Helicosporidium Infection Experiments | 1 | 2 | 3

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Infection Experiments:
For oral treatments, third instars were single starved over night and thereafter presented 2-µl droplets of Nile Blue cyst suspension at a known concentration: 0 (for control), 1x10³, 1x10⁴, 5x10⁴, 1x10⁵, or 2x10⁵ cysts/larva. After 4-7 h, larvae that consumed the whole droplet were weighed and transferred to single 1-oz cups with artificial insect diet. Treated larvae were incubated at constant conditions (27 ± 1 °C, 70% RH, 12-h photoperiod) and checked for mortality every other day. To determine infection rates, control and test larvae were bled by needle puncture within 3-8 d after oral treatment (larval weight ≥ 100 mg) and the presence of Helicosporidium in the hemolymph of test larvae was recorded using light microscopic optics with differential interference contrast (Leica DM R, Leica Microsystems Wetzlar GmbH, Wetzlar, Germany). Insects diagnosed as not infected were checked again 1-3 d after pupation.

For injection treatments, 5 µl of aqueous cyst suspension were injected into the hemocoels of late fifth (S. exigua, T. ni) or sixth instars (H. zea) at a concentration of 2x10⁵ cysts/larva. Control insects were injected with sterile water. Larvae were weighed, transferred to single 1-oz cups containing artificial diet, and incubated at constant conditions (27 ± 1 °C, 70 % RH, 12-h photoperiod). Five to eight days after injection, the presence of cysts in the hemolymph was recorded as described for the oral treatments. By this time all larvae had pupated.

In all experiments, pupae were weighed and sexed, and insect development and mortality were followed throughout adult emergence. Three to five replicates with differing numbers of larvae (n = 7-28) were conducted for each control and treatment.

Helicosporidium Infection Experiments | 1 | 2 | 3