And it came to pass, in the summer of 1981, that a plague of larvaedescended upon trees in the northeastern U.S.
Almost overnight, the hairy grubs hung like strange fruit from leafybranches and twigs of forests, orchards and suburban gardens. In aweek, these finger-length, leaf-devouring gypsy moth larvae couldtotally denude an apple tree.
"The gypsy moth [Lymantria dispar] is a very tough insect tocontrol," said biochemist Francis Rajamohan, of Ohio StateUniversity in Columbus. "It's a major pest in the U.S. and Canada,"he told BioWorld Today, adding that in the U.S. alone, this voraciousinsect threatens 311 million acres of forest land with defoliation, atan estimated economic cost of $140 million a year.
Salvation takes the form of a ubiquitous soil bacterium, Bacillusthuringiensis, (B.t.) which synthesizes a crystalline toxin lethal toinsect life.
That toxin, said Rajamohan, "is probably a kind of survivalmechanism, which allows the bacterium to go on multiplying, afterbeing eaten by an insect."
Also in 1981, the U.S. Environmental Protection Agency approvedthe intracellular exotoxin protein synthesized by B. thuringiensis, var.Israelensis, for killing mosquito larvae. On the downside, B.t.'sinsecticidal power lasted only a few days, requiring repeatapplication, and promoting insect resistance. Researchers have beenat work ever since to overcome these drawbacks in the otherwiseecologically attractive bug killer. Meanwhile, they are extending itsrange to other species of insect pest, notably, nematodes, roaches andhouse flies.
Among the U.S. companies now marketing the insecticidal toxin areAbbott Laboratories, N. Chicago; Sandoz Inc., San Diego; EcogenInc., Langhorne, Pa.; Monsanto Corp., St. Louis.
Rajamohan said he "developed some process optimizationtechnology for the control of mosquitoes, using Bacillus sphericus.It's now produced commercially by the Indian firm of SouthernPetrochemical Industries, Madras."
He is first author of a paper in today's Proceedings of the NationalAcademy of Sciences (PNAS), dated Dec. 10, 1996. Its title: "Proteinengineering of Bacillus thuringiensis d-endotoxin: Mutations atdomain II of Cry[stal]IAb enhance receptor affinity and toxicitytoward gypsy moth larvae."
Toxin Hits Bugs In Their Guts
"B.t. works," Rajamohan explained, "by binding to receptors in thelarval midgut. "So we took that gut, ground it up and prepared avesicle _ actually, the microvillae projecting outside the gut'smembrane. That's where we got the receptors."
The university's laboratories of biochemistry and biophysics,directed by molecular geneticist Donald Dean, have been doing site-directed mutagenesis to find out which amino-acid residues of thetoxin bind to those receptors, and which disrupt the gut membrane'spermeability.
Before turning to the gypsy moth, they investigated the tobaccohornworm (Manduca sexta) and the cotton bollworm (Heliothisviriscens). Now the lab is checking out a still more important croppest, the cabbage looper (Tricoplucia ni).
"Most companies with which we are in contact," Rajamohanobserved, "are more interested in the looper and the cotton wormthan in the gypsy moth." The labs are now aiming to combine theirspecies-specific toxin mutants into a single molecule for attacking arange of major insect pests.
"We want to make this into a kind of super-bug killer," he said,"which the university would then seek to patent."
Their strategy is three-fold:
* "To develop a novel toxin that has the potency to kill those insectsinstantly, thus reducing their resistance against it.
* "Combine the different regions of the various amino acids essentialfor killing one particular insect with another region that is essentialfor killing another insect.
* "That way, make a hybrid toxin, which can kill two or three insectsin one application."
Three At One Blow Is Aim
In pursuit of this goal, Rajamohan and his co-authors have created, sofar, three mutants of the wild-type B.t. toxin, by amino-acidsubstitutions in structural loops of the 3-D crystal protein: This triplemutant replaces _ at various positions in the one molecule _asparagine with alanine, alanine with glycine, leucine with serine.
The alanine and glycine switches increased toxicity toward gypsymoth larvae eight-fold, and enhanced binding affinity to the midgutfour-fold. Toxicity to gypsy moth neonates went up 36-fold,compared to wild-type.
"These residue changes," Rajamohan pointed out, "make the toxinsgo to the receptors in an easier way, and increase the binding affinity.Once it does so, it kills the insect much more quickly." He observed:"It's about five or six times more active in Manduca sexta."
Then they ran another experiment, testing replacement of aphenylalanine residue in the wild-type B.t. toxin with one afteranother of all 20 essential amino acids. "We found one region,"Rajamohan recalled, "where putting other residues all reducedreceptor binding, and toxicity as well. So we know for sure thatNature selects the best residue for that region."
The PNAS article concluded: "These second-generation mutantproteins would be successfully used to express in transgenic plants,and to delay the B. thuringiensis resistance in insects." n
-- David N. Leff Science Editor
(c) 1997 American Health Consultants. All rights reserved.