DNA tool breaks this hitchhiker’s disguise

Khapra beetle (Trogoderma granarium) is one of the five highest-priority threats to the Australian grains industry. Internationally, it is considered one of the most damaging pests of stored products and is of quarantine significance. Economic analysis has estimated that the establishment of Khapra beetle in Australia could cost the grains industry at least $500 million annually in trade restrictions.

Identifying a Khapra beetle in a sample of grain can be difficult, requiring extensive training and experience. This is because the beetle will often be a little worse for wear when discovered. The grain acts like sandpaper and sloughs off the beetle’s hairy cover and most of the identifying features. This assumes that the beetle makes it at all – Khapra beetle larvae usually eat their dead adults for dinner.

It can also be difficult to identify the Khapra beetle from other, less harmful Trogoderma species and related Dermestid insects. There are more than 100 described and possibly 50 undescribed species in the Trogoderma group, which includes the Khapra beetle. There are 52 described and relatively harmless Trogoderma species endemic to Australia.

For the past three years, senior entomologists at the Department of Agriculture and Food, Western Australia, (DAFWA) have been working on the CRC for National Plant Biosecurity Trogoderma diagnostic project. There are three elements to the project: developing protocols for DNA identification of different Trogoderma species, establishing a national Trogoderma laboratory and creating an international Trogoderma reference collection.

The overall aim of the project is to protect Australia’s valuable grain export market by identifying unwanted Trogoderma visitors as quickly as possible to facilitate eradication and to distinguish them from similar native, but non-destructive, pests found in grain leaving our shores.

Working on the Trogoderma diagnostics project, DAFWA’s Mike Grimm initiated the development of a new set of protocols for molecular laboratory testing in 2007. These protocols are establishing real-time PCR (DNA) procedures that will allow species confirmation in a matter of hours.

Mr Grimm says Khapra beetles are a major threat because they are able to survive in many products and situations and will eat anything they can get their teeth into – grains, spices, herbs, dried fruit, meat, wool and many other products coming from overseas – so they can easily ‘hitch a ride’.

“Their capability to stay inactive in larval stage for years means even empty containers may have a healthy population of Khapra beetles capable of infesting anything loaded in it,” he says.

While there has only been one Khapra beetle incident in Australia (found by owners in furniture imported in a shipping container and successfully eradicated), Mr Grimm says the risk is great.

“What we are talking about is protecting the hard work by Australian farmers in producing a valuable commodity for this country,” he says. “Misidentification of exotic pest species poses just as much of a threat to our industry through the imposition of trade barriers as having Khapra beetle in this country.”

Mark Castalanelli has been working on the new DNA protocols for the CRC as part of his PhD project, through Curtin University’s Western Australia Biomedical Research Institute.

The extraction technique he has developed in Australia allows DNA to be taken without physical damage to the specimen. In the past, it had to be crushed and ground to extract DNA. By keeping the specimen intact, visual morphological identification can take place.

“We can’t have DNA identification without morphological identification,” Mr Castalanelli says. “One backs up the other and are equally important when deciding if an intercepted beetle sample is Khapra beetle or not.

“Once we are able to positively identify the Khapra beetle we need to test as many Trogoderma, Trogoderma-related and other stored grain pest species as possible to exclude the false positive results,” he says.

There is still a lot of work to be done since the protocol needs to be verified by independent laboratories; it is already being evaluated by the United States Department of Agriculture. Following its successful acceptance, the protocol may be included in the international Khapra beetle identification protocol.

In conjunction with this work, the CRC has established a national Trogoderma laboratory and international reference collection, which Mr Grimm says is the first facility of its kind for this pest in the world. “Other countries have offered their support in bringing it together,” he says.

Trogoderma laboratory project manager Dr Oonagh Byrne, a molecular entomologist with DAFWA, says the new diagnostics laboratory will be world leading through its use of a suite of tools, including DNA screening with non-destructive DNA extraction methods and imaging using photomontage and the web-based Pests and Diseases Image Library (PaDIL), and Lucid keys (a computer-based identification system).

“The diagnostic equipment purchased to date – the real-time PCR machine and the automated liquid handling system – are widely used in other DNA diagnostics, which is beneficial for other species and many people know how to use them,” she says. But the equipment is the easy part.

Also required are the new testing protocols, accrediting the laboratory and building the collection of species characteristics – through DNA profiling, the morphological validation and development of Lucid keys and an image library of species from all over the world. It is a multi-faceted approach that will take time to complete.

As part of this project, diagnostic entomologist and Trogoderma specialist Andy Szito is building the international reference collection of Trogoderma specimens by visiting collections around the world. Having travelled in Europe and the United States on his mission, this year he will visit France and Russia to view significant collections and he hopes to visit Asia and the Indian subcontinent next year.

After examining thousands of specimens, Mr Szito has morphologically identified more than 100 Trogoderma species, which will back up the DNA profiles.

Locally, the first national Trogoderma trapping program is complete with pests trapped at 64 grain storage sites around Australia. The samples will be the basis of taxonomic studies and the development of DNA markers of native and non-native storage pest Dermestids, including a closely related but less serious pest, the Warehouse beetle. The trapping program will also establish the biogeographic diversity of the different species. A second trapping program will begin later in 2010.

Dr Byrne says many of the specimens held in collections are old. “While useful for morphological validation and development of Lucid keys, they are often unstable for DNA analysis – meaning DNA profiling is more likely from freshly sourced specimens from the trapping program and from sources such as the Australian Quarantine and Inspection Service.”