Molecular Identification of Arthropods

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

• 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’

• 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

blast.ncbi.nlm.nih.gov/

• Quality of submitted sequences: « garbage »

• Lack of sequences of all animals: Barcode of life is yet to do