Smart Materials for Equipment-Free Molecular Identification of Insect Pests and Viral Vectors

The whitefly species complex Bemisia tabaci represents some of the most economically important agricultural and horticultural insect pests globally. Its members cause significant physiological damage directly through feeding, and indirectly on the photosynthetic capacity of plants through the large quantities of honeydew produced encouraging sooty mould growth on leaves. The greatest economic impact is, however, caused by its members collectively vectoring > 300 different species of plant virus. These include Tomato yellow leaf curl viruses, Cucurbit yellow stunting disorder, Cucumber vein yellowing virus, Cassava brown streak viruses and Africa Cassava mosaic viruses. These viruses result in significant economic losses through damage to high value crops across the Mediterranean (tomatoes, cucumbers), southern USA (tomatoes, cucumbers, sweet potato) and Africa (cassava, sweet potato). While B. tabaci is not yet established within the UK it is recognised as a serious and growing threat to the horticultural industry. The UK is currently a regulated B. tabaci pest-free area and a central tool to maintaining this status is rapid and accurate identification of B. tabaci.

Yellow sticky traps are a key component of the Defra recommended measures for monitoring for incursions and outbreaks of B. tabaci in the UK. Insects stuck on traps are difficult for non-entomologists to identify due to similarities with non-quarantine species such as Bemisia afer and the common glasshouse whitefly Trialeurodes vaporariorum, especially if damaged during trapping. As a result, traps are sent to laboratories for identification by specialist entomologists using either low magnification microscopy, molecular diagnostic tools or a combination of both. The logistical burden of collecting traps and the costs associated with the laboratory analysis, reduces the number of sticky traps used in routine surveillance operations, diminishing its value as a surveillance method. As a result, incursions of B. tabaci are usually observed by inspectors when outbreaks become severe. This makes containment and eradication more difficult and costly and runs the risk that the pest spreads to the point of being difficult or impossible to contain.

Here we propose a novel, smart material - inspired by yellow sticky traps - that comprise an adhesive, biofunctional polymer to trap whiteflies and release DNA passively, that is in turn layered on top of a biosensor core capable of discriminating B. tabaci from other whitefly species and producing a visual signal. These materials can be printed on to disposable paper strips for use in different settings such as shipping containers or contained growth facilities.

We propose a novel, smart material to trap and identify Bemisia tabaci. The device comprises an adhesive, biofunctional polymer to trap whiteflies and release DNA passively and a biosensor core capable of discriminating B. tabaci from other whitefly species. The device relies on the ability of hydrogels to absorb samples into the device through each functional layer in pre-defined order.

The core combines helicase activity with BstI polymerase and Nb.BbvCI nickase activity to unwind dsDNA and trigger a strand displacement amplification. The output is a generic, short single stranded DNA that can unwind a molecular beacon. To provide a thorough understanding of the cascade we will use mathematical models and statistical Design of Experiments to guide the experimental work. The cascade will be assessed for its specificity to the target sequence through challenge with variant oligonucleotides, and oligonucleotide sequences derived from non-quarantine whitefly species.

The outer gel is functional both through the adhesive nature of the polymer chassis and the inclusion of proteolytic enzymes. We will assess the adhesive properties of several adhesive hydrogels for sample collection. We will immobilise recombinant proteases, specifically Proteinase K (which is used to release DNA from insects in current molecular assays, leaving the exoskeleton for confirmatory testing) in the most appropriate polymer and confirm proteolytic activities using the fluorescent substrate DQ Gelatin. To test the ability of the gel to release nucleic acids from insect samples we will apply samples and confirm liberation of nucleic acids using PCR and qPCR.

Finally, the project combines both elements together using non-contact printing of hydrogel biosensors on to a paper support, followed by an adhesive gel overlay providing exquisite control of biosensor location and reducing the volume needed for the device.