Infectivity of Plasmodium parasites to Aedes aegypti and Anopheles stephensi mosquitoes maintained on blood-free meals of SkitoSnack – Parasites & Vectors

Source mosquitoes and initial production of colony replicates

Source populations of 500–600 mosquitoes were obtained from Ae. aegypti Liverpool [4, 39, 40] and An. stephensi Nijmegen (sda500) [41, 42] stocks maintained in the Laboratory of Malaria and Vector Research (LMVR) Insectary at the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Rockville, MD, USA. Mosquitoes of all life stages were housed inside a walk-in insect environmental chamber (Conviron Controlled Environments Limited, Winnipeg, Manitoba, Canada) at 27 °C, 75% humidity, and a 12 h light/dark cycle. To start each of the individual Ae. aegypti replicates, approximately 200 dried eggs from the source population were placed into a 8 oz paper cup containing 100 ml distilled water and vacuumed hatched for 30 min. The hatched larvae were transferred to a shallow plastic pan (Cambro 12CW148 Camwear 2.5″ deep polycarbonate food pan, Webstaurant Store, Cumberland, MD) with 1 l of 25 °C distilled water. To start each of the individual An. stephensi replicates, approximately 200 freshly laid eggs were rinsed and put back into the shallow plastic pan containing 1 l of fresh 25 °C distilled water for hatching. The larvae of both species were maintained with powdered or pelleted Tetramin^® Tropical Flakes fish food (Spectrum Brands Pet, LLC, Blacksburg, VA, USA) ad libitum until they reached pupal stage. Pupae were transferred into a small paper cup filled with approximately 200 ml of distilled water and placed inside a 1-gallon mosquito container, which was then closed by a screen at the top. Adults from the pupae were fed through the screen by a cotton wool ball soaked with 10% Karo^® dark corn syrup (ACH Food Companies, Inc., Chicago, IL, USA) until preparation for BB or SS feeding (described below).

SkitoSnack protocol

The powdered ingredients for SS were combined, mixed thoroughly, and stored in a capped plastic Erlenmeyer flask at room temperature (25 °C) as previously described [37] (Additional File 1: Table S1). To prepare the meal, deionized water was added to 0.7 g of powder to the final volume of 3 ml and vortexed for 3–5 min until fully dissolved. The brown-colored meal was pipetted into a glass feeder at 37 °C and offered, within 3 h of the meal preparation, to adult mosquitoes that had been starved for 16 h by replacing the 10% Karo^® soaked cotton wool balls with water-soaked ones.

Bovine blood- and SkitoSnack-maintained mosquito colonies

Five-to-six-day-old mosquitoes, which had been allowed to mate in the cages, were starved at least 16 h as described above and then fed a meal of BB containing citrate dextrose solution (15:85 ratio to blood) as an anticoagulant (Lampire Biological Laboratories, Inc., Pipersville, PA) or a meal of SS. The mosquitoes were offered the meal for 1 h through a water-jacketed artificial membrane feeding system [43, 44] fitted with Parafilm M (Bernis Co. Inc. Nennah, WI) on 40-mm-diameter glass feeders (#1588–40, NDS Technologies, Vineland, NJ) and connected to a 37 °C SAHARA S7-heated bath circulator (ThermoFisher Scientific, Waltham, MA). Aedes aegypti females lay their eggs on a damp substrate; therefore, to collect their eggs, a paper cup filled with approximately 200 ml of distilled water and lined with filter paper was placed in each 1-gallon mosquito container immediately after feeding. On the 5th day after feeding, the filter paper was removed from the cup and dried for 24 h inside the environmental chamber. On the next day, the dried Ae. aegypti eggs were vacuum-hatched for 30 min.

Anopheles stephensi females deposit their eggs directly into the water; therefore, to collect their eggs a paper cup filled with 200 ml of distilled water was placed in each cage immediately after feeding. Anopheles stephensi larvae were present inside the cup on the 6th day after feeding.

Larvae of both species were transferred to shallow plastic pans and maintained as described above. After the adults from the initial 200 eggs were used to establish each replicate, the population of subsequent generations was maintained at approximately 400 mosquitoes with a balanced proportion of females and males.

Collection of Ae. aegypti and An. stephensi engorgement and hatch rates data

Groups of approximately 20–40 females were selectively aspirated after attraction to the side of each gallon container by the warmth of a hand on the outside surface, transferred to plastic pint containers, and offered a BB or SS meal via an artificial membrane feeding system for 30 min. After feeding, the mosquitoes were anesthetized by placing the container inside a −20 °C freezer for 1.5 min. The mosquitoes were placed in a petri dish on ice. Engorged (fed) females were counted to determine engorgement rate (calculated by dividing the number of engorged females by the total number of females offered the meal) and separated to assess the number of eggs to be laid by each individual fed female.

