Culex pipiens, Linnaeus, 1758

Ferguson, Laura V., Hillier, N. Kirk & Smith, Todd G., 2013, Influence of Hepatozoon parasites on host-seeking and host-choice behaviour of the mosquitoes Culex territans and Culex pipiens, International Journal for Parasitology: Parasites and Wildlife 2 (1), pp. 69-76 : 71

2.5. Host-seeking behaviour of infected and uninfected C. pipiens

Trials with C. pipiens were conducted in the same manner as described for C. territans with the following modifications. C. pipiens were permitted to feed on infected or uninfected garter snakes in separate cages at the beginning of the trial. Mosquitoes were then exposed to an uninfected green frog or an uninfected garter snake at 15 days PF and 30 days PF. Mosquitoes were exposed to frogs for 2 h, the maximum time permitted as per animal care protocols, and to snakes for up to 12 h. A total of 10 mosquitoes were initially placed in a feeding cage during trials in which mosquitoes were exposed to green frogs, and a total of 30– 40 female mosquitoes was initially placed in a feeding cage during trials in which mosquitoes were exposed to garter snakes. Mosquitoes were dissected at the end of experiments to count mature oocysts of H.sipedon in the haemocoel.

Behaviour of C. pipiens was not observed during exposure to snakes. Instead, responsiveness was measured as engorgement success, indicated by mosquitoes that had fed by the end of the trial.

2.6. Preference of mosquitoes for infected and uninfected green frogs: interrupted feeding, paired-choice trials

A total of 30 female C. territans , starved of sugar for 12 h, were placed in a feeding cage in a room with natural light entering through windows. The room was maintained at 24 ° C and a relative humidity of 50%. Two wild-caught adult green frogs were placed in the cage on either side of a mesh net that extended half-way up the height of the cage. This arrangement allowed mosquitoes to fly over each side of the cage but prevented contact between frogs. When a mosquito landed on a frog and began to probe, it was considered to have made a choice, and the mosquito was aspirated from the cage and placed in a separate holding container. Two frogs of each of the following parasitaemias were used in these trials: uninfected (0%), low (0.1–1%), moderate (1–2%) and high (2– 3%). Each trial was repeated three times for each of the following six pairs of frogs: high parasitaemia versus uninfected, moderate parasitaemia versus uninfected, low parasitaemia versus uninfected, high parasitaemia versus moderate parasitaemia, high parasitaemia versus low parasitaemia, and moderate parasitaemia versus low parasitaemia.

Paired- choice trials were repeated using experimentally infected green frogs described previously. Two infected frogs, including one female with 10% parasitaemia, and one male with 2% parasitaemia, were paired separately with two uninfected frogs, including one of each sex, and trials were repeated four times. All possible pairs of infected versus uninfected frogs were tested. The number of mosquitoes to land on each frog and the time for each mosquito to land was recorded.

2.7. Preference of mosquitoes for infected and uninfected green frogs: uninterrupted feeding, single-choice trials

A total of 15 female C. territan s, starved of sugar for 12 h, were introduced into each of two feeding cages, one containing a frog infected with H. clamatae , and one containing an uninfected frog, during daylight hours, and cages were placed in natural light. Three infected frogs, two male (parasitaemia of 2% and 0.8%) and one female (parasitaemia of 10%), and three uninfected frogs, two female and one male, were used in these trials. A total of six replicates were completed for these trials.

Mosquitoes were permitted to feed to repletion on frogs, and the total number of mosquitoes that fed fully was recorded. The time for each mosquito to land, the time between bites of different mosquitoes, the time to appearance of blood in the abdomen, and the time to appearance of pre-diuresis was recorded. However, data concerning pre-diuresis could not be reliably collected and was not used in analysis. Trials lasted one hour or until the last mosquito had finished a blood meal, with a maximum of 90 min.