Molecular identification of arthropods Oleg MEDIANNIKOV © by author ESCMID Online Lecture Library Molecular identification? • PCR followed by the sequencing of the amplicons • Comparison of the obtained sequence with the records in the database • Taking the decision on whose gene was amplified © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library … of arthropods? Why? Arthropod Infestation (parasitic Vectors diseases) Pentastomida© by author Crustaceans Arachnidae Insects Myriapoda ESCMID Online Lecture Library … of arthropod vectors? Arthropod vectors Acariens Ticks Mites Insects Scrub Lice: typhus, recurrent fever, trench fever Soft Hard typhus Fleas: plague, murine typhus Mosquitoes: malaria, yellow fever, Saint-Louis encephalitis, dengue, West Nile, Chikungunya, Spotted fevers, Lyme Borreliosis Rift valley, Japanese encephalitis, dirofilariasis disease, Crimean-Congo© by author (relapsing Biting midges (Cératopogonidae) Mansonella, hemorrhagic fever, tick- veterinary pathogens fever) borne encephalitis, Sandflies: leishmaniasis, pappataci fever ESCMID Online LectureTsetse flies: sleeping Library sickness anaplasmose, ehrlichiose, Triatoma: Chagas disease babesiosis, tularemia, Q Lutzomia flies: bartonellose fever Bloodsucking Diptera: filariases Etc… Other medically important arthropods (vectors or infesting) • crayfish (paragonimiasis) • ants (dicrocoeliasis) • Cyclops (Guinea worm) • Scorpions, spiders, Solifugae (wind scorpions) • bedbugs © by author • House dust mites ESCMID Online Lecture Library … but why? 1. Knowledge is the power 2. Clinical application Presence of pathogenic agent, Capacity to transmit the pathogen © by author ESCMID Online Lecture Library … what to amplify/sequence? Mitochondrial DNA: 1. Quantity (100-10,000 separate copies of mtDNA per cell) 2. Quality (no recombination, haploid) 3. House-keeping genes, but high© by author mutation rates. ESCMID Online Lecture Library Human mitochondrion © by author ESCMID Online Lecture Library http://www.barcodeoflife.org/ © by author ESCMID Online Lecture Library … what to amplify/sequence? • COI so called « Folmer » primers (around 658 bps): – LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG-3‘ – HCO2198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3' • Redesigned primers: – Redesigned forward primer jgLCO1490 5’- TITCIACIAAYCAYAARGAYATTGG-3’ – Redesigned reverse primer jgHCO2198 5’-TAIACYTCIGGRTGICCRAARAAYCA-3’ • Ribosomal RNA – Mitochondrial (12S, 16S) – Cytoplasmic (28S, 5.8S, ©18S andby 5S). author May have 300- 400 repeats • Cytochrome B ESCMID Online Lecture Library Rools for successful identification • Good quality specimen = good quality amplicon = good quality sequence • Proper alignment. Do not forget to cut off the primers’ sequences • Exhaustive search© (BLAST) by author ESCMID Online Lecture Library • Decision taking blast.ncbi.nlm.nih.gov/ © by author ESCMID Online Lecture Library BLAST results © by author ESCMID Online Lecture Library BLAST problems • Quality of submitted sequences: « garbage » • Lack of sequences of all animals: Barcode of life is yet to do © by author • Delicate identification: no genetic criteria for ESCMIDmost taxons. Online To be developped Lecture. Library .