Blocking insect digestion to control pests

A study on the intestinal function of insects enhances researchers’ understanding of these animals and could lead to innovative methods for combatting disease and controlling agricultural pests.

By Fábio Reynol

Agência FAPESP – A number of diseases that affect humans, such as dengue fever, Chagas disease and leishmaniasis, and diseases that affect pests that destroy crops, such as cotton, sugarcane and banana, share something in common: they are all caused by insects. 

 

An extensive study carried out at the Universidade de São Paulo Chemistry Institute (IQ/USP) used a unique approach to gain knowledge about different insects, namely, investigating their intestinal function. The study opened up new pathways for innovative pest control methods. 

 

The study is part of a Thematic Project which lasted from 2008 to 2012 and was funded by FAPESP through its Thematic Project Research Support program. 

 

The project was coordinated by IQ/USP professor Walter Ribeiro Terra and the main researcher and vice-coordinator Clelia Ferreira and is a continuation of previous Thematic Projects on the same topic that have been carried out since 1991. The newest project began in 2012 and is expected to end in 2017.

 

Among the main discoveries of the project concluded this year was that hematophagous mosquitoes of the order Diptera all share special trypsins that are fundamental for their ability to digest protein. “This makes this type of trypsin a possible target for the control of all mosquitoes in this group,” said Terra.

 

It is a particularly significant target because Diptera encompasses the genera Anopheles, Aedes and Culex, which include insects that are vectors of diseases such as malaria, yellow fever, dengue fever and filariasis. 

 

According to Terra, inhibiting the dipteran trypsin could be an effective way to control these diseases because it would block the insects’ digestive processes. To that end, the study also involved a search for chemical inhibitors of the enzymes found. 

 

The methods used included the generation of computer models of three-dimensional images of the molecules. Inhibitor molecules are tested virtually in a digital 3D model of the enzyme to identify candidates that fit into the largest number of spaces or active sites.

 

“The more active sites a reagent binds to, the stronger and more efficient the inhibitor will be,” Terra told Agência FAPESP, explaining that 3D molecular modeling is widely used in the pharmaceutical industry.

 

The blocked enzyme cannot rearrange itself and therefore cannot carry out its function (breaking down other molecules) in the digestive process. The mosquitoes then cannot absorb the nutrients they need and subsequently die. 

 

Physiological study of Rhodnius prolixus, the triatomine vector of Chagas disease, has always been difficult, and observing its intestinal function was an obstacle for researchers. 

 

Terra’s team got around this problem by finding a similar insect, Dysdercus peruvianus, commonly known as the chinch bug, which feeds on cotton. Analysis of the transcriptome (the portions of the genome that encode proteins) of this insect revealed details that might also be relevant for the triatomine bug and thus could provide targets for the control of this insect. 

 

The sugarcane industry could also benefit from this study. Cathepsin L, a digestive enzyme typical to many beetles, was isolated from Sphenophorus levis, a beetle whose larvae attack the root system of sugarcane plants. The enzyme was cloned, expressed and characterized using synthetic substrates and inhibitors. The 3D structure of the same enzyme found in Tenebrio molitor, the beetle known as the mealworm, was also resolved. 

 

“The greatest challenge in determining the three-dimensional structure is the crystallization of the protein because if it doesn’t crystallize, we can’t get the structure,” said Terra, clarifying that many proteins cannot be coaxed into forming crystals, making their three-dimensional visualization impossible.

 

Structure of development

 

The IQ-USP Thematic Project focused on one particular structure in the insects’ intestinal systems, the peritrophic membrane. 

 

This minuscule tube is known to be related to digestive efficiency, but its function remains poorly understood. Some of its hypothetical functions were tested in model insects, and it was discovered to play an important role in insect development. 

 

Insects in which the peritrophic membranes had been inhibited didn’t develop as well as their normal counterparts. Some plants produce natural substances that attack this membrane and keep them from being devoured by the insects. “This makes this structure an important target for innovative control processes,” observed Terra. 

 

The Thematic Project also led to significant advances in knowledge about the evolution of species. Aside from its role as a possible target for the control of domestic houseflies, the Cathepsin D enzyme is also present in humans and other animals with very acidic digestive systems that can process bacteria-rich foods.

 

“The interesting thing about this discovery was the proof that the same evolutionary adaptation occurred twice, independently, in the fly and in humans,” said Terra. 

 

Another important advance was related to insect morphophysiology. One study of the chinch bug Podisus nigrispinus, which preys on other insects, showed that what was previously referred to as extra-oral digestion being carried out by this insect is in fact a dispersion of the tissues of its prey as a result of the action of a salivary substance. Digestion itself happens inside the insect’s intestine. 

 

The discovery, published in the Journal of Insect Physiology, received a special mention from one of the magazine’s editors. “He wrote that the results of this study should lead us to rethink our ideas about digestion that happens outside the body,” said Terra, pointing out that the team was especially proud of this acknowledgment. 

 

The project also identified lysozyme as a critical enzyme in digestion by flies that attack fruit, as well as found that trehalase is crucial for crop pest caterpillars and determined that beta-glucanases, which are absent in mammals, are involved in digestion and immune function in insects. All of these molecules represent potential targets for insect control. 

 

Over 1,300 citations

 

The results of the four years of studies are reported in 20 publications and four book chapters. The lab work from the project has been cited 1,357 times in the scientific literature over this period. Three master’s theses, six doctoral dissertations and two post-doctoral theses were written as part of the Thematic Project. The project received five FAPESP undergraduate scholarships, one doctoral scholarship and two post-doctoral scholarships. 

 

The Thematic Project also promoted partner studies with a number of national institutions, including the Universidade Federal de Santa Catarina (UFSC), the Universidade Federal de Lavras (UFL), the Universidade Federal de São Carlos (UFSCar), the National Science and Technology Institute (INCT) for Molecular Entomology, of which IQ-USP is a member, and the Luiz de Queiroz Graduate School of Agriculture (Esalq), also part of USP.

 

The group is also participating in an international consortium for the genetic sequencing of Rhodnius prolixus, the results of which are still under analysis; according to Terra, the results of this endeavor should lead to many practical applications.