Molecular and morphological characterization of third instar Palaearctic horse stomach bot fly larvae (Oestridae: Gasterophilinae, Gasterophilus).

Species of Gasterophilus are obligate parasites of equids and may induce severe, even lethal myiasis. However, identification of the third instar Gasterophilus larva at the species level is still problematic predominantly due to a shortage of diagnostic morphological features and incomplete molecular libraries. Testing the suitability of three different molecular markers showed that the traditional 650 bp barcode region near the 5' terminus of the cytochrome c oxidase subunit I (COI) served as a better tool for species-level identification than a 663 bp region near the 3' terminus of COI and a 554 bp region near the 5' terminus of the large subunit ribosomal RNA. We found that barcoding discriminates G. intestinalis, G. nasalis, G. nigricornis and G. pecorum but not G. haemorrhoidalis and G. inermis. A comparative morphological study using scanning electron microscopy was conducted to promote the identification of the third instar larvae. Photographs of fresh mature third instar larvae are provided for all species, and the remarkable green body colour of third instar G. nigricornis is fully documented for the first time. Two morphological keys are provided, one is suitable for quick identification, and the other based on ultrastructural details is provided for further comparative morphological investigation. A new term 'oral plate' instead of 'mandible' was proposed for a pair of sclerites of uncertain homology emerging from the secondary mouth opening. Our data shows that DNA barcodes cannot replace morphology for identification of third instars of Gasterophilus species, and a scaffold is provided for an integrated taxonomic reference system, which will contribute to monitoring gasterophilosis for equid welfare and protection, and also facilitate further studies in functional anatomy, phylogenetic analyses and host-parasite co-evolutionary investigations of Gasterophilus.