Molecular Methods for Distinguishing Between Wild and Commercial Cotton Variants of

Anthonomus grandis

Raul Ruiz-Arce

USDA APHIS PPQ Mission Lab (ML)

Molecular methods development at ML

TW/BW diagnostics at ML ◦ Description & performance of three methods

◦ Summary for captures 2011-2015

Recent advancements and future work

Overview of Facilities, Expertise, and Diagnostics at USDA APHIS

Mission Lab - Edinburg, TX

4 Molecular Scientists

3 Entomologists

1 GIS Specialist

3 Biologists

1 Plant Pathologist

18 Technicians

3 Office Administration

1 IT specialist

2 Interns

Citrus health response programs ◦ Detection and ID citrus pests

◦ Citrus greening biocontrol

Mexican fruit fly SIT support ◦ Strain development & QC

◦ Colony health

Molecular diagnostics – Conventional/NGS* ◦ *Fruit flies – ID, Source estimation

◦ BW/TW – variant ID

◦ Helicoverpa – ID methods Old/New World Bollworm

Organelles

Membranes

DNAs

Their importance

to molecular work

DNA molecule

Sample preservation

Contaminants

Harvesting DNA

Why: Pest$ take a big bite We’re a small world Quality food is important

What do they tell us: Confirm ID Differences among species Where pests originate History and movement Speciation

3 temperature ◦ Denaturing

◦ Annealing

◦ Extension

Indigenous to C America/S Mexico

Found in Cuba & Haiti in the 1800s

First reported in Texas 1892

S America (1949-1990s)

Morphological - Burke (1968) and Burke et al (1986)

Molecular ◦ Roehrdanz (2001) ◦ ML RAPD-PCR 1990s – 2000s

Southeastern boll weevil (BW) ◦ Host – commercial cotton (Gossypium hirsutum)

Thurberia weevil (TW) Host – wild cotton (G. thurberi), occurs in N Mexico &

SW US

Captures ◦ Trapped weevils initiate response

Challenge in using older methods ◦ Morphology (Burke, 1968)

◦ Molecular (Roehrdanz, 2001)

Opportunity for Improving management ◦ ID source of infestation

◦ Helps in decision for management

◦ Reduction to resources

Molecular Methods (Barr et al., 2013)

Sequencing COI Barcode (Folmer et al 1994)

SCAR (seq characterized amplified region)

Classical Taxonomy (perf by C. Reuter)

Morphometrics (Burke, 1968)

n=158 weevils 30 sample sites 10 Wild cotton 20 Comm cotton

503 base pair fragment

Reference database (n=158), 31 unique seqs

◦ N=84, 14 sites in 7 MX states

◦ N=74, 16 sites in 4 US States

AN2 T C A A C G C G T C A C A C T G G A A A A G T T G T A A A A A C G C A A A T A C T T A T A A A A

AN3 . . . . . . . . . . G . . . . A . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . .

AN4 . . G . . . . . . . G . . . . . . G . . . . . . . . . . . . . . . A . . . . . . . . . . . . . .

AN6 . . G . . A . . . . G . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . .

AN7 . . G . . . . . . . G . . . . . . G . . . . . . . . . . . . . . . . . . . . . . C . . . T . . .

AN8 . . G . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . A . . G . . . . . . . . . . .

AN14 . . G . . . . . . . G . . . . . . G . . . . . . . . . . . . . . . A G . . . . . . . . . . . . .

AN15 . . . . . . . . . . G . . . . . . . G . . . . . . . . . . . . . A . . . . . . . . C . . . . . .

AN16 . . G . T . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

AN17 . . G . . . . . . . G . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . .

AN18 . . G . . . . . . . G . . . C . . . . . . . . . . . . . . . . . . A . . G . . . . . . . . . . .

AN19 . . G . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

AN20 . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

AN21 . . G . . . . . . . G . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . .

AN22 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . .

AN23 . . G . . . . A . . G . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . .

AN24 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . .

AN26 . T G . . . . . . . G . . . C . . . . . . . . . . . . . . . . . . A . . G . . . . . . . . . . .

AN5 C T G . . . T . C T . . . T . . . . . . . . C C A . . . . . . . . . . . . . . T . . . . . . . T

AN1 G . . G . A . . . T G A . . . A A . . . . . . . A C . C . . G . . . . . . . T . C . . . . G G .

