Making sense of chemical communication in the red flour beetle

Project 20081: Biosensor detection of stored grain pests Making Sense of chemical communication in the red flour beetle: finding candidate pheromone receptors in Tribolium castaneum Kelly Hill, Bradley Stevenson, Alisha Anderson, Sylwek Chyb, Richard Glatz

biosecurity built on science Cooperative Research Centre for National Plant Biosecurity

Background  Grain exports in Australia:

- A$5 billion worth of grain - ‘Nil tolerance’ for insects in grain

 Early detection of infestation can prevent contamination and avoid over-use of phosphine  Current methods of detecting pests are labourintensive (sampling) or time-consuming (traps)  Tribolium castaneum (red flour beetle):

- International stored product pest - Sequenced genome - Annotated Olfactory Receptors (Engsontia et al. 2008)

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Insect detection in the grain industry -

Manual sampling Acoustic detection X-ray imaging Near Infrared Spectroscopy Headspace analysis (volatiles)  GC-MS  E-nose – detecting damage - Recent reports of the fabrication of conducting polymer sensors used to detect volatiles associated with stored grain spoilage (Hossain et al 2012)

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Insect detection in the grain industry -

Manual sampling Acoustic detection X-ray imaging Near Infrared Spectroscopy

- Headspace analysis (volatiles) • GC-MS • E-nose Recent reports of the fabrication of conducting polymer sensors used to detect volatiles associated with stored grain spoilage (Hossain et al 2012)

• Biosensors biosecurity built on science

Why a Bio-sensor? ď&#x201A;§ Sensitivity - Small insects in a large space - Early detection

ď&#x201A;§ Specificity - Many odours associated with grain - Receptors specific to a particular volatile would enable an increased signal above noise - Targeted control

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Aims ď&#x201A;§ Identify biological detectors and biological signals associated with Tribolium beetles, which could subsequently be utilised to develop a sensitive pest biosensor for use in grain storage ď&#x201A;§ Produce cell lines or isolated proteins which could be utilised to develop a transduction system that could relay volatile detection events to an end user

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Methodology  Insect culture  Behavioural studies  Electrophysiology studies - Detection of target volatiles with Y-shaped olfactomoter

 Finding candidate odourant receptors - Quantitative PCR used to monitor differences between transcript quantities of receptors and OBPs found in males compared to females

 Calcium assays - Used to monitor receptor activation when expressed in Sf9 insect cells.

 Investigate Odourant Binding Proteins (OBPs) - IMAC purification of bacterially expressed OBPs - DMD capture assay using solid phase microextraction and GC-MS

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Key Findings  Biological Signals- Confirmed target volatile for T. castaneum detection (4,8-dimethyldecanal)  Biological Detectors- Identified candidate receptors and odourant binding proteins for detection of T. castaneum  Produced proteins of interest in recombinant systems  Provided evidence of DMD binding to Odourant Binding Proteins

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Infestation volatile chemicals  4,8-dimethyldecanal (DMD) - Aggregation pheromone

O

 1-pentadecene (C15:1)

- Solvent for other chemicals and possibly an aggregation pheromone O

 Methyl-1,4-benzoquinone (MBQ)

- Aggregation, stress signal, and defence O

 Inter-specific pheromones

- Dominicalure (Ryzopertha dominica aggregation pheromone)

O

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Response to volatiles

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Female T. castaneum show behavioural response to low concentrations of DMD 4,8-dimethyldecanal (DMD)

O

10

Median response value

male female larvae f l

0

0.3 pmol

25 pmol 63 pmol

-10 0

1

10

100

1000

DMD in 1.5 mL/s airflow [pmol]

 Both Larvae and Adults have previously shown to be highly attracted to DMD  Median response of adult unmated T. castaneum to synthetic DMD in Y-tube olfactometer.  Response calculated as the number of beetles that crawl towards the DMD minus the number that crawl towards the blank sample (in groups of ten beetles).

Error bars indicate the inter-quartile range from 8 replicates.

0.3 x 10-12 moles (55 x 10-12 g) biosecurity built on science

Are relative transcript levels different between males and females? 0.03

**

Male Female

**

0.02

Comparative Quantitation (TcRPS6)

0.01

0.003

**

0.002

0.001

*

**

*

0.000

ORD

ORC

ORA

ORB

ORE biosecurity built on science

ORA involvement in DMD perception? â&#x20AC;˘ EAG recordings demonstrated a response to all tested chemicals â&#x20AC;˘ Response to DMD was eliminated with the removal of the terminal club

Expression of ORA in the Male antenna

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Odourant Binding Proteins A different class of detector

â&#x20AC;˘ Insect odorant-binding proteins (OBPs) are thought to function as the first step in molecular recognition and the transport of semiochemicals *

1.75

Male Female

Comparative Quantitation (TcRPS6)

1.50

1.25

1.00

0.75

0.50

0.25

0.00

OBPC

OBPB

OBPD

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Producing the Detectors  Receptor cell lines: -

Full Length cDNA Sf9 insect cells Baculovirus Stable transfection

50μ m

GFP-ORB

 pIB or pIZ vectors

 OBP protein production: - Expressed in E. coli - IMAC purification

Coommassie stained PAGE A

B

C

D

Anti-His Western A

B

C

D

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Evidence of DMD binding to OBPs

OBPD

OBPC

OBPB

OBPA

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Summary 

Signals:

- Larvae and adult male and female beetles are attracted to DMD - Female beetles have higher sensitivity to the pheromone DMD 

Detectors:

- 3 male-biased OR genes - 1 female-biased OR gene - 1 male-biased OBP 

Production:

- Expression of ORs in Sf9 insect cells  ORs can be successfully expressed using stable transfection vectors and using the baculovirus expression system

- OBPs have been expressed using E. coli 

Detection:

- Indication of a role for ORA in DMD response - OBPs show binding to DMD biosecurity built on science

Future Target Detectors

Target Signals

Signal transduction and output

Insect receptors and OBPs trialled in Fluorescent and Electrical platforms

Target signals tested in Cybernoseâ&#x201E;˘

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Poster 3

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Thanks to…  CSIRO Eco Sci

 SARDI

- Richard Glatz - Tamara Cooper - Everyone at SARDI Ento

 Trécé Inc. (Oklahoma, USA)

-

Bradley Stevenson Sylwek Chyb Alisha Anderson Cécile Faucher Lijun Cai Stephen Trowell Amalia Berna Michelle Michie Thomas Wallenius James Darby Wolf Wonjura

Email - Kelly.Hill3@sa.gov.au biosecurity built on science

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Making sense of chemical communication in the red flour beetle

Published on Sep 4, 2012

Plant Biosecurity CRC

Stored product insect pests, such as the red flour beetle (Tribolium castaneum), use their olfactory network to detect volatile chemicals with high sensitivity to perform a range of ecological functions such as finding mates, indicating overcrowding and finding suitable food sources. The sensitivity and specificity of their olfactory system relies on odourant receptors (ORs). For grain-infesting beetles like Tribolium, infestations are currently assessed by visual inspection of grain samples, which can be unreliable. We therefore aim to determine which receptors the beetles use to find each other and investigate the viability on integrating these receptors into a commercial sensing system capable of detecting low concentrations of compounds produced by the beetles.