Towards modernizing the taxonomy of Mediterranean Culicoides using classical morphology, mtDNA barcoding, and MALDI-TOF MS protein profiling.

Culicoides biting midges (Diptera: Ceratopogonidae) are a highly successful group of small (1-3 mm) hematophagous flies, infamous for the role they play as biological vectors for numerous pathogens of veterinary significance. The principal aim of the national animal disease surveillance program of Israel is to be able to rapidly sort and identify live field-captured insects including Culicoides for arbovirus screening. In this exploratory study, three identification methods-classical morphology, DNA barcoding, and MALDI-TOF MS-were applied simultaneously to individuals of 10 Culicoides species that commonly attack livestock in Israel. The strengths and limitations of the three methods are compared and evaluated. In essence, the CO1 barcoding and MALDI-TOF MS results closely matched those of classical morphology. Furthermore, at a higher level and in strong accordance with recognized subgenera, the 10 species, in the reconstructed phylogenies, coalesced into multiple deeper-branched monophyletic clades. However, some discrepancies between the molecular and protein profiling results did occur and proved difficult to assess in terms of taxonomic significance. This difficulty underscores how tricky it is to establish clear species limits when methods involving borderline cutoff values and similarity indices are used as a taxonomic aid. An added shortcoming of the pluralistic triple-method approach is that a significant percentage of the species-level depositions in the GenBank and BOLD databases are misidentified, hindering structured comparison and interpretation of the morphological and molecular results obtained. Aspects of the unresolved taxonomy of various biting midge assemblages within the Mediterranean basin, including minor changes to the Israeli Culicoides checklist, are discussed in light of the methods applied. It is observed that the direct access that classical morphology provides to the external environment (or species niche) is indispensable to the full and correct interpretation (and application) of concomitant molecular and protein profiling results. The Culicoides taxonomy of the future ought to be fully integrative, during which the assimilation of modern methodological advances should strengthen-rather than undermine-the morphological foundations laid down during the 260-year Linnaean epoch.

The 'Culicoides obsoletus group' in Italy: relative abundance, geographic range, and role as vector for Bluetongue virus.

As Bluetongue virus (BTV) spread in Italy following its first incursion in 2000, it soon became apparent that, besides Culicoides imicola, additional species of the subgenus Avaritia were involved as vectors, namely one or more of the species that belong to the so-called 'Culicoides obsoletus group', which comprises C. dewulfi, C. chiopterus, C. obsoletus sensu stricto, C. scoticus and C. montanus; the three last named species are considered generally as forming the Obsoletus complex. This study presents the findings made over the last decade and more, within the Italian entomological surveillance program for Bluetongue. It describes the integrated morphological and molecular approach used to identify the species of the 'C. obsoletus group', maps in detail their relative abundances and geographic ranges in Italy, clarifies the hitherto unknown comparative seasonal abundances of C. obsoletus s.s. and C. scoticus in a site in Central Italy, and provides further details on the potential vector status of five species of the 'C. obsoletus group', with emphasis on C. obsoletus s.s., C. scoticus and C. montanus. Unlike the situation in Northern Europe, Culicoides dewulfi and C. chiopterus are uncommon to rare in Italy. In contrast, the Obsoletus complex occurs abundantly throughout Italy, with C. obsoletus s.s. being the most prevalent and ecologically adaptive of the three species making up the complex. A longitudinal study conducted at a site in Central Italy revealed that: (i) species of the Obsoletus complex prefer horses to sheep; (ii) their parity rates range from 10% (March) to 56% (November); (iii) throughout the year C. scoticus is consistently more abundant than C. obsoletus s.s.; (iv) abundances in both, C. obsoletus s.s. and C. scoticus, peak in May-June, with the peak of the latter species being more evident. Bluetongue virus was first isolated from wild caught midges of the Obsoletus complex in 2002. Thereafter, pools of selected parous midges collected across Italy, and during multiple outbreaks of BT, have been found consistently PCR-positive for the virus. More recently, viral RNA has been detected in field specimens of C. dewulfi, C. obsoletus s.s., C. scoticus and C. montanus.

