mosquito barcoding new

DNA Barcode Analysis of Mosquito Species from

Pakistan

Muhammad AshfaqUniversity of Guelph, Canada

Outline

I. DNA barcoding & its use in species identities

II. DNA barcoding mosquito species from Pakistan

1. Species analysis

2. Distribution analysis

• Why?

AND

• How to identify the biological units?

“In conservation biology it is important to decide on the taxonomic unit deserving Attention”

Biological Species Concept (BSC)

I. DNA barcoding and its use

I. Taxonomic identification Morphological characters Breeding Biology Host plants etc

II. Molecular identification Use of DNA nucleotide data Protein analysis etc

What is molecular identification?

Use of DNA sequence data for individual or species identification

by

• Sequence comparisons

• Constructing phylogenetic trees

• Other available tools

Why molecular identification?

• Clear genetic basis

• Deformed/broken samples are OK

• DNA from fossilized specimens can be used

• Identification of immature stages, or concealed stages possible

• Quick and unbiased

• Reliable

Target genes of DNA analysis

• Mitochondrial

• Ribosomal RNA

• Nuclear protein-coding genes

• Satellite DNA/ SSRs

• Introns

• Rare genomic changes

Problems/ issues

• Different target genes from the same species

• Different regions of the same gene

• No standardized comparisons

• No single database to perform sequence comparisons

• Species consensus/ conclusions – difficult

Why not to standardize the molecular identification?

The Mitochondrial genome

COI

850 bp658 bp

The candidate gene?

Why mtDNA?

• Ease of isolation

• High copy number

• Lack of recombination

• Conservation of sequence and structure across metazoa

• Range of mutational rates in different regions of the molecule

Why COI?

• Relatively well studied at the biochemical level• Size and structure conserved across all aerobic

organisms• Mix of variable and conserved regions• Largest of the three CO subunits• Broad spectrum of substitutional rates

Characteristics of Barcode Regions

• Flanked by conserved regions• Easy to amplify• Low intraspecies variability• Discontinuous variation between species• Long enough to work in all groups• Short enough for single reads

A DNA barcode is a short gene sequence taken from standardized portions of the genome, used to identify species

DNA barcoding: towards an inventory of life

Using DNA Barcodes Establish reference library of barcodes from

identified voucher specimens If necessary, revise species limits Then:

Identify unknowns by searching against reference sequences

Look for matches (mismatches) against ‘library on a chip’

Before long: Analyze relative abundance in multi-species samples

1. Databasing2. Labeling3. Imaging4. Tissue sampling5. DNA extraction6. PCR7. PCR check8. Sequencing reaction9. Sequencing cleanup10. Sequencing11. Trace editing & submission

Analytical chain

MethodsMethods

BOLD: Barcode of Life Data System

GOAL: Assemble the sequence library – rapidly and inexpensively to identify the organisms

Barcoding: a global initiative

iBOL nodes

Barcoding species from Pakistan

Canadian Centre for DNA Barcoding

NIBGE

Funded by: HEC

:iBOL

Using barcode data for species IDs

• Nucleotide identities/matches

• Distance analysis

• Barcoding gap

• Cluster analysis

MAIMB592-09|HM424125|Isoptera (W)

MATER039-11|Odontotermes obesus (S) MAIMB595-09|HM424126|Isoptera (W)

MAIMB608-09|HM891583|Isoptera (W)

MAIMB586-09|HM424124|Isoptera (W)

MAIMB616-09|HM891589|Isoptera (W)

MAIMB588-09|HM891573|Isoptera (W)

MATER040-11|Odontotermes gurdaspurensis (S) MAIMB648-09|HM424131|Isoptera (W)

MATER050-11|Odontotermes sp. (S) MAIMB598-09|HM891578|Isoptera (W)

MATER019-10|HQ991626|Odontotermes lokanandi (S) MAIMB591-09|HM891575|Isoptera (W)

MAIMB590-09|HM891574|Isoptera (W)

MATER011-10|HQ991622|Coptotermes travian (S) MAIMB638-09|HM891596|Isoptera (W)

MAIMB626-09|HM891591|Isoptera (W)

MAIMB635-09|HM891595|Isoptera (W)

MAIMB629-09|HM891593|Isoptera (W)

MAIMB653-09|HM891600|Isoptera (W)

MAIMB640-09|HM891597|Isoptera (W)

MATER005-10|HQ991620|Microcerotermes sp. (S) MATER014-10|HQ991624|Angulitermes dehraensis (S) MATER032-11|Microtermes obesi (S)

MAIMB645-09|HM891598|Isoptera (W)

MAIMB627-09|HM891592|Isoptera (W)

MATER033-11|Microtermes unicolor (S)75100

9953

100

10072

89

97

57

46

100

100

64

54

100 99

99 68

99

69

0.02

Cluster analysis

Barcoding gap or no gap!