AN9 G . . G . A . . . T . A . . . A . . . . G . . . A C G C . G G T . . . G . C T . C . . C . G . .

AN10 G . . G . A . . . T G A . . . A . . . G . . . . A C G C G . G . . . . . . . T . C . . . . G G .

AN11 G . . G . C . . . T . A . . . A . . . . . . . . A C G C . . G . . . . G . . T . C . . C . G . .

AN12 G . . G . A . . . T . A . . . A . . . . . . . . A C G C . . G . . . . G . . T . C . . C . G . .

AN13 A . . G . A . . . T G A . . . A . . . . . . . . A C G C G . G . . . . . . . T . C . . C . G G .

AN25 G . . G . A . . . T . A . . . A . . . . . . . . A C . C . . G . . . . G . . T . C . . C . G . .

AN27 G . . G . A . . . T . A . . . A . . . . G . . . A C G C . G G T A . . G . C T . C . . C . G . .

AN28 G . . . . A . . . T . A G . . A . . . . . . . . A C G C G . . . . . . . . . T . C . G C . G G .

AN29 G . . G . A . . . T G A . . . A . . . . . . . . A C G C G . G . . . . . . . T . C . . . . G G .

AN30 G . . G . A . . . T G A . . . A . . . G . A . . A C G C G . G . . . . . . . T . C . . . . G G .

AN31 G . . G . A . . . T G A . . . A . . . . . . . . A C . C G . G . . . . . . . T . C . . . . G G .

443

The list of variable nucleotide positions for the 31 COI haplotypes recovered from the Anthonomus spp. specimens. The diagnostic positions distinguishing Thurberia and boll weevil highlighted.

SNPs consistent with boll weevil

TW

BW

* Sonora, Chihuahua, Coahuila, Durango, AZ

Developed from excised band fixed to BW

- = not BW

+ = BW

Neg C

Femur Ratio (length:width) (Burke, 1968) ◦ 3.0-3.4 TW

◦ 3.6-4.0 BW

ID

Morphometrics

SCAR

COI profile

Correct*

89.4%

82.9%

94.3%

Identification results for n=158 with different methods (Barr

et al 2013).

*considered correct when the results agreed w/host

C. Reuter (Phoenix, Az) Morp ID then,

Shipped to ML

Day 1

• Intake

• DNA Isolation – minimally invasive

Day 2 PCR/Electrophoresis

Day 3 Send off for sequencing

Day 5+ Interpret data/complete report

Capture Date

n Collection Site

Reported ID

March 2011 30 Ojinaga, Chihuahua

BW

March 2011 30 Delicias, Chihuahua

BW

10 May 2012 3 Sonoyta, Sonora

TW

1 Oct 2014 3 Caborca, Sonora

TW

22 Oct 2014 8 Caborca, Sonora

TW

7 July 2015 1 Delicias, Chihuahua

NOT A. grandis

16 July 2015 2 Nuevo San Lucas, Chi

NOT A. grandis

17 July 2015 1 Delicias, Chihuahua

NOT A. grandis

Provides early warning ◦ Results to managers within ~5-7days

Helps improve management strategies ◦ Identifies hot spots/feedback

Reduction to resources ◦ TW ID = no spraying, trade is not impacts, etc

◦ BW ID = target only problematic areas

1. Sequence and assemble the CBW genome

2. Resolve the population genomic structure of A. grandis in northern Mexico

3. Infer movement/dispersal of CBW in northern Mexico and southern Texas

4. Identify source populations of potential reintroductions in southern Texas

5. Additional marker development

https://mail.google.com/mail/?ui=2&ik=d747726f63&view=att&th=132452fe56568661&attid=0.1&disp=inline&zw

Mediterranean fruit fly (Ceratitis capitata)

• Frequent US infestations (captures)

• Identified informative markers (conserved & fine scale)

• Informative reference database

• Results: Effective tool for determining the source of introductions

• Improve pest management strategies

• Minimize future infestations

• Identify hot-spots

*

*Photograph by: Scott Bauer, USDA

DNA Marker set A

DNA marker set B

GEO DIST =

=

=

Source = “Below Regional-level”

Source = “Regional-level”