The spatio-temporal occurrence of Culicoides biting midges was determined in Switzerland over 3 years at 12 locations with one trap per site, at altitudes from 270 to 2,130 m. The six trapping locations (up to 670 m; Table1) were part of a national monitoring system that included an additional 12 trapping sites in Switzerland and one in Liechtenstein up to a maximal altitude of 870 m. These 13 traps were only run from October until May to determine the vector-free period (i.e. less than five parous midges per trap night) which is of high relevance when restriction measures for the movement of livestock are applied during an outbreak. Thus, for these two countries, the vector-free period lasts from week 47–49 to week 12–14, depending on weather conditions (for vector-free periods in other European countries see). In order to also identify putatively 0vector-free areas, which are conceivable as retreat areas for animals during outbreaks of midge-borne diseases, six traps were placed at locations not covered by the national monitoring, particularly at higher altitudes. Monitoring activities in other European countries focussed on locations below 1,400 m (highest altitudes investigated: 1.400 m in Sicily; 1,190 m in Austria; 1,184 m in Central Italy). The highest record of Culicoides in Switzerland was 1,600 m (from a zoological study). We determined high abundances at the high altitude locations (1,800, 2,130 m; Table1). In accordance with earlier preliminary studies[37, 38], midges of the Pulicaris group consistently prevailed at high altitudes, as is the case at high latitudes in Scandinavia[5, 39]. Nevertheless, biting midges of the Pulicaris group occasionally (single farms, few catches) occurred in higher numbers than the largely dominating Obsoletus group species in Central Europe[40, 41]. However, in only one of our total 1,202 catches from the nine locations below 1,110 m altitude (Table1) were the Pulicaris group species the most abundant (not shown). Species identification of 100 randomly selected midges per locations (by MALDI-TOF MS, see below) revealed a vast predominance of C. obsoletus (total 67.6%) and C. scoticus (24.8%) at the nine low altitude locations and of the cryptic species C. grisescens II (67.3%) at the three sites above 1,500 m. Highly interestingly, single specimens of C. obsoletus and C. scoticus were identified in the Alpine region and, vice versa, of C. grisescens II in the lowlands (Table3), putatively due to the (albeit rare) long range dispersal of these tiny insects and references cited therein. Further, whereas C. obsoletus was dominating in eight of the nine lower altitude locations, C. scoticus accounted for 95 of the 100 midges at the ninth location (‘Dittingen’, Table3). As these analyses were done with insects from a single catch per location, obtained in summer, further analyses are required to understand the spatio-temporal population dynamics of the various species.
We observed a considerable range of the total number of collected biting midges between the locations. Such a variability between sites/farms is well known from many other studies e.g.[20, 38, 43–45]. In a previous study in Switzerland, for example, the total number of biting midges collected over one season at two farms only 4 km apart and located at the same altitude differed by the factor 24. Major factors influencing the abundance of midges are particularly topoclimate, land use and soil (as proxy for larval breeding sites)[35, 46–48]. However, when comparing the number of midges we collected at the locations over three consecutive summers (June – September), statistically significant differences were neither observed for the total number nor for the vast majority of analyses of the three Culicoides groups (Table2). Thus, collection of midges over the four summer months during one single year sufficed to obtain a representative picture of their abundances at a given location.
Our study was running over 3 years, adding up to a maximum total number of 156 weekly trappings per location. At six of the 12 locations, more than 145 catches were made (Table1), and the data from these locations were used to depict the seasonal dynamics (Figure1) and to analyse yearly differences in abundance (Table2). The lower number of trappings at the other locations was due to technical problems (unnoticed failure of light bulbs/ventilator); missing/incorrect labelling of catches; independent decisions taken by farmers to skip trapping during adverse (mainly cold) weather conditions, and by simply forgetting to operate the traps by the farmers. A somewhat surprising and unique picture was obtained from the highest altitude trapping location (2,130 m) where, throughout two winters low midge activity was observed (Figure1). We speculate that midges from the inside population escaped through shakes in the wooden wall of this barn. Such indoor populations of biting midges can reach considerable sizes[49–52] albeit these populations are also strongly reduced in winter and own unpublished results from another trapping location.
MALDI-TOF MS has come of age for high throughput, accurate and reproducible identification of medically relevant microorganisms (bacteria, yeasts, filamentous fungi) at low costs and minimal preparation time. Only recently, this proteomic approach has become available for Culicoides identification, relying on a validated reference database of biomarker mass sets from 15 Culicoides species, all but one (C. imicola) being indigenous to Switzerland. Analyses of 1,200 biting midges with MALDI-TOF MS confirmed the method’s reliability, as 98.9% of the specimens yielded high quality spectra, and 97.8% of the midges could be assigned to one of the species (Table3) included in the database. Obviously, the database covers the most abundant species of Central Europe. It is not clear how many indigenous Culicoides species exist in Switzerland. A compilation based on published data lists 35 established valid species. In comparison, 51 species have been listed for north-eastern France, a region which has thoroughly been studied (Delécolle, personal communication). However, several new species as well as specimens that could not unequivocally be identified by morphology have recently been reported from Switzerland[22, 23, 37, 45]. In addition, cryptic species have been reported[21, 22], and the genetic identification of two midges from the highest altitude trapping site as C. segnis-like (with 96% sequence identity to a C. segnis GenBank entry) indicates that the taxonomy of Culicoides midges remains an unfinished story.
No biomarker mass sets existed for 14 specimens, which therefore could not be assigned to a species. These 14 insects belonged to six species as identified by PCR/sequencing (Table3), including five C. fascipennis. As the biomarker mass set for a species in general is derived from the reference spectra of at least five genetically confirmed specimens, a C. fascipennis-specific biomarker mass set comprising 29 masses (not shown) could be derived and added to the database.
Poor quality mass spectra, yielding no information with regard to species or group affiliation, were obtained for 13 (1.1%) specimens. The main source for this failure is most probably insufficient homogenization, which was done by a hand-held homogenizer, and automated sample preparation is desirable. With the already very high rate of good quality spectra (98.9%) obtained using a ‘quick and dirty’ preparation, it seems doubtful whether the evaluation of laborious refinements of pre-analytical processes might be a worthwhile expedient approach to further increase the efficiency of MALDI-TOF MS analyses.
Thus, MALDI-TOF MS is a new tool available for high throughput Culicoides species identification. This method is particularly economic for approaches requiring detailed and quantitative information on the midge fauna (to gain a rapid overview on the species present; to follow their spatio-temporal occurrence; to identify morphologically similar or indistinguishable species, e.g. C. obsoletus and C. scoticus) whereas PCR performed on DNA from pools of midges remains the method of choice for tracking down one or a few species of interest.
The foundation for MALDI-TOF MS analyses is the availability of a database with reference biomarker masses from carefully confirmed reference specimens. As the creation and maintenance of such a database is a tedious task, a centralised structure seems to offer an efficient solution. The database we rely on was created in collaboration between our group and a private company (Mabritec SA, Riehen, Switzerland) and, as shown in this work, enlarges when being utilized. Thus, this database might be of value as the core of an eventual comprehensive Culicoides database.