Hematin

Various parameters of reproductive fitness of females were assessed following an uninfected or infected bloodmeal, or an infected meal given to mosquitoes that had been additionally stressed. Stresses were designed to simulate conditions in the field by (1) maintenance at the lower temperature of 20°C for 12 h each night, (2) removing glucose for 12 h each night, and (3) subjecting the cage to vigorous cage movements to induce flight for 2h on days 1 and 2. Starvation and lowered temperature stresses were applied simultaneously every night and flight was induced during the daytime. Tests were carried out on the Keele strain prior to any selection and on the 12th or 13th selection generations of all lines. The number of larvae produced by each mosquito was also monitored at generation 9.
Mass sibling-mating took place on emergence, and insemination rates were checked in 30 females on days 4–6 by examining the spermathecae. Another 120 females were removed from the stock cages and fed on an uninfected or P. y. nigeriensis-infected mouse. S, R, and C lines were fed on the same mouse to minimize variations in gametocyte density or blood factors such as hematocrit. Approximately 100 fully engorged females from each group were kept individually until hematin had been excreted (usually day 2 after feeding), then those still alive were moved to oviposition pots. Eggs were counted within 24 h of oviposition. Egg hatching success was determined by assessing the proportion of larvae that hatched from each egg batch. On day 4 post-hatching, 40 larvae from each female were chosen randomly and reared to pupation in standardised conditions. Larval survival, first and last day of pupation, percentage pupation, and, in the initial experiments, imago wing lengths were noted and significant differences determined by a chi-squared test.
On day 6–7 after feeding, females were dissected and ovaries were examined for retained eggs. Midguts of infected females were examined for the presence and density of oocysts, and wing length was measured as an indicator of mosquito size (Ahmed et al. 1999 ). Bloodmeal size was expressed in terms of hematin excreted with reference to bovine hematin standards (Hogg and Hurd 1997 (link)).

Cyclooxygenase activity of ovine COX-1 or human COX-2 was assayed by a method that quantifies conversion of [1-¹⁴C]arachidonic acid to [1-¹⁴C]prostaglandin products. Reaction mixtures of 200 µL consisted of hematin-reconstituted protein in 100 mM Tris-HCl, pH 8.0, 500 µM phenol, and [1-¹⁴C]arachidonic acid (50 µM, ~55–57 mCi/mmol, Perkin Elmer). For the time-dependent inhibition assay, hematin-reconstituted COX-1 (44 nM) or COX-2 (66 nM) was preincubated at 25°C for 17 min and 37 °C for 3 min with varying inhibitor concentrations in DMSO followed by the addition of [1-¹⁴C]arachidonic acid (50 µM) for 30 s at 37 °C. Reactions were terminated by solvent extraction in Et₂O/CH₃OH/1 M citrate, pH 4.0 (30:4:1). The phases were separated by centrifugation at 2000g for 2 min and the organic phase was spotted on a TLC plate (EMD Kieselgel 60, VWR). The plate was developed in EtOAc/CH₂Cl₂/glacial AcOH (75:25:1) at 4 °C. Radiolabeled products were quantified with a radioactivity scanner (Bioscan, Inc., Washington, D.C.). The percentage of total products observed at different inhibitor concentrations was divided by the percentage of products observed for protein samples preincubated for the same time with DMSO.