Non-taxonomic biochemical tools for
enhancing decision making at the border
– assessing viability
Ilia Iline, Craig Phillips (presenting author), Max
Novoselov (AgResearch, New Zealand),
Alan Flynn, Dave Voice and Sherly George
(MAF Biosecurity New Zealand)
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Abstract
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Non-taxonomic biochemical tools for enhancing decision making at the border
Ilia Iline1, Craig Phillips1,2, Max Novoselov1, Alan Flynn3, Dave Voice4 and Sherly George3
1AgResearch, PB 4749, Christchurch 8140, NZ; and B3 (www.b3nz.org)
2Presenting author (craig.phillips@agresearch.co.nz)
3MAF Biosecurity New Zealand, PO Box 2095, Auckland, NZ
4MAF Biosecurity New Zealand, PO Box 24, Lincoln, NZ
In addition to the daunting task of making taxonomic identifications of organisms that are intercepted at the border,
regulatory authorities must also evaluate other characteristics of intercepted organisms such as their viability.
When the intercepted organisms are sessile (e.g. eggs, pupae, scale insects) the challenge of rapidly and
objectively assessing viability is significant, and the costs of incorrectly interpreting viability can be high. For
example, if regulated scale insects associated with a consignment of imported produce were incorrectly assessed
as being alive, the consignment may be unnecessarily rejected and either destroyed, fumigated or returned to the
exporter. Alternatively, if the scale insects were incorrectly assessed as being dead, they could be allowed to
cross the border into a new region or country, thus risking the establishment of a new pest. Viability assessments
of sessile organisms are generally made using morphological criteria that demand high levels of user-experience
to obtain reliable results. Moreover, morphological observations are often difficult to quantify or otherwise record in
an objective manner, and this can cause problems when border authorities need to defend their viability
assessments. This presentation describes a series of rapid, sensitive, inexpensive, simple-to-use biochemical
tests that have been developed primarily for use by border authorities to make viability assessments of sessile
organisms. The tests are also proving useful in other applications such as validating the efficacy of treatments that
have been applied to consignments prior to export. The tests perform well across a diverse range of arthropods,
and give easily interpreted, contrasting colour reactions depending on the viability of the organism; these can
readily be quantified using a spectrophotometer or by measuring pixel values. The biochemical data have also
shown that the physical appearance of sessile organisms can sometimes be misleading with respect to their
viability. This work illustrates how relatively small research projects can help to increase the speed, reliability,
objectivity, transparency, defensibility and overall efficiency of border biosecurity processes.
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Quarantine inspections at the border
lymantriid
eggs & larvae
on imported
used car
• Border authorities often detect organisms
in or on imported goods & conveyances
• (In NZ) infested items are held at border
while MAFBNZ evaluates risks posed by
the intercepted organisms
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Quarantine inspections at the border
• Strong commercial imperative not to slow trade
– Many imports (e.g. fresh produce) are perishable
– MAFBNZ assesses risks within 1 working day
• Detection of high risk organisms can mean
imported items must be (re-)treated, destroyed,
or returned to the exporter
– Major financial implications
pentatomid eggs on
imported fresh beans
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Evaluating risk
• First two questions asked
1. Is the organism alive?
2. What species is it?
• Most realise that answering #2 can be
difficult or impossible
• Less well recognised that answering #1
is also often problematic
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Insect eggs are often detected on
imported fruit and vegetables
Nitidulidae & Lonchaeidae
Drosophilidae
1mm
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Insect eggs are often detected on
imported fruit and vegetables
Blattodea
Coccinellidae(?)
1mm
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Assessing viability - rearing
• Rearing / hatching is an obvious possibility, but involves
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–
–
Unacceptably long waiting periods
Significant labour costs
Specialist facilities, including containment labs
Detailed biological knowledge
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Assessing viability using
morphological criteria
• Scoring morphological criteria has been the most
practical & frequently used method, but
– Demands high levels of experience & expertise to obtain reliable
results
– Results difficult to quantify & defend
– Morphology can be misleading
Apparently dessicated
weevil eggs that
subsequently hatched
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The challenge for research
• Develop a viability assessment method that is
– Fast
– Objective
– Easy to use
– Inexpensive
– Quantifiable & defensible
– Sensitive enough for very small organisms
• The marker used must degrade quickly after death
– Too slow = false positive
• Insensitive to presence of host plant material
– Risk of false positives
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A biochemical approach
• The main idea
– Many biomolecules (e.g. metabolic enzymes)
should decline in concentration after the death
of an organism
– Develop a biochemical stain that indicates
high (alive) or low (dead) concentrations of
certain biomolecules
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Results to date
• A very straightforward test
– Crush the organism in the stain
– Wait for 3 – 7 minutes at room temperature
– Look for a staining reaction
• Several different staining systems have been developed
• Some differences in suitability to different applications
– E.g. differences in sensitivity, & in post-death degradation rate
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Test results are quantifiable
• Colour response can be quantified using a
spectrophotometer, or a digital camera + image analysis
software
Aspidiotus nerii
(oleander scale)
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Tests can be conducted in tubes or on
slides
Hemiberlesia rapax (greedy scale)
Aspidiotus nerii (oleander scale)
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Evaluating the validity of
biochemical test results
• Alive versus heat inactivated
• Treated versus untreated
– Hydrogen cyanide, methyl bromide, phosphine, cool storage
• Biochemical c.f. morphological c.f. rearing
• To date, 9142 evaluations conducted on invertebrates
spanning 18 Orders, 41 Families, 62 species and all
insect life stages
• Minimal test responses to host plant material of apple,
kiwifruit, nectarine, orange, potato & rock melon
– But positive responses to fungi associated with rotting plant
material
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MAFBNZ currently assessing
suitability of test for its application
• Begin using biochemical test alongside current
morphological assessments to gain confidence and
ensure results are robust
• Implement quality control systems
• Aim to have use of the test accredited (IANZ 17025)
Even relatively small
research projects can help
to increase the overall
efficiency
of border biosecurity
processes
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Future steps & challenges
• Publication
• Overcoming technical limitations to preparing tiny
organisms for testing (handling, crushing, staining)
• Development and production of a test kit
• Commercialisation versus open-source
– currently protected by 2 provisional patents
• Other applications (e.g. meeting export pre-clearance
requirements, testing efficacy of treatments,
maintenance of cultures)
• Uptake by overseas quarantine authorities
• Lots of ideas for additional diagnostic tests…
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Acknowledgements
Thanks to Mark McNeill, Nicola Richards,
Gill Worth (AgResearch, Lincoln), and
Lalith Kumarasinghe, Barney Stephenson,
Kelly McAlpine and Rory Morrison
(MAFBNZ) for assistance with various
aspects
The work was funded by B3 and MAFBNZ
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