Molecular identification of Stylops advarians (Strepsiptera: Stylopidae) in western Canada.

Strepsiptera are an enigmatic order of insects with extreme sexual dimorphism which makes it difficult to "match-up" free-living adult males with parasitic conspecific females of the Stylopidia, and free-living females of the Mengenillidae using morphological characters. Species identification is further complicated for the Stylopidia because adult females are endoparasitic and neotenic. Therefore, we used DNA sequencing of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) to confirm the species identity of adult strepsipterans that were morphologically identified as Stylops advarians. These specimens, collected from Saskatoon (Saskatchewan, Canada), included one adult male, and eight females, the latter of which had been collected from solitary bees (Andrena milwaukeensis). Also included in the analyses were three pools of first-instar larvae that had emerged from three of the females. The results of the molecular analyses revealed that all specimens had an identical cox1 sequence, and belonged to a clade, with total statistical support (bootstrap value of 100%), that contained specimens of S. advarians from New York and Maine (USA). Hence, the results were consistent with the morphological identification of S. advarians. This study demonstrates the usefulness of a molecular approach for the identification of endoparasitic adult female and larval strepsipterans, life cycle stages that lack significant morphological characters for species identification.

Molecular Differentiation of Four Species of Oropsylla (Siphonaptera: Ceratophyllidae) Using PCR-Based Single Strand Conformation Polymorphism Analyses and DNA Sequencing.

It is often difficult to distinguish morphologically between closely related species of fleas (Siphonaptera). Morphological identification of fleas often requires microscopic examination of internal structures in specimens cleared using caustic solutions. This process degrades DNA and/or inhibits DNA extraction from specimens, which limits molecular-based studies on individual fleas and their microbiomes. Our objective was to distinguish between Oropsylla rupestris (Jordan), Oropsylla tuberculata (Baker), Oropsylla bruneri (Baker), and Oropsylla labis (Jordan & Rothschild) (Ceratophyllidae) using PCR-based single strand conformation polymorphism (SSCP) analyses and DNA sequencing. A 446 bp region of the nuclear 28S ribosomal RNA (rRNA) gene was used as the genetic marker. The results obtained for 36 reference specimens (i.e., fleas that were morphologically identified to species) revealed no intraspecific variation in DNA sequence, whereas the DNA sequences of the four species of Oropsylla differed from one another at two to six nucleotide positions. Each flea species also had a unique SSCP banding pattern. SSCP analyses were then used to identify another 84 fleas that had not been identified morphologically. DNA sequencing data confirmed the species identity of fleas subjected to SSCP. This demonstrates that PCR-SSCP combined with DNA sequencing of the 28S rRNA gene is a very effective approach for the delineation of four closely related species of flea.