Locomotion

Data previously gathered comparing with acceleration data for animals during activity on a treadmill at Buenos Aires Zoo [12] were reanalyzed to supplement the work on humans. Species used were; coypu (Myocastor coypus) (4 individuals), larger hairy armadillo (Chaetophractus villosus) (1 individual), Muscovy duck (Cairina moschata) (1 individual), greylag goose (Anser anser) (2 individuals), Magellanic penguin (Spheniscus magellanicus) (2 individuals) and rockhopper penguin (Eudyptes chrysocome) (1 individual). Briefly, animals were equipped with acceleration data loggers, attached variously, before being exposed to a treadmill with the tread moving at a range of speeds between 0 and 2.52 km/h, the upper limit dependent on their capacities. The animals were given rests between the higher speeds where the predominant behavior was locomotion however at the lower speeds the animal typically exhibited a range of behaviors including searching, scratching and lying. An open circuitry respirometry system was used to measure . Full details of the protocol are given in Halsey et al. [12] .

OFT was performed to evaluate the locomotor activity of the prenatally e-cig exposed or control mice both male and female at PD45 (adolescent) and PD90 (adult) following our previously published study [47 (link), 48 (link)]. Versamax software (Accuscan Instruments., Columbus, OH) was used to automatically calculate the total distance traveled by the mice. Briefly, mice were introduced to 16″ × 16″ unobstructed glass chamber and their activities were monitored and recorded for 1 h. The first 10 min of 1 h was excluded as the acclimatization period. All experiments were performed between 8 and 10 am. Fecal boli was counted for each mouse after completing of the OFT to measure stress/anxiety level following published literatures [49 (link), 50 (link)].

The A. aegypti isolate was obtained from the Department of Parasitology and Entomology at the Liverpool School of Tropical Medicine, UK, in 1972. TRS Labs, Incorporated obtained this isolate from the University of Georgia in 1980. During the years that the mosquitoes were maintained at University of Georgia and then at TRS Labs, Incorporated, both laboratory colonies were refreshed with eggs from the other colony.
Post-treatment mosquito infestations were performed on days 1, 7, 14, 21, 28 and 35. For each infestation, dogs were sedated with Dexdomitor at 0.04 mL/kg and Butorphanol at 0.02 mL/kg, or Dexdomitor at 0.04 mL/kg and Butorphanol at 0.02 mL/kg plus Antisedan at 0.15 mL/kg to prevent mosquito-bite hypersensitivity reactions, and placed into individual infestation chamber into which 50 ± 5 unfed female adult A. aegypti mosquitoes were released. After 60 ± 10 min of exposure, all live mosquitoes were removed from the infestation chamber and the dogs were then carefully taken out of the chamber to allow for removal of the dead mosquitoes. All dead mosquitoes collected from the infestation chamber were then counted. All fed live and moribund mosquitoes in the infestation chamber were aspirated into separate incubation cartons (one chamber per animal) using a vacuum pump, and were counted and evaluated for feeding status. Other mosquitoes (dead, and live unfed mosquitoes) were discarded. The live fed mosquitoes were kept in an incubation carton which had a nylon screen mesh top. On the tops (lids) of the incubation cartons, the mosquitoes had cubes of sugar and cotton soaked with sugar water at their disposal. Dead mosquitoes were counted at 12 ± 2 h, 24 ± 2 h and 48 ± 2 h after exposure to the animals (study 1) or at 24 ± 2 h, 48 ± 2 h, 72 ± 2 h, 96 ± 2 h, and 120 ± 2 h after exposure (study 2). Dead mosquitoes were counted after they had been removed from the incubation carton at each time point, while the live/moribund mosquitoes remained in the carton until after the last observation had been made.
During the counts, the mosquitoes were categorized as live, moribund, or dead and as fed or unfed. A mosquito was considered live when it exhibited normal behaviour, such as being capable of walking or flying. A mosquito was considered moribund if it was unable to perform normal locomotion and exhibited clear signs of neurological disruption, such as showing a lack of balance or being unable to fly in response to external stimuli. The feeding status of live or moribund mosquitoes was determined with the naked eye according to distension of the abdomen and the presence of blood in the abdomen. Dead mosquitoes were assessed for feeding status by placing each of them on tissue paper and squashing the abdomen with a spatula or other suitable object to assess if a blood meal had been taken.