Molecular identification of arthropods

Oleg MEDIANNIKOV

ESCMID Online Lecture Library

© by author

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

ESCMID Online Lecture Library

© by author

ESCMID Online Lecture Library

© by author

… of arthropods? Why?

Arthropod

Infestation (parasitic diseases)

Vectors

Crustaceans

Pentastomida

Arachnidae Myriapoda

Insects

ESCMID Online Lecture Library

© by author

Arthropod vectors

Acariens

Insects Ticks Mites

Soft Hard Lice: typhus, recurrent fever, trench fever Fleas: plague, murine typhus Mosquitoes: malaria, yellow fever, Saint-Louis encephalitis, dengue, West Nile, Chikungunya, Rift valley, Japanese encephalitis, dirofilariasis Biting midges (Cératopogonidae) Mansonella, veterinary pathogens Sandflies: leishmaniasis, pappataci fever Tsetse flies: sleeping sickness Triatoma: Chagas disease Lutzomia flies: bartonellose Bloodsucking Diptera: filariases Etc…

Scrub typhus

Spotted fevers, Lyme disease, Crimean-Congo hemorrhagic fever, tick-borne encephalitis, anaplasmose, ehrlichiose, babesiosis, tularemia, Q fever

Borreliosis (relapsing fever)

… of arthropod vectors?

ESCMID Online Lecture Library

© by author

Other medically important arthropods (vectors or infesting)

• crayfish (paragonimiasis)

• ants (dicrocoeliasis)

• Cyclops (Guinea worm)

• Scorpions, spiders, Solifugae (wind scorpions)

• bedbugs

• House dust mites

ESCMID Online Lecture Library

© by author

… but why?

1. Knowledge is the power 2. Clinical application

Presence of pathogenic agent, Capacity to transmit the pathogen

ESCMID Online Lecture Library

© by author

… 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

mutation rates.

ESCMID Online Lecture Library

© by author

Human mitochondrion

ESCMID Online Lecture Library

© by author

http://www.barcodeoflife.org/

ESCMID Online Lecture Library

© by author

… 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 and 5S). May have 300-400 repeats

• Cytochrome B

ESCMID Online Lectu

re Library

© by author

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)

• Decision taking

ESCMID Online Lecture Library

© by author

blast.ncbi.nlm.nih.gov/

ESCMID Online Lecture Library

© by author

BLAST results

ESCMID Online Lecture Library

© by author

BLAST problems

• Quality of submitted sequences: « garbage »

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

• Delicate identification: no genetic criteria for most taxons. To be developped. ESCMID Online Lectu

re Library

© by author