Species complexes: confusion in identifying the true vectors of malaria and other parasites
Professor Susan Paskewitz
The screen versions of these slides have full details of copyright and acknowledgements 1
Species Complexes: Confusion in Identifying the True Vectors of Malaria and Other Parasites
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Professor Susan PaskewitzDepartment of Entomology
University of Wisconsin
Historical malaria in Europe
Hyperendemic
Mesoendemic
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Hypoendemic
Epidemic
Anopheles mosquitoes and malaria
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Species complexes: confusion in identifying the true vectors of malaria and other parasites
Professor Susan Paskewitz
The screen versions of these slides have full details of copyright and acknowledgements 2
• European malaria was mostly coastal
• But the proposed vector, Anopheles maculipennis, was often dense inland, where malaria was rare
Anophelism without malaria
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• This puzzling pattern was called “anophelism without malaria”
Clues to the puzzle
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Distribution of Anopheles atroparvus
PresentAbsent
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Species complexes: confusion in identifying the true vectors of malaria and other parasites
Professor Susan Paskewitz
The screen versions of these slides have full details of copyright and acknowledgements 3
• 1926 Falleroni
• 2 colors of eggs in An. maculipenniscollections in Italy
• Dark grey eggs = An. labranchiae
• Silvery white eggs = An. messeae
More clues to the puzzle
7Distribution of An. labranchiae
Identification of blood meal sources
8• The precipitin test
The Anopheles maculipennis complex
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Species complexes: confusion in identifying the true vectors of malaria and other parasites
Professor Susan Paskewitz
The screen versions of these slides have full details of copyright and acknowledgements 4
Key biological characteristics
• Host preference and feeding behavior
• Insecticide resistance
• Vector competence
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• Larval habitat
• Phenology
What is a species complex?
• Species are reproductively isolated gene pools
• Group of species that are closely related
• No consistent morphological characters for identification even by specialists
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even by specialists
• Sometimes called “cryptic species”
• “Subgroup” vs. “Species complex”
• Ochlerotatus detritus
• Anopheles nuneztovari
• Anopheles funestus
• Anopheles subpictus
• Anopheles annulipes
Examples of species complexes
• Culex pipiens
• Ochlerotatus communis
• Anopheles gambiae
• Anopheles quadrimaculatus
• Anopheles punctipennis
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• Anopheles nili
• Anopheles nivipes
• Anopheles marshallii
• Anopheles leucosphyrus
• Anopheles albitarsis
• Anopheles fluviatilis
• Anopheles pseudopunctipennis
• Anopheles dirus
• Anopheles minimus
• Anopheles sundaicus
• Anopheles farauti
• Anopheles barbirostris
Species complexes: confusion in identifying the true vectors of malaria and other parasites
Professor Susan Paskewitz
The screen versions of these slides have full details of copyright and acknowledgements 5
Other complexes
• Blackflies (Simulium damnosum)
• Sandflies (Lutzomyia longipalpis)
• Midges (Culicoides imicola)
• Tsetse (Glossina morsitans)
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( )
• Body and head lice (Pediculus)
• Soft tick (Ornithodoros moubata)
• Tick (Rhipicephalus sanguineus)
• Tick (Hyalomma marginatum)
How are they identified?
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How are they identified?
Anopheles gambiae complex
• “sensu stricto” vs. “sensu lato”– A. gambiae sensu stricto
– A. arabiensis
– A. merus
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– A. melas
– A. quadriannulatus A
– A. quadriannulatus B
– A. bwambae
– A. comorensis
Species complexes: confusion in identifying the true vectors of malaria and other parasites
Professor Susan Paskewitz
The screen versions of these slides have full details of copyright and acknowledgements 6
Identification of gambiae complex
• Identification of differences in larval habitats
16Anopheles merusAnopheles melas
Genetic crosses
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• George Davidson Genetic crosses of field collected mosquitoes in laboratory revealed absence of fertile offspring (F1 males are sterile)
Fig. 1The normal male reproductive system
of A. gambiae (group B) from Pala, Upper Voltat=testis; a=accessory gland
Fig. 2The male reproductive system, showing very reduced
testes, from a hybrid produced by crossing male A. gambiae from Lagos, Nigeria (group A) and female A.
gambiae from Pala, Upper Volta (group B)t=testis; a=accessory gland
Cytogenetics
• Mario Coluzzi and colleagues
• Polytene chromosome banding patterns
18• Each species can be identified by its unique sequence of bands
(like a bar code!)
