Experiments using blood-feeding insects reared under identical laboratory conditions can control for factors such as physiological status, which might influence host-seeking behaviour . However, for N. neivai, as for many other species of sandfly, laboratory rearing is difficult to maintain over more than a few generations , with the small number of insects produced being a limiting factor in conducting behavioural studies.
The results of this study demonstrate that, even though sandflies were taken from the field, and were of unknown age and nutritional status, female N. neivai showed consistently higher activation and attraction responses to 1-octen-3-ol in laboratory bioassays than an air control. In nature, octenol is released from a diverse range of sources, including fungi  and vertebrate breath [29, 30]. This semiochemical, previously identified as attractive to N. neivai in field studies , also proved to be an excellent positive control for wind tunnel experiments which seek to assess responses to other potential sandfly attractants.
Often, the number of sandflies captured from the field is not sufficient to conduct all the necessary experiments in planned behavioural studies. The results of the current work showed that groups of N. neivai used to assess baseline responses to air flow in the wind tunnel can be reused without influencing the number of flies responding to octenol, as compared to a separate group of flies with no test experience. More studies are required to determine whether the same might be generally applicable following exposure to other semiochemicals, or if any form of learning or habituation occurs in N. neivai, as reported for mosquitoes  and other species of sandfly [32, 33].
The time at which experiments are conducted is another potential source of variation in sandfly responses in laboratory bioassays: most insects follow daily cycles in activity, in part governed by endogenous circadian rhythms . In nature, N. neivai is typically most active during nocturnal hours , but no previous study has examined how time of day might affect N. neivai responses to kairomones under laboratory conditions. Bioassays with L. longipalpis have been successfully performed at different times of the day (e.g. the start of the scotophase  and 0900–1300 ). The results here showed no difference in N. neivai responses to octenol during different parts of the day, suggesting that laboratory experiments with field collected insects need not be restricted to night and evening. However, similar experiments with other compounds should be performed to determine the general applicability of this observation. We should also make clear that all of the bioassays conducted for experiment 3 were carried out during the same 24 h period, with the lights on even during the night session (19:00–22:00), such that the insects had no exposure to a scotophase before the bioassay. It seems therefore that under the described conditions there was no effect of any underlying circadian rhythm on N. neivai host odour-seeking behaviour: previous work in L. longipalpis suggests that such rhythms in locomotory behaviour do occur in sandflies, and may be modulated by blood intake .
Lactic acid elicited activation responses in the wind tunnel, but was only weakly attractive: octenol both activated a greater number of N. neivai, and was significantly more attractive. A component of human breath , attraction to lactic acid has been well studied in mosquitoes, and in general is more attractive when presented synergistically in combination with other host odour components, such as CO2, carboxylic acid, ammonia or acetone [38–42]. In sandflies, attraction to lactic acid has only previously been assessed in the field, and was not found to be attractive to L. longipalpis or N. intermedia. More studies are needed to assess sandfly attraction to lactic acid when presented with other host odour components, including octenol.
The BG-Lure, which releases a mixture of lactic acid, ammonia and caproic acid, was not attractive to N. neivai. This lure was developed to mimic human odour and has been shown to attract A. aegypti. Interestingly, while lactic acid alone did attract a small number of sandflies, there was no response whatsoever to the BG-Lure. Potentially, this might be because either the amount of lactic acid released is too small to evoke even a minimal response, or that the other chemicals released by the lure have some masking or repellent effect.
This study made use of two different methods of releasing test chemicals within the wind tunnel. Experiments 1, 2 and 5 were performed using micro-reaction vials, following the same methodology that has been previously used in the field . While octenol released from these vials did activate and attract sandflies in the wind tunnel, measurements of the weight of these vials before and after experiments suggest considerable variation may exist in their individual release rates, even when two vials are prepared and set to release under identical conditions. One potential source of this variation could be the amount of string in contact with the octenol in each vial.
Experiments 3 and 4 (performed at a later date) made use of filter paper as a release substrate. The volume of octenol was not reapplied onto filter paper during the bioassays because the responses of the sandflies did not change along the test period. Results of post-hoc analysis comparing between experiments suggest that a greater proportion of N. neivai were both activated and attracted by octenol released from the filter paper than from the vials, perhaps indicating a faster release rate from filter paper, and hence a greater response. However, this hypothesis requires stricter testing in order to draw firm conclusions.