Mosquitoes rearing
Rearing and maintenance of the mosquito colony was performed in a secure insectary maintained at 26 °C and a relative humidity of 80 %, with alternating 12 h cycles of light and dark. The secure insectary consists of four rooms. Infected mosquitoes are kept in locked incubators, which are separated from the outside by four doors. Access to the secure insectary is strictly restricted to authorized and trained personnel. The facility is fully sealed. The SMRU laboratories, including the insectary are part of the Mahidol University, Faculty of Tropical Medicine. Every 3 days mosquitoes were allowed to lay eggs on a Whatman filter paper wrapped in a conical shape and placed in a plastic bowl filled with 60 mL of distilled water.
The eggs were collected and rinsed with 1 % NaOCl, and then with distilled water on a new filter paper, using a modified Nalgene 500 mL disposable filter attached to a vacuum so as to minimize the contamination by microsporidia [
14]. The eggs were then placed under a blue light bulb until hatching, which occurred within 2 days. One day later a sprinkle of fine TetraBits Complete
® food was added to the bowl. First instar larvae were transferred to plastic trays (25 cm × 36 cm × 6 cm) filled with 1.3 L of drinking water. The optimal number was around 60–70 larvae per tray. The water was changed every 2 days in order to minimize the risk of fungal or bacterial contamination. Trays with many dead larvae were discarded in order to select healthy mosquitoes. The larvae were fed twice daily with ground TetraBits Complete
®, which had been cooked in an oven overnight at 121 °C and thereafter kept sealed in plastic bags in a refrigerator at 4 °C. On development, pupae were collected from the trays with a sterile syringe and transferred to small bowls filled with distilled water that were then placed inside cages until the emergence of adult mosquitoes. These were fed with cotton pads soaked with 5 % vitamins (MULTILIM syrup, Atlantic Laboratories Corp. Ltd, Bangkok, Thailand) and 10 % sugar solution. Every 4 days female mosquitoes were fed with human blood; either directly from an arm or by using a Hemotek
® membrane-feeding system using drawn heparinized blood. The number of adults that were maintained per 30 cm × 30 cm × 30 cm cage did not exceed 500. In order to maintain optimal humidity, wet clean towels were placed on the top of the cages that were then covered with a black plastic sheet.
Mosquito colony
The A. cracens colony was considered stable 2 years after it was first initiated in the SMRU insectary. On average, 1,500–2,500 pupae were collected weekly and 6,000–10,000 adult mosquitoes were produced each month. Two cages of uninfected females were always kept to maintain the colony, and were fed every 3 days with uninfected blood. For each feeding experiment, it was estimated that only one-third of the mosquitoes actually ingested P. vivax-infected blood, those that did not feed were principally males. Mosquitoes were not recycled in the colony.
Blood samples
Non-pregnant adult patients presenting with clinical symptoms (i.e., fever, chills, headache) and with a blood smear positive for
P. vivax gametocytes were considered eligible for recruitment. After obtaining written consent, and prior to receiving anti-malarial drug treatment, 5–10 mL of heparinised venous blood were obtained and placed immediately in a water bath at 37–38 °C in order to avoid microgametocyte exflagellation and the emergence of macrogametes. Within 1 h post-collection, the blood samples were transported in thermos flasks filled with water at 37–38 °C from the clinics to the SMRU insectary. On reception, the samples were centrifuged at 1,800
g for 5 min at 37 °C. Plasma was replaced with warmed AB
+ serum, and the mixture transferred within 10 min to the insectary. The gametocytaemia was determined by counting the number of gametocytes per 500 white blood cells (WBC) by microscopic examination of Giemsa-stained thick blood smears, assuming an average of 8,000 WBC per µl as per WHO guideline [
15].
Sporozoites count
Between days 15 and 21 post-infection all the remaining fed mosquitoes were collected in small cups, anesthetized by placing at 4 °C and then killed in 70 % ethanol. After rinsing twice with sterile medium (Leibovitz or RPMI), the salivary glands were dissected under a stereomicroscope, and then pooled together in a 1.5-mL Eppendorf tube with medium, in which they were crushed with a sterile pestle. After centrifugation, 10 µL of a sporozoites suspension was placed into a KOVA® Glassic ® Slide 10 with grids or into a Neubauer chamber haemocytometer. A phase-contrast microscope set to 40× magnification was used to enumerate the sporozoites. The average number of sporozoites counted per grid was multiplied per the dilution factor and multiplication factor of the chamber in order to calculate the sporozoites per µL.