Towards the PCR-based identification of Palaearctic Culicoides biting midges (Diptera: Ceratopogonidae): results from an international ring trial targeting four species of the subgenus Avaritia.

BACKGROUND: Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are biological vectors of internationally important arboviruses. To understand the role of Culicoides in the transmission of these viruses, it is essential to correctly identify the species involved. Within the western Palaearctic region, the main suspected vector species, C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus, have similar wing patterns, which makes it difficult to separate and identify them correctly. METHODS: In this study, designed as an inter-laboratory ring trial with twelve partners from Europe and North Africa, we assess four PCR-based assays which are used routinely to differentiate the four species of Culicoides listed above. The assays based on mitochondrial or ribosomal DNA or microarray hybridisation were tested using aliquots of Culicoides DNA (extracted using commercial kits), crude lysates of ground specimens and whole Culicoides (265 individuals), and non-Culicoides Ceratopogonidae (13 individuals) collected from across Europe. RESULTS: A total of 800 molecular assays were implemented. The in-house assays functioned effectively, although specificity and sensitivity varied according to the molecular marker and DNA extraction method used. The Obsoletus group specificity was overall high (95-99%) while the sensitivity varied greatly (59.6-100%). DNA extraction methods impacted the sensitivity of the assays as well as the type of sample used as template for the DNA extraction. CONCLUSIONS: The results are discussed in terms of current use of species diagnostic assays and the future development of molecular tools for the rapid differentiation of cryptic Culicoides species.

Schmallenberg virus in Culicoides spp. biting midges, the Netherlands, 2011.

To determine which species of Culicoides biting midges carry Schmallenberg virus (SBV), we assayed midges collected in the Netherlands during autumn 2011. SBV RNA was found in C. scoticus, C. obsoletus sensu stricto, and C. chiopterus. The high proportion of infected midges might explain the rapid spread of SBV throughout Europe.

Novel environmental factors influencing the distribution and abundance of Culicoides imicola and the Obsoletus Complex in Italy.

Bluetongue (BT) is an orbiviral disease of ruminant livestock. In Europe, the insect vectors implicated in BT virus transmission are Culicoides imicola, C. obsoletus, C. scoticus, C. pulicaris and C. dewulfi. C. imicola - an Afro-Asiatic species - is believed to account for approximately 90% of BT virus transmission; the latter four species are endemic to the Palaearctic region and are becoming increasingly important as the zone of BT virus infection in Europe moves northwards. As Culicoides distribution and ecology is critical in the spread of BT, it is essential to map their geographic ranges and to determine the ecological and climatic factors influencing their occurrence. Since 2000, more than 50 000 light trap collections have been made across Italy and the presence/absence and abundance of C. imicola ascertained; 3000 of these collections were also screened for two species (C. obsoletus, C. scoticus) of the Obsoletus Complex. To gain further insights into vector breeding habitats, spatial analysis techniques and statistical methodologies were used to investigate the association between the trap collection (municipality [polygons] and geographic point level) data and environmental and climatic variables. For the first dataset, 100 municipalities with the largest collections of C. imicola and 100 of the Obsoletus Complex, were selected and the average values of six independent climatic/environmental variables were calculated for each polygon. Factor analysis identified two principal factors explaining 84% of the total variability in climatic and environmental variables; 87.5% of trap collections were correctly classified by a discriminant analysis model. C. imicola was found to predominate in more sparsely vegetated habitats. Obsoletus Complex species were associated with a more densely vegetated habitat. For the second dataset, the presence/ absence of C. imicola at 172 sites in the region of Calabria (southern Italy) was investigated using 13 climatic, environmental and pedological variables. Factor analysis identified four factors; 89.5% of trap collections were correctly classified by a discriminant analysis model. Proximity to water-holding, clay soils was the most important factor explaining the markedly disjoint distribution of C. imicola in the 'toe' of Italy. Soil type may explain the patchy distribution of C. imicola elsewhere in Mediterranean Europe.