Kansas State scientist’s study paves ways for new insect control methods

A Kansas State University researcher's work has shed new light on which of two genes present in many insect species should be targeted for new insecticides that will be safer for humans and the environment than those currently used.

K-State entomology professor Kun Yan Zhu, along with a team of scientists at K-State, the Mayo Clinic, Oklahoma State University and China Agricultural University, uncovered the functions of two different acetylcholinesterase or AChE genes in the red flour beetle, which is a serious, globally-distributed insect pest of stored grains and grain products found in grain storage facilities, flour mills, grocery stores, warehouses and homes.

The research team's findings were published Feb. 12, 2012 in PLoS ONE and Feb. 27, 2012 in Scientific Reports, both are online scientific journals.

Acetylcholinesterase (AChE) is a crucial enzyme involved in cholinergic neurotransmission in the nervous system of insects and other animal species. The enzyme breaks down the neurotransmitter acetylcholine at the synaptic cleft (the space between two nerve cells) after acetylcholine has served the function of signal transmission. This keeps the synaptic cleft clear so that next signal transmission can occur.

Commonly used organophosphate insecticides, which are related to nerve agents, are known as anticholinesterases because they are potent inhibitors of AChE. Inhibition of AChE prevents the breakdown of acetylcholine by the enzyme. As such, acetylcholine continues to cause nerve signal transmission, which leads to overstimulation of the nervous system and eventual death of an animal.

For years, AChE was known to be encoded by a single gene in vertebrates and insects. But by 2002, in a breakthrough discovery, a team of researchers led by Zhu found a paralogous or second AChE gene in the greenbug, which is an aphid species that often causes serious damage to sorghum, wheat and other small grains in the world. Since Zhu group's finding of a second AChE gene in the greenbug, scientists have quickly learned that many insect species have two different AChEs.

That earlier work, described in a scientific journal as "a breakthrough in this toxicological riddle...," led to the latest work to determine which of the two genes studied in the red flour beetle is responsible for cholinergic neurotransmission. In turn, that tells the scientists which should be the target for anticholinesterase insecticides and is responsible for insecticide resistance conferred through the target gene mutations.

"After a paralogous AChE was reported in the greenbug in 2002, many scientists identified the paralogous AChE genes in other insect species. It became clear that most insect species possessed two different AChE genes," Zhu said. "So, the question was what the functions of each gene were. Our study provided the first strong evidence that the paralogous gene is responsible for cholinergic neurotransmission, whereas the first AChE gene which scientists have known about for years and now is called orthologous gene does not appear to play an important role in cholinergic neurotransmission." Orthologous genes are the ones originated from speciation events, whereas paralogous genes are the ones diverged from gene duplication events.

"The red flour beetle was chosen for the more recent research largely because of its robust RNA interference (RNAi) responses," Zhu said. "RNAi is a specific and effective approach for loss of function studies in virtually all species of large complex organisms, including animals, plants and fungi. Once RNAi is triggered, it destroys the messenger RNA, or mRNA, of a particular gene. This prevents the translation of the gene into its product. By silencing each of the two AChE genes in the red flour beetle, we would be able to learn the function of each gene by examining the insect responses to the RNAi."

The scientists found that silencing the paralogous gene in the red flour beetle larvae led to 100 percent mortality and increased larval susceptibility to well-known anticholinesterase insecticides, including carbaryl and malathion. In contrast, silencing the orthologous gene did not lead to insect mortality and increased insecticide susceptibility, but delayed insect development and reduced female egg laying and hatching.

"As consequences," Zhu said, "we observed relatively lower numbers of pupae and adults in the offspring, and determined that such RNAi effects for the orthologous gene were carried through the female rather than the male." Thus, the orthologous gene seems to play non-cholinergic functions but is important for insect growth, female reproduction and embryo development.

That finding reinforces the idea that the AChE encoded by the paralogous gene should be a target of anticholinesterase insecticides, rather than the AChE encoded by the orthologous gene, for the most insect species possessing both AChE genes. However, for a few insect species, such as house fly, which possess only orthologous AChE gene, their control by anticholinesterase insecticides should still relay on the inhibition of AChE encoded by the orthologous gene.

"Based on some unique structures of the enzyme encoded by the paralogous gene, which are not found in mammals and other vertebrates, researchers could potentially develop insect specific and environmentally safe insecticides for control of agricultural insect pests, human and animal disease vectors, and residential insect pests," said Zhu, who has been studying insecticide toxicology since 1987. Indeed, recent studies led by Yuan-Ping Pang at Mayo Clinic have demonstrated that AChE encoded by the paralogous gene is a promising target for developing such insecticides for safer insect pest control.

Other collaborators on the recent project were Yanhui Lu, who was a visiting scholar in Zhu's K-State laboratory from 2008-2010 from China Agricultural University (CAU) in Beijing.
She works now at Huazhong Agricultural University in China, Xiwu Gao of CAU, Yoonseong Park, at K-State, Yuan-Ping Pang, Mayo Clinic, and Haobo Jiang, Oklahoma State University. Two of Zhu's former graduate students Xin Zhang and Jianxiu Yao, are now postdoctoral research associates in the Division of Biology and the Department of Plant Pathology, respectively, at K-State.