Researchers studying how insect predators such as the convergent lady beetle (Hippodamia convergens) prey on pest insects may want to discern whether they feed on eggs, larvae, or adults of their prey. While observing these interactions can be difficult, a new method for marking prey with a vertebrate protein and testing for it in the gut contents of predators could now allow these stage-specific feeding events to be more easily detected and studied. (Photo credit: Russ Ottens, University of Georgia, Bugwood.org)

For researchers on the hunt for beneficial insects that will prey on insect pests, the search can at times be like looking for the proverbial needle in a haystack. A parasitoid wasp, for instance, might target the eggs of a single species but leave its larvae and adults alone. Or vice versa. Or some other combination. If experimental tests can’t be equally specific, then these ecological interactions can be difficult to unravel.

Polymerase chain reaction (PCR) assays can be useful in this effort, but they typically detect an individual species’ DNA. Used in gut content analysis, PCR can show an insect predator preyed on a particular species but not which life stage. An alternative, however, known as ELISA (enzyme-linked immunosorbent assay), can open doors for some creative experiments, and a pair of entomologists in Arizona have conducted a successful proof-of-concept test that shows ELISA can be used in gut-content analysis to detect stage-specific feeding. Their methods are detailed in a report published last week in the open-access Journal of Insect Science, in the publication’s new “Protocols” section.

“The major advantages of this technique over the conventional PCR assay approach is that it can be modified to study all sorts or feeding activities, without the need to develop a pest-specific gut assay, and that it is much easier and less expensive than PCR,” says James Hagler, Ph.D., research entomologist at the U.S. Department of Agriculture-Agricultural Research Service in Maricopa, Arizona, and co-author on the paper with the University of Arizona’s Ayman Mostafa, Ph.D.

ELISA has been deployed in entomological research since the 1990s, frequently as an advanced form of mark-release-recapture and mark-capture dispersal studies. (Just last year, researchers employed it in a new, non-lethal method for marking and tracking bees.) Hagler has used ELISA for studying insect feeding behavior for many years, and he dubbed the practice the “universal food immunomarking technique” in a review published earlier this year in Annals of the Entomological Society of America.

Here’s how it works: The food item (plant or prey) is sprayed, fed, or otherwise exposed to a unique vertebrate protein marker (e.g., rabbit or chicken blood serum). Then, insect predators that may have consumed the food are collected and analyzed via a chemical test that reacts with the marker protein if present.

Hagler and Mostafa put this method into action to see if they could detect predation on eggs of the western tarnished plant bug (Lygus hesperus) and whether it could be deployed in a feeding-choice study. They marked L. hesperus eggs with rabbit and chicken protein—applied with a fine-tipped paint brush—and offered them in petri dishes to two predator beetle species, Collops vittatus and Hippodamia convergens (also known as the convergent lady beetle). In the second part of the study, Hagler and Mostafa placed L. hesperus eggs in two different setups, one exposed on the surface of an artificial stem and one concealed beneath a thin film to mimic the western tarnished plant bug’s egg-laying behavior (beneath the surface of plant tissue). The eggs in each exposure were marked with different proteins. Then the same predator species were placed in a cage with the eggs and allowed to feed as they chose.

To test whether the universal food immunomarking technique could be applied in a feeding-choice experiment, researchers placed western tarnished plant bug (Lygus hesperus) eggs in two different setups, one exposed on the surface of an artificial stem and one concealed beneath a thin film to mimic the bug’s egg-laying behavior (beneath the surface of plant tissue). The eggs in each exposure were marked with different vertebrate proteins. Then two beetle predator species were placed in a cage with the eggs and allowed to feed as they chose. The gut contents of the predators were then tested for the protein markers. A diagram illustrates the experimental setup on the left; photos A and B show the egg exposures. (For illustrative purposes, egg densities in the photographs are greater than the densities used in the predator feeding choice study.) (Image originally published in Hagler and Mostafa 2019, Journal of Insect Science)

Using ELISA in gut-content analysis of the predators within three hours after feeding was successful in detecting the protein marks, and the feeding-choice study showed the predators fed far more on the exposed eggs. The latter result supports they view that L. hesperus lays its eggs beneath plant tissue to avoid predators, and it also illustrates the utility of the UFIT and ELISA techniques in such an experimental design, says Hagler.

“The beauty of the method is that you can design ‘manipulative’ studies using the technique,” he says.

Hagler and Mostafa’s tests were conducted in the lab and in greenhouse cages, but Hagler says he wants to take it out in the field next, for instance to place marked sentinel L. hesperus eggs on plants enclosed in large field cages and then collect predators within the cages to see if they ate the marked eggs.

“Also, I plan to conduct field cage studies where I mark the Lygus eggs with rabbit protein, nymphs with chicken protein, and adults with rat protein,” he says. “Then we’ll examine the predators for the presence of all three marked prey items.”

In the meantime, Hagler says he published the proof-of-concept paper in the Journal of Insect Science now to share it with fellow researchers, to provide them with a new potential tool in their toolbox. The UFIT method, he says, “can be used to study all types of arthropod feeding activities.”