Of1 mice

During the national surveillance plan implemented in 2016 to establish the geographical distribution of Ae. albopictus in Morocco, five ovitraps less than 500 m apart were placed on a street of the Agdal neighborhood in Rabat (33°59'20.9′′ N, 6°51′07.9′′W). Ovitraps were checked for eggs once a week from May to November 2016 and were brought back to the laboratory to be stored in humid chambers (relative humidity of 80%) before being sent to Institut Pasteur in Paris to perform the vector competence studies.
After hatching, larvae were split into pans of 200 individuals and supplied every 2 days with a yeast tablet dissolved in 1L of dechlorinated tap water. All immature stages were reared at 26±1°C. Emerging adults were maintained at 28±1°C with a 16L:8D cycle, 80% relative humidity and supplied with a 10% sucrose solution. Females were fed twice a week on anaesthetized mice (OF1 mice, Charles River laboratories, France). Resulting F2 adults were used for vector competence assays. It should be noted that variations of oral susceptibility to an arbovirus can be considered negligible in fewer than five laboratory generations [30 (link)].

Cryostabilates were thawed in a water bath at 37°C and diluted in PSG to 1 trypanosome per field (± 10^5.7 cells ml⁻¹). Two-hundred μl volumes were injected IP in two naïve 20–30 g female OF-1 mice (Charles River, Belgium). Starting from three days post infection (DPI), parasitaemia was monitored daily and harvested at first peak parasitemia, typically at day 4 to 5 post-infection, as described above. Volumes of 0.5 ml of the blood were run over a mini Anion Exchange Centrifugation Technique (mAECT) column to separate the trypanosomes from the blood [39 (link)]. The trypanosomes eluted from the column were washed twice with 5 ml ice-cold PSG by centrifugation at 1500 g for 15 min. After the last centrifugation, the supernatant PSG was discarded and the trypanosome sediment was re-suspended in 100 μl of PSG. Part of this suspension was used for in vitro culture adaptation. The remainder was centrifuged at 1500 g for 5 min and the sediment was frozen at -80°C until DNA extraction. The isolates used for in vivo isolation and expansion and the corresponding T. evansi type A and B specific PCR result on their corresponding buffy coat DNA are indicated in Table 1.

Three Aedes mosquito colonies were used: (i) A. aegypti Paea (Papeete, Tahiti), (ii) A. aegypti collected in Pazar in the northeast of Turkey in February 2016 by Vincent Robert (IRD, France), and (iii) A. albopictus collected in the city of Nice in the south of France in May 2011 by Pascal Delaunay (CHU de Nice). All colonies were derived from field-collected eggs. Eggs were immersed in dechlorinated tap water for hatching. After hatching, larvae were split into pans of 150 individuals and supplied every 2 days with a yeast tablet dissolved in 1 liter of dechlorinated tap water. All immature stages were reared at 26 ± 1°C. Emerging adults were placed in different cages and were maintained at 28 ± 1°C with a light/dark cycle of 16 h/8 h at 80% relative humidity and supplied with a 10% sucrose solution. To produce eggs, females were fed three times a week on anesthetized mice (OF1 mice; Charles River Laboratories, Inc., Saint-Germain-Nuelles, France). The F3 and F10 generations were used for the infectious blood meals for A. aegypti Pazar and A. albopictus Nice, respectively. A. aegypti Paea has been maintained in the laboratory since 1994 (16 (link)).

Three-month-old pregnant female OF-1 mice were obtained from Charles River Laboratories (Les Oncins, France). All animal studies were approved by the institutional guidelines and by the European Community for the Use of Experimental Animals. Gravid female OF-1 mice were obtained by in-house mating. The date of the presence of a vaginal plug was taken as day 0.5 post coitus (dpc). The gravid females were maintained in the animal facility. They were injected at day 6.5 or day 11.5 dpc of gestation, and after imaging they were sacrificed at 7.5 or 12.5 dpc via a lethal injection of Doletal (Figure 1). These gestational ages correspond to the peak of angiogenic processes in the placenta. At least three mice were used for each gestational age examined. This group of animals was only used for imaging. To assess the level of expression of the complex α_vβ₃ in the placenta, we used another set of gravid mice that were sacrificed at 10.5, 14.5, and 17.5 dpc. This group of animals was used as a control group and was not injected by Angiolone-Alexa-Fluor. The choice of the gestational dates to study the expression of α_vβ₃ in the placenta was based on the fact that 10.5 dpc corresponds to the placental angiogenic peak and represents the first trimester of pregnancy in women, 14.5 dpc corresponds to the second trimester, and 17.5 dpc will represent the third trimester.

OF1 mice were provided by Charles River Laboratories, Inc. (Lyon, France), kept under controlled temperature (22 ± 2 °C), humidity (40–60 %), and light (12-h cycles) and treated in accordance with the European Community Council Directive (86/609/EEC) on animal welfare. Under ketamine/xylazine anesthesia, stab wound lesions were made in the parietal cortex with a scalpel blade as described previously [19 (link)].

Eggs were collected from ovitraps placed in the city of Brazzaville (Congo) on the ORSTOM campus in august 2011 and sent to the Institut Pasteur in Paris where they were reared in standardized conditions. After morphological identification, Ae. aegypti and Ae. albopictus adults were placed in different cages and reared at 28 ± 1°C, at relative humidity of 80% and a light:dark cycle of 16 h:8 h. A constant supply of 10% sucrose was provided to adults. To produce eggs, females were fed three times a week on anesthetized mice (OF1 mice from Charles River laboratories, France). Oral infection experiments were performed using mosquitoes from the F2-F4 generations. The F12 generation of Ae. albopictus Providence (ALPROV) collected in 2007 on La Réunion Island was used for comparison.

Experiments were performed on OF1 mice (Charles River, France) in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals in an accredited establishment (Institut National de la Santé et de la Recherche Médicale, Unité 1256) according to the UE guidelines 2010-63-UE and to French governmental decree 2013-118. Mice were maintained under standard laboratory conditions on a 12 h light/dark cycle with access to food and water ad libitum, and environmental enrichment with wooden pieces. Since none of the animals used were kept alive after cell collection, no animal suffering was recorded and no analgesia was necessary.
For both adult (OP cultures) and newborn (primary cultures) mice, neuronal tissues were obtained after gas anesthesia using isoflurane diffusion. Then animals were killed by decapitation. A total of 10 adults and 20 newborns from three different litters were used.