Computational protocol: Blood serum and BSA, but neither red blood cells nor hemoglobin can support vitellogenesis and egg production in the dengue vector Aedes aegypti

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[…] Insect culture. Ae. aegypti (Rockefeller strain) eggs were submerged in tap water and connected to a vacuum pump for 20 min to deoxygenate the water and induce hatching. Larvae were raised on cat food (Special Kitty; Walmart, Bentonville, Arkansas, USA) and water was changed as needed. Pupae were separated by hand using a handheld screen and placed in a separate container with clean water. Pupae containers were placed inside 30 × 30 × 30 cm cube-shaped mosquito cages for emergence with 20% sucrose solution as carbohydrate source. During the rearing process mosquitoes were held in an insect environmental chamber at 26.5 °C, 70% relative humidity (RH) and 16:8 h light/dark cycle. Adults were at least 4 days old before given a meal.Meal treatments. Prior to feeding experiments, sucrose solution was withheld from mosquitoes for at least 16 h. Starved females were separated from males and equally distributed into smaller 15 × 15 × 15 cm cube-shaped mosquito cages and fed using an artificial lab-made feeding system. RBCs or blood serum was fractionated from whole defibrinated bovine blood (Hemostat Labs, Dixon, California, USA). Whole blood and serum was given alone and RBCs were washed with phosphate buffered saline (PBS). The whole blood was centrifuged and the serum supernatant was pipetted off without disturbing packed RBCs. Serum was collected and stored at −20 °C. RBCs were washed 3 times by resuspending in one volume of PBS, centrifuging and pipetting off supernatant. Washed RBCs were stored at 4 °C. All centrifugations were done at ∼1,275 × g for 20 min at 4 °C. Females were each fed a meal consisting of different protein components. BSA (Research Products International, Mt. Prospect, Illinois, USA) or Hb (Sigma-Aldrich, St. Louis, Missouri, USA) (200 mg/mL) meal was dissolved in either PBS, Aedes physiological saline (APS), sodium PBS (NaPBS) or potassium PBS (KPBS). Chemical concentrations and components are listed in . A 50 mM ATP (Sigma-Aldrich, Seelze, Germany) solution was made and immediately stored at −20 °C. ATP solution was thawed right before feeding experiment and kept on ice during preparation.Membrane feeding system. A membrane feeding system was constructed from available laboratory materials (). A cylindrical feeding receptacle was made from a 50 mL centrifuge tube that was cut at the 45 mL marking. One open end of the receptacle was sealed with parafilm stretched to near breaking point to serve as a membrane for mosquito piercing mouthparts. The parafilm membrane was rubbed on sweaty human skin to stimulate host-seeking behavior. 1 mL of each prepared meal solution was heated in a 37 °C water bath for 15 min, then pipetted into the feeding receptacle and placed, parafilm side down, on top of each 15 × 15 × 15 cm cage. ATP was added to a final concentration of 1 mM as a phagostimulant in all treatment meals. Re-usable heat packs were microwaved for 2 min and placed on top of each feeding receptacle to keep solution warm and to aid in attracting mosquitoes. A microcentrifuge tube rack was placed on each side of the feeding receptacle to provide support for the packs. Females were provided with a meal for 1 h then immediately collected with a battery operated aspirator and anesthetized on ice. Fully engorged females were counted and weighed together on a balance. The females were then relocated to a different 15 cm × 15 cm × 15 cm cube-shaped mosquito cage, provided with a water-soaked cotton ball and a 20% sucrose-soaked cotton ball on top, and kept at 26.5 °C, 69% RH for 48 h. Each female was then individually placed into a 50 mL centrifuge tube containing a water-soaked cotton ball and filter paper substrate for egg deposition for additional 24 h. Seventy-two hour post-fed dissections were carried out and the number of fully-developed retained oocytes and the number of deposited eggs were determined.Egg viability test. To determine egg viability of the deposited eggs, an en masse viability test was performed. Groups of 20 individual females were starved for at least 16 h and fed whole blood, serum, 200 mg/ml BSA in APS, and an iron-enriched BSA in APS solution (200 mg/ml BSA in APS + 0.5 mg/mL Iron (III) chloride). The iron concentration was selected because it reflects the average iron content present in whole blood of normal individuals (). The females were allowed to lay eggs at 72 h post blood meal for 24 h. Ninety-six hours post blood meal, the eggs of each group were removed from the cage and kept on a moist filter paper for the next 48 h to allow them to mature. After 48 h the eggs were counted and stored in paper envelopes for another 24 h in an insect environmental chamber. Three days after deposition, the eggs were placed in 100 mL of deionized water and connected to a vacuum pump for 30 min to induce hatching. Vacuumed eggs were transferred to shallow plastic trays and provided with Special Kitty food pellets and placed inside an insect environment chamber. Egg viability was determined by counting the number of larvae present 5 days after the eggs being placed in water.Data analysis. A Kruskal–Wallis analysis of variance was conducted between feeding trials of the same meal to detect any significant variation. If no significant variation was discovered, then the trials were grouped and compared to other protein meals. The proportion of females that fed was calculated by dividing the number of fully engorged females by the total number of females given the meal. The Kruskal–Wallis test was performed to determine significant differences among multiple groups, and the Mann–Whitney U test was used to compare pairwise treatment meals. In the analysis of egg deposition numbers of all treatment meals, the high percentage of zero values for the RBC and Hb treatments, and consequent high number of ties, precluded the use of the U test for pairwise comparisons. Therefore, data for whole blood control and all other treatments were recoded as “yes” for individual females that laid eggs and “no” for individual females that did not. A contingency table analysis, Fisher’s exact test, was used to determine significant differences between each meal. For egg viability, the proportion of viable eggs was calculated by dividing the number of larvae present after 5 days (indicates the hatch rate) by the total number of eggs deposited for each treatment meal. A total of four replicates was conducted. Each decimal fraction was transformed into arc sine data (arc sine of the square root of each decimal fraction) and analyzed by a one-way analysis of variance (ANOVA) test to detect statistical significant differences between treatment meals with the Holm–Sidak posttest for pairwise comparisons versus the whole blood control. Some statistical tests were performed on JMP 12.0 (SAS Institute, Inc., Cary, North Carolina, USA) software and others on SigmaPlot 12.0 (Systat Software, Inc., San Jose, California, USA). Graphs were made using SigmaPlot 12.0 and Adobe Illustrator Creative Cloud (Adobe Systems, Inc., San Jose, California, USA). […]

Pipeline specifications

Software tools SigmaPlot, Adobe Illustrator
Applications Miscellaneous, CAGE analysis
Organisms Aedes aegypti, Homo sapiens
Diseases Chikungunya Fever
Chemicals Iron