The introduction and transmission of BTV and SBV in northern Europe by Culicoides species has highlighted the requirement for a clearer understanding of the relationship between vector species and their hosts. Host location is an essential part of Culicoides biology as females of the majority of species require a blood meal in order to mature egg batches. This interaction is crucial in driving the transmission of arboviruses between susceptible hosts. Previous investigations of Culicoides host location in northern Europe have primarily focused on the nuisance biting species C. impunctatus. With the implication of C. obsoletus and C. pulicaris group species in the transmission of BTV and SBV (Carpenter et al. 2006a, De Regge et al. 2012), it is clear that there is an urgent need to fill the gaps in our knowledge concerning the host location behaviour of these species. A clearer understanding of these behaviours would not only assist in understanding the transmission of these pathogens, but could also provide the opportunity for the development of novel tools for surveillance and control. In addition, novel, convenient surveillance methods based on LED-baited light-suction traps had not previously been tested for Culicoides in the Palaearctic region. These were seen as potentially providing an interim means of more accurately sampling the genus in northern Europe. The work presented in this thesis therefore provides a systematic investigation of responses of Culicoides species to host animals; response to specific host-derived olfactory stimuli; and differential response to visual cues.
The substantial data sets that were generated as part of this thesis were important in providing a more realistic representation of Culicoides activity on
194 livestock holdings than previous work. Samples collected were identified to species level through the use of multiplex PCR with 21,045 individuals subjected to molecular analysis and an overall amplification success rate of 93.6%. The importance of identifying to species level is shown by the significant differences observed at this level throughout the studies conducted. Detailed analyses were also carried out to include meteorological data collected throughout sampling periods as this is known to be a key factor in determining Culicoides flight activity (Sanders et al. 2012).
Investigation of differential responses of C. obsoletus group females to hosts was conducted through a series of studies involving the direct collection of Culicoides from hosts. Previous work has been carried out to investigate responses of Culicoides to hosts, but the aims of these studies were primarily to establish biting- rates on hosts rather than host preference (Carpenter et al. 2008c, Gerry et al. 2009, Viennet et al. 2011). One study has attempted to investigate host preferences by collections on a range of host species but the number of Culicoides collected was very low (Viennet et al. 2013). In the present study, three separate investigations were conducted to assess the differential responses of Culicoides to different breeds of sheep, sheared and unsheared sheep, and the effect of cattle on biting rates on sheep.
No previous investigation has examined differential responses of Culicoides to breeds of sheep despite BT being documented as affecting some breeds more severely than others. In the work carried out in this thesis 16,170 Culicoides were collected in this trial and it was demonstrated that C. dewulfi and C scoticus exhibit differential attraction to breeds, even when the breeds are closely related. For both of these species significantly fewer females were collected on the pure sheep breed
195 compared to the cross. Of the C. dewulfi collections numbers of un-pigmented and pigmented females were significantly lower on the pure breed while in the C. scoticus collections there was a significant reduction in the number of blood fed individuals on the pure breed. It is not clear what drives these differences, one explanation for the differences in attraction of the breeds could be due to different odour profiles of the breeds, differences were found in the concentrations of some chemicals released from the breeds (J. Cook, personal communication). In a separate study, however, using the odour of the sheep breeds in isolation from other host cues, no differences were observed in attraction and the numbers collected in that work were far lower than the collections made on the host. This difference could be due to the different physical attributes of the two breeds, although little is known regarding the response to visual cues in Culicoides. The phenomena of intra-breed variation in host preference is not unique to Culicoides and has been described in other vector groups (Birkett et al. 2004, Jensen et al. 2004). Following the discovery of breed preference a practical continuation of this work would be to investigate the differential attractiveness of other, more commercially important, sheep breeds.
The investigation into differential attraction and feeding on sheared and unsheared sheep collected significantly greater numbers of blood fed C. obsoletus on sheared sheep compared to unsheared with 4.7 times as many collected in sheared sheep. No significant differences were noted in any other species including C. scoticus which was the most abundant during this trial. In addition, no significant difference was observed for total numbers of Culicoides females of any species on the hosts which was surprising as it was hypothesised that unsheared sheep would be likely to have a different odour and thermal profile and increased respiration rate that would lead to increased attractiveness to host seeking individuals. This indicated that
196 shearing of sheep did not influence the number of Culicoides initially attracted to the host. The difference in feeding success of C. obsoletus is of significant interest due to the ubiquitous nature of this species on livestock holdings across northern Europe and its status as a putative vector of BTV and SBV (Carpenter et al. 2006a).
The reasons why not shearing failed to impact upon the other members of the C. obsoletus group species remains unknown and could be related to differences in feeding efficiency and feeding site selection. This area would be of significant interest to pursue in detail during future studies. The findings of the current study demonstrate that choosing not to shear could have a mitigating effect in the event of an arbovirus outbreak where there are large populations of C. obsoletus, however the likely trade-off with reducing the efficacy of insecticides on unsheared sheep (Venail et al. 2011) would need to be assessed as this could lead to an overall heightened risk of transmission. Another consideration for such action would be the fact that C. scoticus is also found in high abundance on farms and has been shown to replicate BTV to high levels in the laboratory (Carpenter et al. 2008a). While sheep may be protected to some degree from BTV transmission from C. obsoletus bites if left unsheared the risk remains for transmission from C. scoticus which demonstrated no significant reduction in blood feeding between sheared and unsheared sheep.
Grazing cattle in close proximity to sheep has been reported as a means of protecting sheep from Culicoides bites in South Africa (Du Toit 1962, Nevill 1978). No entomological investigation of the effect of this husbandry had been carried out prior to the current study. Surprisingly, considering the South Africa trial, the results convincingly demonstrate that in the case of Palaearctic Culicoides species, grazing cattle with sheep would provide no protection. The impact of cattle being held in close proximity lead to a doubling of Culicoides collections on sheep. One limitation
197 of the study design was that the cow corral was in very close proximity to the drop trap and in a natural grazing situation cattle and sheep would be likely to have more distance between them. Follow up work with the cow either in the same field but at a greater distance from the sheep or in a neighbouring field would provide useful information as to how this alters the responses of Culicoides. An interesting observation of this trial was the numbers of C. dewulfi, in the sheared and unsheared trial that was conducted immediately prior to this one where there was a low abundance of C. dewulfi but this changed completely once the cow trial started, emphasising the close association of this species with cattle.
Olfaction is known to be an important component of host location for haematophagous Diptera including Culicoides species (Gibson and Torr 1999). Three studies were conducted to investigate the olfactory response of Culicoides on farms. CO2 is known to be an attractant for many haematophagous species and
collections of the north American BTV vector, C sonorensis, are typically in suction traps supplemented with this kairomone (Mullens 1995). The dose response work presented here demonstrates that C. nubeculosus displays a significantly greater response to 1,500 ml/min CO2 than to 500 or 1,000 ml/min and that beyond 1,500
there is no significant increase in collections. The findings are in accordance with a previous study at the same site which reported no significant difference in response to 500 and 1,000 ml/min CO2 (Harrup et al. 2012). This species is not typically
collected in large numbers in light-suction trap surveillance and the current work indicates that this could be a result of low response to the standard surveillance tool rather than to a low abundance of the species. Similar behaviour is seen in C. sonorensis and both species are members of the Monoculicoides sub-genus and this response to olfactory cues over visual cues may be a common trait for this group.