After counting, the engorged females were individually placed into 50-ml conical centrifuge tubes containing a filter paper placed over a water-soaked cotton wool ball in the bottom. Each tube was capped with containment netting, and a second cotton wool ball wetted with 10% Karo^® syrup was placed on top (changed daily). The individual Ae. aegypti females were kept in their separate egg collection tubes for 4 days. On day 5, the females and water-soaked cotton wool balls from the bottom were discarded, and each clutch of eggs was collected on a filter paper. The eggs of each clutch were counted, and the individual papers were returned to their respective tubes to air dry overnight. On day 6, each dried egg paper was placed at the bottom of its respective tube, and 25–30 ml of distilled water was added to ensure that the paper was submerged. The tubes were vacuum hatched for 30 min and observed for larvae development.

Anopheles stephensi mosquitoes lay their eggs in water; therefore, the engorged counted females were placed individually into a disposable paper pint container with an egg collection cup (filter paper funnel placed inside a 30-ml plastic medicine cup filled with 15 ml distilled water). Each pint container was closed with a mesh top and sealed with a cardboard ring. The individual An. stephensi females were kept in their separate containers for 3 days for egg laying. On day 4, the females were removed, and the eggs of each clutch were counted and rinsed into a paper cup filled with 100 ml distilled water, where they were allowed to hatch.

For both mosquito species, on the day of hatch, and every other day afterward, a sprinkle of finely powdered Tetramin^® Tropical Flakes fish food was added to each tube or cup to ensure the growth of larvae. The larvae of the two species were counted 5–6 days post hatch. Average number of larvae per hatched egg was calculated by dividing the number of larvae 5–6 days after hatch by the total number of counted eggs.

Plasmodium gallinaceum infections of Ae. aegypti mosquitoes

All animal experimental procedures were performed under protocols approved by the National Institute of Allergy and Infectious Diseases (NIAID) Animal Care and Use Committee. Animals were purchased from NIH-approved sources and transported and housed according to Guide for the Care and Use of Laboratory Animals [45].

Plasmodium gallinaceum strain 8A [46, 47] was maintained by continuous passage in 4- to 5-week-old white leghorn chickens (Gallus gallus). Approximately 60–100 Ae. aegypti mosquitoes 3–8 days old were transferred from respective replicate colonies into a new 1-gallon mosquito container and starved for at least 1 h prior. The mosquitoes were then allowed to feed directly on a ketamine/aceproprazine-sedated P. gallinaceum-infected chicken (10–20% parasitemia) through a mesh screen for 20 min. Immediately after feeding, approximately 30 fully engorged females were transferred into a new 1-gallon mosquito container and provided a cotton wool ball soaked with 10% Karo^® dark corn syrup daily.

Quantification of P. gallinaceum oocysts and sporozoites in Ae. aegypti mosquitoes

Seven to 8 days after infection, the female mosquitoes were transferred by a battery-powered aspirator (Clarke no. 13500) to a half-gallon mosquito container and placed inside a −20 °C freezer for 1.5 min. The cold-anesthetized females were drowned in 70% ethanol for 2 min and then washed with 1 × phosphate-buffered saline (1 × PBS; 10 mM PO₄³⁻, 137 mM NaCl, 2.7 mM KCl, pH 7.4). With the aid of a stereomicroscope (Olympus 5Z61, Olympus America Inc., Center Valley, PA), the female midguts were dissected with tweezers into 1 × PBS and stained for 30 min with a solution of 0.1% mercurochrome in distilled water, and oocysts were counted at 200 × magnification (20 × objective, 10 × oculars).

Fourteen to 15 days after infection, the remaining Ae. aegypti females were −20 °C anesthesized and drowned in 70% ethanol for 2 min, and the salivary glands were removed and collected in 50 µl of 1 × PBS. The number of lobes collected per female was recorded and pooled for each replicate. The lobes were milled for 1 min with a plastic disposable pestle, and 10 µl of each sample was pipetted and counted using a disposable hemocytometer as recommended by the manufacturer (Incyto C-Chip hemocytometers, SKC, Inc., Covington, GA).

