Species complexes: confusion in identifying the true ... · • Polytene chromosome banding patterns 18

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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• 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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• 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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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




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




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!)




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




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




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




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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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




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 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




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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Anopheles sundaicus complex

43Java

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




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




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


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Thank you

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Thank you




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Species complexes: confusion in identifying the true ... · • Polytene chromosome banding patterns 18