Low doses of insecticide trigger neurodegeneration in insects

Insect populations are consistently declining around the world and intense use of insecticides is suspected to play a role. This week, a study published in the Proceedings of the National Academy of Science USA by researchers at the University of Melbourne, Baylor College of Medicine, Texas Children’s Hospital and other institutions shows that very low doses of imidacloprid, the world’s most used insecticide, trigger neurodegeneration and disrupt body-wide functions, including energy production, vision, movement and the immune response, in the laboratory fruit fly, Drosophila melanogaster.

Given that imidacloprid’s mechanism of action is similar among insects, and that other insecticides also seem to trigger similar cellular responses, these findings have potential implications for millions of insect species, most of which are beneficial as pollinators, pest control, nutrient recyclers and food sources for other animals.

“Researchers have studied the effects of high doses of insecticides, but the consequences of exposure to low doses have not been investigated in detail,” said co-author Dr. Hugo Bellen, Distinguished Service Professor of Molecular and Human Genetics at Baylor and member of the Jan and Dan Neurological Research Institute at Texas Children’s Hospital.

“Although many studies have shown that low doses of insecticides can affect insect behavior, they have not uncovered whether insecticides trigger changes at the cellular and molecular levels,” said first author Dr. Felipe Martelli, currently a research fellow in the School of Biological Sciences at Monash University in Melbourne, Australia. “The goal of this work was to have a better understanding of the effects of low doses of the common insecticide imidacloprid at the cellular, physiological and behavioral levels.”

During the development of this project, Martelli worked in the laboratory of Dr. Philip Batterham, corresponding author of this work and professor in the faculty of science at the University of Melbourne, as well as in the Bellen laboratory at Baylor.

It all starts in the brain

The researchers began by studying the effects of imidacloprid in fruit fly larvae. In the field, the insecticide is sprayed at concentrations up to 100 parts per million (ppm). In the lab, they tested lower doses, identifying 2.5 ppm as a dose that reduced the movement of fruit fly larvae by 50 percent after a 2-hour exposure, an indication of toxicity to the larvae.

Although being exposed to 2.5 ppm of imidacloprid for 2 hours did not seem to have a long-term effect on the larvae’s development into adults, the exposure did trigger a cascade of events with widespread effects.

Previous research has shown that imidacloprid binds to nicotinic acetylcholine receptors in the central nervous system. These nicotinic receptors are conserved or similar among insects, suggesting that the findings in the fruit fly likely apply to other insects with similar receptors.

“We discovered that imidacloprid did bind to the nicotinic receptors in the larvae’s nervous system, causing a long, enduring influx of calcium ions into the neurons. Having too much calcium inside the neurons damaged the mitochondria, the energy-producing structures of the cell. This led to the accumulation of significant amounts of reactive oxygen species (ROS), or free radicals inside the brain that triggered a cascade of damaging events that spread to many other tissues,” Martelli said.

ROS are highly reactive molecules that can damage lipids, DNA and proteins with harmful consequences for the organism. The body responds to ROS with antioxidants to neutralize them, but in this case, there is an imbalance between the ROS and the antioxidant responses, leading to oxidative stress. In larvae, the effect of imidacloprid started in the brain where it triggered oxidative stress and damage that disrupted the lipid landscape in metabolic tissues, altering lipid or fat distribution in various organs, including liver and kidneys.

“Lipids are much more than energy storage. They play many important roles in the body, from being a crucial part of the integrity of cell membranes to working as messenger molecules or in hormone synthesis,” Battenham said. “In addition to lipid alterations, we also observed that imidacloprid triggered changes in the activity of genes related to metabolism, energy production, detoxification and the immune response. The overall physiology of the larvae was systemically impaired.”

Other experiments showed that simultaneously treating larvae with imidacloprid and the antioxidant NACA significantly reduced the alterations triggered by the insecticide, further supporting the role of oxidative stress as a mediator of the damage caused by imidacloprid.

Low doses of imidacloprid also affect adult fruit flies

Martelli worked for six months in the Bellen lab studying the effect of low doses of imidacloprid on adult fruit flies.

“Bellen’s is one of the leading labs working with the fruit fly to study the genetic causes of neurodegenerative diseases. Our work has shown that imidacloprid and other insecticides lead to neurodegeneration, and we anticipated that this collaboration would bring new insights into the mechanism,” Martelli said.

The researchers found that adult flies that were chronically exposed (25 days) to low doses (4 ppm) of imidacloprid became blind and also developed movement problems that affected their ability to climb, suggesting that the insecticide was triggering a neurodegenerative process. These flies also had a shorter life span than those not treated with the insecticide.

“When we looked closer at the light-sensing cells in the adult fly’s retina, we found that glial cells, which provide support and protection to neurons, had progressively accumulated vacuoles and a significant number of defective mitochondria, indications that the glia were dying,” said Bellen, who also is an investigator at the Howard Hughes Medical Institute at Baylor.

“As we had observed with the larvae, administering the antioxidant NACA together with imidacloprid also significantly lessened the damage caused by the insecticide. In the adults, NACA improved the flies’ longevity and the damage to the glia, when compared with the insecticide alone,” Martelli said.

“It is concerning that even at low doses, insecticides can cause neurological damage, disrupt energy production and compromise the immune system of insects,” Batterham said. “Those problems can make it more challenging for insects to adapt to other stresses, such as climate change or infections. Our findings emphasize the importance of better understanding the mechanisms of action of insecticides, in particular on beneficial insects.”

Other contributors to this work include Ute Roessner, Thusitha Rupasinghe and Trent Perry (the University of Melbourne), Zuo Zhongyuan and Julia Wang (Baylor College of Medicine), Ching-On Wong (Rutgers University), Nicholas E. Karagas (University of Texas Health Sciences Center, Houston) and Kartik Venkatachalam (Texas Children’s Hospital).

This work was supported by a Victorian Latin America Doctoral Scholarship, an Alfred Nicholas Fellowship and a University of Melbourne Faculty of Science Traveling Scholarship. Further support was provided by University of Melbourne, the Howard Hughes Medical Institute, NIH (NIA) and a National Collaborative Research Infrastructure Strategy initiative under Bioplatforms Australia Pty Ltd.