Olfactory learning in blood-feeding insects, such as mosquitoes, could play an important role in host preference and disease transmission. However, standardised protocols allowing testing of their learning abilities are currently lacking, and how different olfactory stimuli are learned by these insects remains unknown. Using a Pavlovian conditioning paradigm, we trained individuals and groups of Aedes aegypti mosquitoes to associate an odorant conditioned stimulus (CS) with a blood-reinforced thermal stimulus (unconditioned stimulus; US). Results showed, first, that mosquitoes could learn the association between L-lactic acid and the US, and retained the association for at least 24 h. Second, the success of olfactory conditioning was dependent upon the CS – some odorants that elicited indifferent responses in naïve mosquitoes, such as L-lactic acid and 1-octen-3-ol, were readily learned, whereas others went from aversive to attractive after training (Z-3-hexen-1-ol) or were untrainable (β-myrcene and benzyl alcohol). Third, we examined whether mosquitoes9 ability to learn could interfere with the action of the insect repellent DEET. Results demonstrated that pre-exposure and the presence of DEET in the CS reduced the aversive effects of DEET. Last, the nature of the formed memories was explored. Experiments using cold-shock treatments within the first 6 h post-training (for testing anaesthesia-resistant memory) and a protein synthesis inhibitor (cycloheximide; to disrupt the formation of long-term memory) both affected mosquitoes9 performances. Together, these results show that learning is a crucial component in odour responses in A. aegypti, and provide the first evidence for the functional role of different memory traces in these responses.

SUMMARY: Vertebrate blood is essential for the growth and the reproduction of haematophagous insects. Provided that hosts play the double role of food sources and predators, feeding on their blood exposes these insects to a high predation risk. Therefore, it is expected that host seeking occurs only when insects need to feed. In the present study, we analyse how the feeding status affects the response to host-associated cues in the blood-sucking insect Rhodnius prolixus. We show that the responsiveness to host-associated cues, such as CO2 and heat, and that the motivation to feed depend on the time elapsed since a blood meal. Depending on the time elapsed after feeding, the same concentration of CO2 may attract or repel the insects. As far as we know, this is the first time that a host signal has been shown to be repellent for a haematophagous insect. The response to heat is also modulated but no repellence was observed. When blood was replaced by saline solution as food, a significant reduction of the response to both signals was evinced but repellence was not observed. The injection of haemolymph from fed insects into starved insects inhibited the response of the receiver insects to both signals but this was not observed after the injection of saline solution or of haemolymph from non-fed insects. This is the first time that the modulation of feeding behaviour by the feeding status has been analysed in a hemimetabolous blood-sucking insect, fully excluding any effect of other processes, such as reproduction.