Species complexes: confusion in identifying the true vectors of malaria and other parasites
Professor Susan Paskewitz
The screen versions of these slides have full details of copyright and acknowledgements 7
Insecticide resistance in the gambiae complex
• Zimbabwe, 1977
• Organochlorine
• WHO susceptibility test results
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• Species ID results
• Insecticide replaced
Techniques used for investigation of species complexes
• Cuticular hydrocarbons
• Scanning electron microscopy
• Allozyme analysis
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y y
• Nucleic acid-based analysis
Proteins as markers
• Allozyme electrophoresis
• Examine phenotypes for multiple enzymes
• Rapid and inexpensive
C lt i bi h i l k t i
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• Can result in biochemical keys to species
• e.g., S.J. Miles (1979) key to 5 species of the A. gambiae complex
Species complexes: confusion in identifying the true vectors of malaria and other parasites
Professor Susan Paskewitz
The screen versions of these slides have full details of copyright and acknowledgements 8
Example of allozyme analysis
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
• Lanes 6-8, 18, 19 = A. halphylus
• Lanes 1,3, 5, 9-11, 13-15 = A. triannulatus
• Lanes 2, 4, 12, 16, 17 = possible new species
Disadvantages
• Expression of proteins can vary across life stages
• Proteins are unstable and enzyme activity is lost unless live or stored at -40ºC
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DNA-based methodologies
• PCR (polymerase chain reaction) can amplify a single sequence from a complex mixture of DNA
• Tool for identification/diagnosis of known cryptic species
• Investigation of potential species complexes
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Investigation of potential species complexes
Species complexes: confusion in identifying the true vectors of malaria and other parasites
Professor Susan Paskewitz
The screen versions of these slides have full details of copyright and acknowledgements 9
How PCR works
Adenine Thymine
Base pairs
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Guanine Cytosine
Sugar phosphate backbone
3’ 5’DNA
Step 1: denaturation
95oC
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3’5’
95oC
3’ 5’DNA
50oCPrimer 1
Step 2: annealing
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3’5’
Primer 2
Species complexes: confusion in identifying the true vectors of malaria and other parasites
Professor Susan Paskewitz
The screen versions of these slides have full details of copyright and acknowledgements 10
Step 3: extension
3’ 5’DNA
Primer 172oC
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5’
Primer 2
3’
72oC
Repeated cycles amplify fragment of interest
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Amplification of genomic DNA fragment
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Species complexes: confusion in identifying the true vectors of malaria and other parasites
Professor Susan Paskewitz
The screen versions of these slides have full details of copyright and acknowledgements 11
PCR reaction components
• Template DNA
• Oligonucleotide primers
• Nucleotides (dATP, dGTP, dCTP, dTTP)
T l
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• Taq polymerase
• Reaction buffer
• MgCl2
Advantages to PCR-based diagnostic methods
• Preservation of DNA
• Highly sensitive: small amounts of starting material (e.g., a single leg or small arthropods)
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• Specificity
• Random or targeted amplification of sequences
Distinguishing gambiae complex members by diagnostic PCR
• Identification of DNA to be targeted
• Amplify and sequence target from members of complex
• Compare sequences, identify differences
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Compare sequences, identify differences
• Produce primers encompassing areas where species differ
Species complexes: confusion in identifying the true vectors of malaria and other parasites
Professor Susan Paskewitz
The screen versions of these slides have full details of copyright and acknowledgements 12
Target sequences
• Repetitive or multi-copy DNA
• Highly conserved regions next to divergent regions
• Conserved regions are used to design primers that will amplify divergent regions
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that will amplify divergent regions
– Mitochondrial cytochrome oxidase I
– Nuclear ribosomal DNA
Cytochrome oxidase I gene
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CO1
