Morphological and molecular insights into the diversity of Leptoconops biting midges from a heavily infested Mediterranean area

The genus Leptoconops Skuse (Diptera: Ceratopogonidae) are blood-sucking midges known to pester humans and domestic animals. In certain Mediterranean areas, midges occur in large numbers during summer and limit the use of recreational areas, also raising serious health and social concerns. Despite such impact, the diversity and distribution of Leptoconops in Maremma Regional Park (Tuscany Region, Italy), a heavily infested area, is not well known, and neither molecular nor detailed morphological studies exist. We sampled adult midge females in six areas and used high-resolution digital stereomicroscopy and scanning electron microscopy to identify species and investigate the morphology of structures involved in host searching/recognition (antennae and maxillary palps) and host attack (mouthparts). We also performed energy-dispersive X-ray spectroscopy to characterize the elemental composition of mouthparts. Finally, the cytochrome c oxidase subunit 1 (cox1) gene was amplified and sequenced, to confirm species identification of collected specimens. We identified two species: Leptoconops (L.) irritans Noé and Leptoconops (L.) noei Clastrier & Coluzzi, with the former being more frequently sampled than the latter and closer to sea coast and rivers. The antennal segments appeared slightly more globular in L. noei than in L. irritans. Five types of trichoid, basiconic and chaetic sensilla were found on the antennae, with some differences between the two species. Mouthparts had the labellum visibly larger in L. noei compared with L. irritans. The maxillary palps possessed a pit filled with bulb-shaped sensilla, which appeared denser in L. noei than in L. irritans. Mouthpart cuticle included Calcium (Ca) and Aluminum (Al) at small but significant concentrations (0.3-1.0%) in both species. Our results suggest that the limited but appreciable differences in sensory system between the studied species of Leptoconops and other Ceratopogonidae may reflect different host or habitat preferences, a scenario potentially suggested also by preliminarily data on their distribution in the studied area. The presence of Ca and Al in the cuticle of mouthparts may help host skin drilling during bite activity. Finally, the gene sequences obtained in this study provide a first reference for future investigations on the taxonomy and dispersal patterns of Leptoconops spp. in the Mediterranean area.