Data analysis
All analyses were performed using Stata 12.0 (StataCorp, College Station, TX, USA). Gametocytaemia values were log-transformed to obtain a normal distribution and Student’s t test was used to compare mean log-gametocytaemia between infectious and non-infectious gametocytaemic blood. Results are presented as geometric means of original values for each group. Sporozoite production was evaluated by the arithmetic mean number of sporozoites obtained per mosquito dissected from a given batch. Spearman’s correlation coefficient was used to assess the relationships between gametocytaemia and sporozoite production, and between gametocytaemia and mosquito mortality in infected mosquito batches; Mann–Whitney test was used to analyse if transmitters and non-transmitters differed in their age distribution.
Establishment of the Anopheles cracens colony
The initial attempts to establish a colony at the SMRU were made at the beginning of 2012 from egg batches sent from the insectary of the Department of Parasitology, Faculty of Medicine, University of Chiang Mai (Thailand), to the SMRU on wet filter papers in sealed Petri dishes. A number of factors (type and temperature of the water, number of larvae per tray) needed to be optimized in order to achieve optimal adult production.
As it was not possible to use the same source of water that was used in Chiang Mai, different types of water were tested at the SMRU: natural fresh spring water collected directly from the source, filtered water left for a few days in a tank to let the chlorine evaporate, de-ionized water mixed with spring water, or mineral drinking water. Satisfactory full larval development was obtained only when mineral drinking water was used. The larvae were maintained in tray with 1.3 L of water, and optimal development and minimal mortality were obtained at densities not exceeding 80 larvae per tray. The larvae were fed with fine powdered fish food, with a small amount (0.01 g) sprinkled twice per day. In order to minimize the risk of contamination by fungi and bacteria, aquatic macrophytes were not used, and the larvae were moved to new trays filled with clean water every 2–3days as surface scum became obvious. The optimal water temperature was found to be 25 ± 1 °C, and small lamps with 40-W lights were shone on the trays to maintain this temperature. Under these conditions the cycle of development to pupae lasted about 15 days.
Anopheles cracens have been shown to preferentially feed on human blood. Attempts to feed adults on the blood of white rats, mice, guinea pigs, gerbils or rabbits proved unsatisfactory. Indeed the original colony had been maintained on human blood from volunteers for many decades in the laboratory and it was considered safe to feed the mosquitoes directly on human arms during the initial phases of colony establishment at the SMRU. As the colony grew, direct feeding was replaced with artificial membrane feeding using human heparinized blood. This human blood was kept in the refrigerator, and renewed every week.
The main danger that faced the colony, at SMRU and at Chiang Mai, was microsporidial infection. This was detected by observing spores in the midgut of dissected mosquitoes under a light microscope at 100×. The infection led to high pupal mortality, reduction in egg production and a consequent dramatic decrease in colony size. More importantly, afflicted mosquitoes lost their susceptibility to infection by
Plasmodium and those that fed on malaria-infected blood showed increased mortality, in a similar manner to that reported in the literature [
16,
17]. Thus, strict measures were necessary to control and eliminate any microsporidial contamination. The whole insectary was cleaned with soap and water, and glass shelving was used, as it was easier to clean. The cotton pads soaked with sugar and vitamins solution, and the water to prepare it were autoclaved before use. Aliquots of TetraBits
® fish food were cooked overnight at 121 °C in order to kill the spores, then sealed in clean plastic bags, and kept at 4 °C for not more than 3 days. The food stock was kept at −20 °C until use. Gloves were worn to handle larval and adult food. Given that microsporidial spores can spread from generation to generation through horizontal and vertical transmission (depending on the species) or through cannibalism of infected larvae [
18], the trays were monitored for dead larvae. Dead larvae were removed, dissected and examined for microsporidial spores, and the trays where any were found removed and sterilized.
Mosquito eggs were bleached with 1 % NaOCl for 1 min and then rinsed with sterile water, the larval trays were rinsed with boiled water and 70 % ethanol, and the cages’ netting material was replaced. The pupae were rinsed twice in deionized water before the emergence of adults and the towels covering the mosquito cages were autoclaved and subsequently rinsed daily in boiled water. Full recovery of a colony after microsporidial infection took 12 months to achieve.