Ribosomal DNA
28S 18SIntergenic spacer 5.8S
ETSITS1 ITS2
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ITS1 ITS2
• Usually tandemly linked copies, ranging from 100-1,000 copies in insects
• Intergenic spacer (nontranscribed spacer) separates individual transcription units
• Internal transcribed spacers 1 and 2 are within the transcription unit
Species complexes: confusion in identifying the true vectors of malaria and other parasites
Professor Susan Paskewitz
The screen versions of these slides have full details of copyright and acknowledgements 13
Species 1
Species 2
Universal primer Species-specific primers
Primer design for diagnostic PCR
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Species 3
Species 4
Example of diagnostic PCR
Fig. 2 Specific PCR products for six A. maculipennis complex species (1 100-bp marker, 2 A. atroparvus, 3 A. sacharovi, 4 A. melanoon, 5 A. messeae, 6 A. labranchiae, 7 A. maculipennis s.s., 8 100-bp marker)
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Validation
• Examine intraspecific variation to verify that overlap does not occur between species
• Lab colonies are a limited sample of actual genetic variability
• Field-collected samples across the geographic range
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• Field-collected samples across the geographic range
Species complexes: confusion in identifying the true vectors of malaria and other parasites
Professor Susan Paskewitz
The screen versions of these slides have full details of copyright and acknowledgements 14
Multiplex PCR
M 1 2 3 4 5 6 7
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500 bp
Species T
P. fal
Species S
P. fal
Identification of new cryptic species and molecular taxonomy
• Molecular tools (rDNA sequences and COI barcoding)
• Chromosomal inversions
• Behavioral characters
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• Morphological characters of some stages
• Phylogenies using multilocus assessments
Evolution of species complexes
• Historical geography
• Human population expansion and agriculture
• Ecological and Ecotypic speciation
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Species complexes: confusion in identifying the true vectors of malaria and other parasites
Professor Susan Paskewitz
The screen versions of these slides have full details of copyright and acknowledgements 15
Anopheles sundaicus complex
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Human expansion
• Savanna vs. forest habitats
• Anopheles gambiae and A. arabiensis
• Temporary sunlit pools
F d h d d ti i l
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• Feed on humans and domestic animals
• Rest in human-made shelters
Humans and agriculture
• A. gambiae s.s.
• Chromosomal forms
– Savannah
– Bamako
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Bamako
– Mopti
– Forest
– Bissau
Species complexes: confusion in identifying the true vectors of malaria and other parasites
Professor Susan Paskewitz
The screen versions of these slides have full details of copyright and acknowledgements 16
Molecular forms
• M form and Mopti form in Mali
• S form and Savanna/Bamako forms in Mali
– Linkage of Mopti/M suggests this form may be reproductively isolated
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may be reproductively isolated
Ecological speciation
• M vs. S larval biology
– Predator pressure
• Reproductive isolation via swarm formation
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via swarm formation
Ecotypic speciation
• Colonization of marginal habitats during periods of population expansion
• Adaptation to marginal conditions
• Adaptive alleles within inversions are protectedEcotypic speciation
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• Adaptive alleles within inversions are protected from recombination
• Can be linked to reproductive behavior
Ecotypic speciation
Species complexes: confusion in identifying the true vectors of malaria and other parasites
Professor Susan Paskewitz
The screen versions of these slides have full details of copyright and acknowledgements 17
Bamako
• Laterite rock pools
• 2Rj inversion
• Incipient or already
Mali
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p ycompleted speciation within the gambiae complex
Species complexes and gene flow
• Genetic vector control
• Resistance to insecticides
Species complexes and gene flow
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Species complexes and gene flow
Thank you
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Thank you