregarine parasite for two generations. of five spore suspensions: 0, 50, 500, 5000, and 50,000
The larvae were fed leaves of the blood flower milk-
spores/ml. Starting at the lowest concentration, each
weed, Asclepias curassavica, or the crown flower milk-
treatment, replicated three times, was applied to the
weed, Calotropis gigantea. The milkweeds were grown
leaves using a hand atomizer (DeVilbiss atomizer 15,
in the greenhouse to provide year-round production of
The DeVilbiss Co., Somerset, PA) until runoff and
food and to eliminate possible contamination by dis-
eased wild butterflies. allowed to air dry before the larvae were introduced. A
total of 15 larvae (5 larvae/replication) were used for
Spore inocula. Unless otherwise indicated, all spore each of the four spore suspension levels and control.
inocula used for the study were harvested from the The larvae were allowed to feed for 48 hr before being
abdomens of infected butterflies that were stored indi- transferred to clean milkweed bouquets and reared
vidually in envelopes at 5°C for less than a month. The using the protocol described earlier.
spores were recovered from infected butterflies using a In the second experiment, third instar larvae were
modified methodology described by Leong et al. (1992). given five levels of spore inocula. The spore level was
The abdomen was placed in a 15-ml centrifuge tube based on exposing each larva to 14.5 spores/mg of body
containing 2 ml of 0.05% (v/v) wetting agent (Tween 20; weight. Because the mean weight of the third instar
J. T. Baker Chemical, Phillipsburg, NJ) in deionized was 45.4 mg 6 0.35 SE, the maximum inoculum was
water and washed for spores by agitating with a vortex 658 spores/larva. Succeeding spore levels were halved
mixer for 1 min, letting stand for 5 min, and reagitating so that each larva received 329, 165, or 82 spores, with
for another minute. The abdomen was removed and the the control larva receiving 0 spores. Nine larvae were
wash suspension was centrifuged for 10 min at 3000 used for each of the five spore levels. In addition,
rpm with a bench-top centrifuge (Beckman GS-6 Centri-
1-year-old spores stored at 5°C were tested to deter-
fuge; Beckman, Palo Alto, CA). The supernatant was
mine their viability. Nine larvae were each given 658 of
discarded, 0.2 ml of the 0.05% wetting solution was
these spores.
added, and the suspension was agitated for 2 sec on a
Each inoculum was applied in 100 µl to a half leaf of
vortex mixer to resuspend the pellet. The number of
A. curassavica. The suspension was air dried, after
neogregarine spores/ml recovered was determined with
a hemocytometer. In all cases, control treatments con- which each larva, confined in a 50-ml plastic vial, was
tained 0.05% wetting agent. given a half leaf with the appropriate spore inoculum.
The cap of the vial was perforated with holes for
Larval rearing. The first to third instar larvae were aeration. Upon consumption of the leaf, the larvae were
maintained on milkweed bouquets. A bouquet consisted transferred to clean milkweed bouquets (3 larvae/
of one or two blood flower milkweed shoots (15 to 20 cm) bouquet, 9 larvae total per inoculum level) and reared
inserted through the middle of a plastic lid for a 473-ml using the same protocol as that described earlier.
drinking cup and secured to the lid with cotton. The
cutting was inserted into an opening of a parafilm- Viability of spores after passage through the larvae.
covered 150-ml beaker filled with water, and the shoot Infectivity of spores after passage through the larval
was adjusted to an upright position. The 473-ml cup, intestine was determined by collecting the feces from
modified by replacing the bottom with a Kimwipe larvae in each of the five inoculum levels in the second
tissue (secured with tape), was then inverted over the experiment described above. The feces from each inocu-
milkweed bouquet and snapped onto the lid. The larvae lum level were pooled and each pooled sample was
were transferred to fresh bouquets every 2 days. placed into a separate 15-ml centrifuge tube. Three
When larvae reached the fourth instar, the lid and milliliters of 0.05% Tween 20 was added and mixed
parafilm-covered beaker were replaced with a new until a suspension was achieved. An aliquot from each
plastic lid cover. The larvae were fed the crown flower inoculum level was examined at low power (103 objec-
MONARCH BUTTERFLY SUSCEPTIBILITY TO A NEOGREGARINE 81