Plasmodium falciparum cultures

Human O + erythrocytes depleted of white blood cells were obtained weekly from Grifols Bio Supplies Inc. (Memphis, TN). The erythrocytes were washed upon arrival with 0.2-µM filtered RPMI 1640 medium (containing 25 mM HEPES and 50 µg/ml hypoxanthine; KD Medical, Columbia, MD) and stored at 50% hematocrit in a 4 °C refrigerator for use within a week from processing. Asynchronous cultures of the P. falciparum NF54 line [48] were maintained at 10-ml volumes in T25 vented flasks (Corning Inc. Life Sciences, Oneonta, NY) at 5% hematocrit with complete RPMI medium [RPMI 1640 medium supplemented with 10 mg/l gentamicin, 0.23% sodium bicarbonate, and 10% O + pooled human serum from 20 donors (Grifols Bio Supplies Inc.)]. Cultures were incubated at 37 °C under a 90% N₂, 5% O₂, and 5% CO₂ gas mixture. Medium was changed daily. Parasitemias were monitored by methanol-fixed thin blood films stained for 15 min with 20% Giemsa solution (Sigma-Aldrich, St. Louis, MO) and maintained between 0.5 and 9% parasitemia.

Induction of P. falciparum gametocytes

Plasmodium falciparum gametocytes were generated by “crash” induction in vitro [49, 50]. For this purpose, cultures of P. falciparum NF54 parasites were initiated as mixed stages in T75 flasks at 0.5% parasitemia and 5% hematocrit in the complete RPMI medium described above. Cultures were maintained with daily medium changes at 37 °C under a 90% N₂, 5% O₂, 5% CO₂ gas mixture and monitored by methanol-fixed, Giemsa-stained thin blood films. Media changes were done on top of a slide warmer unit at 37 °C. When the stage V gametocytemia was prevalent at > 0.5% (days 14–16), the culture was collected for mosquito feeding.

Infection of An. stephensi mosquitoes by P. falciparum gametocytes

All operations with infected live An. stephensi were performed inside a secure, triple-screened insectary. Approximately 20–40 uninfected An. stephensi females were transferred from each replicate colony maintained on BB or SS to a secure mosquito pint container (using a double mesh top secured with a metal ring) and starved for 16 h as described above. In some instances, when sufficient numbers of gametocytes and mosquitoes were available, additional pints of the same generation were prepared to increase the numbers of infected mosquitoes for evaluation (e.g. three pints each of BB- and SS-maintained An. stephensi at generation F8). The P. falciparum-infected blood meal was prepared as a 500-µl mixture containing one part heat-inactivated (56 °C × 30 min) O⁺ pooled human serum at 37 °C and one part of NF54 gametocytes in culture at 37 °C so that a final stage V gametocytemia of 0.1–0.3% was achieved. From this mixture, 250 µl was pipetted into a parafilm-sealed glass feeder at 37 °C as described above and offered to the 16 h-starved An. stephensi for 30 min. Counts of the engorged females were visually estimated, but, for safety, the pint containers were not opened to sort the engorged from non-engorged mosquitoes. After feeding, the pint containers of mosquitoes were placed inside a clear plastic bin (secondary containment) and stored inside the secure insectary. The mosquitoes were provided a 10% Karo^™ dark corn syrup-soaked cotton wool ball daily.

Quantification of P. falciparum oocysts and sporozoites in An. stephensi mosquitoes

Midgut oocysts of the infected An. stephensi were counted 6–8 days post infection, and salivary gland sporozoite assessments were performed 15–22 days post infection. For midgut dissections, the female mosquitoes were transferred via the battery-powered aspirator from each secure pint to a separate container and exposed to chloroform vapor for 1 min in a fume hood. The chloroform-anesthetized females were drowned in 70% ethanol for 1 min and then washed with 1 × PBS. Oocysts were stained and counted as described above. For sporozoite counts, salivary glands were extracted, and the number of lobes collected per female was recorded and pooled for each replicate. The lobes were milled for 1 min with a plastic disposable pestle, and the sporozoites were counted as described above.

Statistical analysis

Data from colony replicates were recorded along with calculated averages or geometric means in Microsoft Excel workbook spreadsheets (Microsoft 365 online version 2208). Statistical modeling analyses were performed in R (version 4.3.0) [51]. Engorgement rates and parasite infectivity by oocysts were evaluated using generalized linear mixed models with binomial and negative binomial families, respectively, using package lme4 [52]. Egg hatch rates were evaluated using a generalized linear model with a quasibinomial link. We tested for potential interactions between meal and generation number in every model, and if an interaction was not found to be statistically significant, it was deleted from the model. In all mixed models, we considered the experimental variations of a given mosquito colony replicate and each individual mosquito to be a random effect and generation and meal to be fixed effects. To test whether an effect of the BB or SS meal significantly varied across generations, a likelihood ratio test (LRT) was performed in linear mixed models, and a deviance test was performed in the quasibinomial model. Sporozoites per mosquito were calculated separately for each colony, and t-test (Ae. aegypti) or weighted linear regression (An. stephensi) on the log-transformed colony rates was used to calculate geometric mean ratios (SS over BB) and confidence intervals. Further details are provided in Additional File 2: Statistical Appendix.