Insects and plasma collection

B. mori and M. sexta larvae were reared on artificial diet at 27°C and a 16 h light: 8 h dark photoperiod as previously described [10, 100]. Hemolymph was collected by narcotizing 4^th or 5^th instars with CO₂ and then bleeding each larva from a cut proleg into a 1.5 mL Eppendorf tube on ice [4]. Glutathione (GSH, 5 mM final concentration) and phenylthiourea (PTU, 0.1 mM final concentration) were added immediately after hemolymph collection to prevent melanization [101]. However, they do not block formation of the IC, which requires additional treatment with the inhibitor Egf1.0 (30 μL of a 0.1 mg/mL Egf1.0 solution to 100 μL of hemolymph) [6]. Treated hemolymph samples were immediately centrifuged at 16,000 x g for 30 sec to pellet hemocytes and the resulting plasma supernatant was transferred to a new centrifuge tube for experimental use. The UGAL strain of A. aegypti was reared in an insectary at 27°C and 60% relative humidity using a 16 h light: 8 h dark photoperiod. Larvae were fed a standard diet consisting of finely ground rat chow (Purina): lactalbumin: brewers yeast (1:1:1) in open aluminum rearing pans containing distilled water. Pupae were transferred from larval rearing pans to plastic cages for emergence, which produced large cohorts of similar sized adults that were fed water supplemented with 5% sucrose (wt/vol) on day 2. To obtain hemolymph, pupae and non-blood-fed adults were cold anesthetized and then perfused with 2.5 μl of TRIS-buffered saline (TBS) containing 100 μM PTU by injection into the thorax using a pulled capillary pipet and collection through a hole in the last abdominal segment. Pooled samples were centrifuged at 9k rpm for 10 min at 4°C to remove hemocytes and other tissues.