A

B

Identity analysis

NCBI

BOLD

Barcode applications

• Resolving cryptic species complexes

• Estimating species diversity

• Constructing barcode reference libraries

• Resolving commercial disputes

• Detecting invasive species

• Analyzing food chain etc etc

II. Barcoding mosquitoes from Pakistan

Mosquito species from the region

• 1971: 100 species from West & East Pakistan

• 1976-77: 43 species from Lahore area

• 1978-79: 30 species from Central Punjab

• 1997: 30 species from Indian Punjab

• 2007: 63 species from India– Culex tritaeniorhynchus was reported as the predominent

species in Punjab, Pakistan

No definite number of mosquito species from Pakistan available

Progress on mosquito barcoding

Genus: Aedes Genus: Anopheles

Genus: Culex

Known Disease Link

• Aedes – Dengue

• Anopheles – Malaria

• Culex – West Nile Virus, Filariasis, Encephalitis

Dengue transmission cycleDengue transmission cycle

West Nile Virus transmission cycleWest Nile Virus transmission cycle

Current studies

Collection Areas

• 300 localities

• 21 districts of Punjab and few localities from Peshawar

• 2-7 specimens barcoded from each locality

• A total of 1425 specimens

• 190 larvae

• 1235 adults

Islamabad

Multan

Lahore

Mosquito collection localities

Target Locations

• Urban areas• Locations in close proximity of

human dwellings, such as:• Marshes and ponds• Water drums• Abandoned locations• Underconstruction buildings• Junkyards• Sub-ground water catchment areas• Gutters• Waste bins near hospitals and

petrol-pumps

Collection Methods

Arial nets Motorized aspirators

Manual catching

Human landing

Pipettes and sieves Traps with CO2 source

Barcode data obtained

Data analysis

• Distance analysis

• Barcode gap analysis

• Cluster analysis

Data analysisSequence alignments

Distance analysis

Histogram of distances Ranked distances

Distance analysis and “barcode gap”

barcode gap

Using ABGD (Automatic Barcode Gap Discovery)

Barcode Gap Analysis (using BOLD)

NN distances

MAMOS171-12| Anopheles culicifacies_A

MAMOS167-12| Anopheles culicifacies

MAMOS173-12| Anopheles annularis

MAMOS147-12| Anopheles splendidus

MAMOS1613-13| Anopheles sp nr dravidicus

MAMOS1227-12| Anopheles stephensi

MAMOS159-12| Anopheles pulcherimus

MAMOS637-12| Anopheles subpictus

MAMOS185-12 | Anopheles peditaeniatus

MADIP347-10| Ochlerotatus pseudotaeniatus

MAMOS086-12| Culicidae

MAMOS1309-12| Aedes albopictus

MAMOS017-12| Aedes aegypti

MAMOS087-12| Aedes sp1pk

MAMOS1230-12| Aedes sp2pk

MADIP300-10| Aedes sp3pk

MAMOS1582-13| Aedes sp4pk

MAMOS1366-12| Mansonia bonneae

MAMOS905-12| Armigeres subalbatus

MAMOS1217-12| Culex tritaeniorhynchus

MAMOS012-12| Culex quinquefasciatus

MADIP334-10| Culex theileri

MAMOS1364-12| Culex perexiguus

MAMOS394-12| Culex fuscocephala

MAMOS207-12| Culex bitaeniorhynchus

MADIP472-11| Culex mimeticus

MAMOS1581-13| Lutzia fuscana

10055

3819

18

49

33

98

50

72

55

50

15

31

23

88

31

30

16

18

15

0.000.010.020.030.040.050.060.07

Anopheles

Aedes

Culex

Cluster analysis of mosquito species

Cluster analysis of Aedes COMPLEX

MADIP347-10| Ochlerotatus pseudotaeniatus

Ochlerotatus pseudotaeniatus (DQ154153)

Aedes iyengari (DQ431717)

MAMOS1309-12| Aedes albopictus

Aedes albopictus (JQ412504)

Aedes caspius (FJ210904)

Aedes vittatus (AY834246)

MAMOS087-12| Aedes sp1pk

MAMOS138-12| Aedes walbus

MAMOS1582-13| Aedes sp4pk

MADIP300-10| Aedes sp3pk

MAMOS1230-12| Aedes sp2pk

Aedes lineatopennis (HQ398909)

MAMOS017-12| Aedes aegypti

Aedes aegypti (HQ688295)

Aedes vexans (AY917213)

MAMOS1366-12| Mansonia bonneae

Aedes fumidus (AY729978)

100

100

100

78

51

14

17

10

1

0.000.020.040.060.08

Aedes aegypti

Aedes albopictus

Aedes sp1pk

Aedes sp2pk

Aedes sp3pk

Aedes sp4pk

Aedes W-albus

Aedes COMPLEX

Ochlerotatus pseudotaeniatus

Species ratio

76%Culex quinquefasciatus

8%

16%

26 species

5%

Genus-wise distribution

Aedes COMPLEX Anopheles COMPLEX

Culex COMPLEX

Conclusions

• DNA barcodes effectively discriminated the 26 mosquito species

• Culex quinquefasciatus was the predominant species in our collections comprising 76% of the total

• There were seven species in Aedes complex which made only a fraction of the collected specimens

• The distribution patterns did not reveal localization of a species in one specific area

• Similarity of members of Aedes complex warrants use of molecular methods for vector population forecasts

Acknowledgements

Dr. Sohail Hameed, Director NIBGE Dr. Paul Hebert, Director BIO, ON, Canada Dr. Sajjad Mirza, NIBGE Several faculty members around Pakistani universities for

providing specimens and help in identifications My lab staff and students Higher Education Commission for funding