Experimental Design of Helicosporidium Infection Experiments

 

Helicosporidium: Cysts were amplified in vivo in orally infected H. zea larvae and purified from homogenates of late instars and pupae by high-speed centrifugation on a linear gradient of Ludox HS40 (DuPond Chemical, Boston, MA). The band containing the cysts was collected, diluted in sterile water, and subjected to several cycles of low-speed centrifugation to remove residual gradient material. After re-suspension in sterile water or in 10% DMSO plus 10% glycerol for cryo-protection, cysts were counted using a hemacytometer and aliquots with a concentration of 1x10⁸ cysts/ml were stored at -70 °C until used. Prior to each experiment, cysts were thawed quickly, washed twice in sterile water, and re-suspended in 0.1 % aqueous Nile Blue A or in sterile water.

 

Insects: Larvae of H. zea and eggs of S. exugia and T. ni were obtained from established colonies at the Center for Medical, Agricultural and Veterinary Entomology, USDA, ARS, Gainesville, Florida. Hatching neonates and larvae were provided with artificial insect diet and maintained at constant conditions (27 ± 1 °C, 70% RH, 12-h photoperiod). Third instars were used for oral treatments; injection treatments were conducted using fifth instars.

 

Infection Experiments: For oral treatments, third instars were single starved over night and thereafter presented 2-ml 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 ³ 100 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 ml 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.

 

Mating Experiments: To examine adult fecundity and vertical transmission of the pathogen, mating experiments were conducted with infected and control adults obtained from injection treatments with S. exigua. One female and one male were transferred into a 16-oz-paper cup. A piece of tissue towel along the edge served as egg laying substrate and a Kimwipe^® soaked in a 10-% sucrose solution in a small Petri dish (60 mm diameter) served as nutrient source. Each cup was held in a separate Perspex box to prevent pheromone interference between different mating trials. Insects were incubated at constant conditions (27±1 °C, 70 % RH, 12-h photoperiod) and the numbers of deposited eggs were recorded daily. When eggs were laid, pieces of artificial diet were added to the cup and humidity was increased by covering the bottom of the Perspex box with wet paper towels. Dead adults were removed from the cup and their infection status was confirmed by examination of tissue smears for the presence of Helicosporidia using light microscopic optics with differential interference contrast (Leica DM R, Leica Microsystems Wetzlar GmbH, Wetzlar, Germany). Two to three days after egg hatch, 50 second instars were transferred to a clean paper cup with artificial diet and allowed to undergo metamorphosis. Upon pupation, the infection status of 25 pupae was determined as described for the infection bioassays. During the course of the experiment, smears of 10 second instars and hemolymph samples of 20 fourth instars, taken from the original mating arena, were also examined for the presence of Helicosporidia.