Morphological and molecular identification of obligatory myiasis-causing species in wild cervids in Croatia

The infestation of ruminants by fly larvae of the Oestridae family can lead to nasopharyngeal or subcutaneous myiasis, which can harm animal welfare and productivity on sheep and cattle farms. The infestation of wild ruminants allows them to serve as reservoirs for subsequent transfer to domestic animals. The fly species most commonly responsible for myiasis have been well studied in many parts of the world, but less so in southeastern Europe. Here, we identified three botfly species among 197 subcutaneous larvae and 138 nasopharyngeal larvae from 36 wild roe deer (Capreolus capreolus) and 9 wild red deer (Cervus elaphus) at 30 locations across Croatia. Instars were stored in 75% ethanol and their species and developmental stage were identified based on morphological and morphometric keys. Subcutaneous larvae in both cervid species were identified as Hypoderma diana, while nasopharyngeal larvae were identified as Cephenemyia simulator in roe deer and Pharyngomyia picta in red deer. Partial sequencing of the gene-encoding mitochondrial cytochrome oxidase subunit I confirmed the taxonomic classification of C. stimulator and P. picta but was unable to confirm that of H. diana for lack of previously published sequences. This study appears to be the first to combine the morphological and molecular characterization of myiasis-causing botflies in wild cervids in southeastern Europe.