Global gene guild gets the buzz on pesticide resistance

It seems that globalisation works for genes too. Over the past half century, a global guild of the geneticist's favourite fruit fly, Drosophila melanogaster, has been using science's global networks to smuggle a souped-up, multi-purpose survival kit to its six-legged subscribers.

It's a gene called Cyp6g1, and it confers resistance not just to the archetypal synthetic insecticide DDT, but to a slew of synthetic slayers.

The list includes some of DDT's organochlorine cousins, and various organophosphates, carbamates and neonicotinoids -- it can even defuse new-wave insect-growth regulators.

Melbourne University geneticist Prof Phil Batterham said the discovery has enormous implications for the development of new insecticides, and for predicting and managing pesticide resistance.

The discovery also helps to explain the phenomenal capacity of insects to mutate and defeat new pesticides, including compounds they may not even have been exposed to in the field. Batterham said it also confirmed that some pesticides inflicted what the military euphemistically terms "collateral damage" on non-target species,

A team of Australian, British, French and US researchers, including Batterham, published its findings in the international journal Science this week.

One of Batterham's graduate students, Dr Phil Daborn, now at the University of Bath, was the lead author for the study.

It was Daborn's Bath team that identified Cyp6g1, a member of the cytochrome P450 gene family, as the sole source of these multiple, diverse resistances in Drosophila. Researchers might have expected that the manifold resistance would depend, not on a single gene, but on multiple, one-for-one resistance genes for each class of pesticides.

The mutation was identified by two of Batterham's postgraduate students, Trent Perry and Michael Bogwitz.

They found that a transposon or mobile DNA element called Accord had inserted itself into the promoter region of the Cyp6g gene -- the promoter contains 'switches' that turn the gene on and off.

Perry and Bogwitz discovered that the Accord transposon actually has another promoter embedded within it.

Diverse array of toxins

Batterham said it was not yet clear if the embedded promoter acted as an inbuilt turbocharger, or whether the mutation disrupted the gene's 'off' switch, leaving it continuously switched on.

But the result of the mutation is a hundredfold increase in the output of the Cyp6g gene's toxin-busting enzyme -- a veritable Swiss army knife of a molecule that can inactivate an astonishingly diverse array of toxins, including a substantial number of the 20th century's most potent synthetic pesticides.

Batterham said the Cyp6g gene was just one of 90 genes assigned to the cytochrome P450 gene family when the Drosophila Genome Project catalogued all of the fruit fly's 13,061 genes.

"Interestingly, Cyp6g has a human equivalent, called cytochrome P450K, which also similarly broad activity," he said.

"The drug industry has to take note of this enzyme, because it produces an enzyme that degrades least 50 per cent of at all commercial drugs. New drugs must not only be pharmacologically effective, they must evade degradation cytochrome P450 enzymes in the liver.

"In Drosophila, we know very little about what all the other genes in the cytochrome P450 family are doing.

"One of the real lessons from this study is that if we want to control insect pests, we must understand their defences better. We're working hard in our research centre to analyse the functions of this family of genes, and other detoxification genes."

Batterham said the study also highlighted the unpredictable and unrecognized impacts of pesticides like DDT on the environment, and on living organisms.

"In the most famous book on environmental science, Silent Spring, Rachel Carson expressed her concerns about the wider effects of DDT on non-target species," Batterham said.

"Here we have an example: Drosophila is not a pest, so it wasn't directly targeted by DDT, yet it has become resistant to DDT, and now exhibits cross-resistance to a wide range of synthetic pesticides that it has never seen in the field, or is yet to see.

"One of the groups of compounds it is resistant to are the nenocotinoids, a class of insect neurotoxins that are used only in the US and Australia, to control fleas in cats and dogs."

No heavy metabolic burden

In the Science paper, the researchers pointed out that the resistance gene has been propagated through the world's Drosophila research laboratories, which routinely exchange and interbreed strains with different genetic traits.

The gene has been maintained intact, in the absence of any selection pressure pesticides within the laboratory, suggesting that the mutation imposes no heavy metabolic burden on the fly that would result in selection pressure against it -- it is now permanently fixed and stable in the Drosophila gene pool.

There was now an urgent need, said Batterham, to determine whether other insects possess similar genes to Cyp6g -- the only other insect pest that has been 'genomed' to date is the mosquito that transmits malaria, Anopheles gambii.

"We're working to isolate P450-family genes from two of Australia's major insect pests, the cotton bollworm, Helicoverpa armigera, which is probably the world's worst crop pest, and the sheep blowfly Lucilia cuprina," Batterham said..

"We're also continuing work on Drosophila, because it's the only insect so far in which we can study the activity of genes by knocking them out."

Batterham said the fact that the Accord transposon was involved in the Cyp6g mutation was unsurprising, because transpons were responsible for about 99 per cent of all mutations in Drosophila fruit flies.

But the discovery underlines the fact that insects are endowed with a formidable array of genetic mechanisms that allow them to defeat pesticides or natural toxins.

Batterham's colleague, Dr David Hackel, had identified the mutation responsible for resistance to one of the Bt bacterial toxins, which are now widely in transgenic crops to protect them against leaf-chewing and stem-boring pests.

"David discovered that the Bt-resistance gene in the US cotton pest Heliothis virescens, was also due to the insertion of a transposable element that knocked out the gene," Batterham said.

"The gene encodes the protein in the insect's gut wall where the Bt toxin attaches and kills the cells of the gut lining -- the mutation deletes the protein, so the insect becomes resistant.

Rational design

"It costs around $100 million for a company to go through all the trials and bring a new pesticide to market," he said. "So much of the screening and testing of new compounds is done with no understanding of what the chemical does to the insect, and what means the insect has to defend itself.

"We've now reached a point where we can look at how insects develop resistance, so we can take account of these defences and design new insecticides to circumvent them.

"It's odd that some of the big corporations that are actively involved in rational drug design for humans don't use rational design for their pesticides."