A fluorescent dye designed for use as an anti-theft tool and aid in forensic criminal investigations also works well for marking insects in mark-release-recapture efforts, a new study shows. Shown here are western tarnished plant bugs (Lygus hesperus) under white light (left) and ultraviolet light (right). The individuals in the top row were marked with a fluorescent dye called SmartWater; those in the bottom row were control specimens “marked” with water. (Image originally published in Hagler et al 2021, Journal of Insect Science)

By John P. Roche, Ph.D.

In many situations—from control of insect pests to the introduction of natural enemies of pests—entomologists need to estimate the dispersal patterns of insect populations. One effective way to do this is via the mark-release-recapture method. In this technique, investigators mark individual insects in the lab, release them into the field, and later sample a number of individuals in the field to check for marked individuals.

Various marking methods have been evaluated for use on different types of insects, and a new study published last week in the open-access Journal of Insect Science reports on the use of a green fluorescent dye originally designed for use as an anti-theft tool in law enforcement.

James Hagler, Ph.D., research entomologist at the U.S. Department of Agriculture-Agricultural Research Service in Maricopa, Arizona, has studied and developed various insect marking techniques throughout his career. Techniques include using Day-Glo dust, ink dyes, or animal protein. In the present study, Hagler—along with USDA-ARS colleagues Allya Hull, Miles Casey, and Scott Machtley—examine marking insects with a fluorescent dye called SmartWater, a mixture of a liquid fluorescent dye and a liquid polymer emulsion. SmartWater is commercially available for use as a forensic and anti-theft product. It is not visible under regular light, but it fluoresces under ultraviolet light, and it can be coded with unique markers so that recovered items can be identified by their exact origin. The dye is long lasting and easy to see under UV, making it potentially ideal for mark-release-recapture studies.

“With traditional markers,” Hagler says, “you almost always have to look at each individual under the microscope for grains of dust, which is time consuming and tedious. SmartWater ‘glows’ under blacklight, so it is easy to detect without the need to handle each individual. We are doing follow-up tests that show you can shine a blacklight over an entire sample of field-collected specimens, and the marked ones stick out like a sore thumb.”

Hagler and his colleagues examined the use of SmartWater “fluorophore” markings in three insect species: western tarnished plant bugs (Lygus hesperus), which are pests to a wide range of crops; convergent lady beetles (Hippodamia convergens), which are the natural enemies of many insect pests; and sweetpotato whiteflies (Bemisia tabaci), which are crop pests and vectors of plant diseases. To test for presence of the fluorophore mark in each experiment, they examined test insects by eye and also quantitatively measured fluorescence using a device called a microfluorometer.

To test a marking technique for mark-release-recapture estimates, investigators need to confirm that the mark meets the assumptions of the technique: that the mark has to avoid being washed off and that the mark cannot harm the insect being marked. Hagler and colleagues tested both of these assumptions in their study.

In this image, panels A and B show western tarnished plant bugs (Lygus hesperus). Panels C and D show convergent lady beetles (Hippodamia convergens). The insects in the top row of each panel were marked with a fluorescent dye called SmartWater; the insects in the bottom row of each panel were control specimens “marked” with water. The two photos on the left (A and C) were photographed with white light; the two photos on the right (B and D) were photographed with UV light. Note in the UV photos that the SmartWater-marked individuals showed green fluorescence, whereas the control individuals did not. (Image originally published in Hagler et al 2021, Journal of Insect Science)

In the first of five experiments in the study, the researchers tested whether SmartWater could be used effectively to mark Lygus bugs and convergent lady beetles. Some individual insects from each species were marked with SmartWater, and control individuals from each species were “marked” only with plain water. They found a high mortality of Lygus bug treated with SmartWater but almost no mortality in lady beetles treated with SmartWater. In terms of visibility of the dye, they found that SmartWater adhered well to Lygus bugs but less well to lady beetles. It was easy to see fluorescence on all SmartWater-marked Lygus bugs, and no marking could be seen on any control Lygus bugs.

In their quantitative analysis using a microfluorometer, they observed high fluorescence on all marked Lygus bugs, but no fluorescence on control Lygus bugs. In lady beetles, dye was detected by sight in about 80 percent of individuals marked with SmartWater, and the microfluorometer detected fluorescence in 93 percent of the SmartWater-marked beetles. However, after three days, only 25 percent of the marked lady beetles showed fluorescence.

In response to the high mortality of Lygus bugs in the first experiment, in their second experiment the researchers tested if they could decrease mortality in marked Lygus bugs. They changed the apparatus that applied the SmartWater from an airbrush to a nebulizer and used the nebulizer to apply either SmartWater or water as a control. The investigators found that using the new nebulizer method increased survivorship of SmartWater-marked Lygus bugs to more than 95 percent.

In their third experiment, Hagler and colleagues tested if a marked insect could transfer a mark to an unmarked insect. They tested for mark transfer in crowded arenas (about 80 L. hesperus bugs in a half-liter container) and uncrowded arenas (10 bugs per container) and found that there was a high amount of transfer of marks to unmarked individuals in crowded arenas, but mark transfer was observed in less than 1 percent of individuals in uncrowded arenas.

Next, they tested if SmartWater could work as a tag for a tiny insect, using whiteflies as test subjects. They found that SmartWater adhered effectively to the whiteflies and was readily discernable by both visual inspection and with a microfluorometer.

In the fifth experiment, the research team tested the amount of SmartWater needed to effectively mark whiteflies, seeking the ideal balance between avoiding mortality and maintaining a visible mark. Different cohorts received either 10-second, 20-second, 30-second, or 60-second applications of either SmartWater fluorescent dye or water. They found that the 60-second application provided the highest levels of fluorescence without causing a significant increase in mortality in the flies.

In this image, the top two insects are sweetpotato whiteflies (Bemisia tabaci) marked with a fluorescent dye called SmartWater and the bottom two insects are control whiteflies “marked” with water. The insects on the left (panel A) were photographed with white light and the white flies on the right (panel B) were photographed under UV light. Note in the UV photos that the SmartWater-marked individuals showed green fluorescence, whereas the control individuals did not. Photos courtesy of James Hagler and Journal of Insect Science. (Image originally published in Hagler et al 2021, Journal of Insect Science)

In sum, Hagler and colleagues found strong evidence that SmartWater is promising as a marking method for mark-release-recapture studies in Lygus bugs, convergent lady beetles, and white flies. But it doesn’t work for every species. “We just finished a [separate] study examining the efficacy of SmartWater on 16 different species,” Hagler says. “The mark is foolproof on most species, but it is not effective on others. The reason is that many insect species have natural autofluorescence. If so, this feature masks out the mark. In short, the fluorophore marks won’t be useful for every species; that can be said for the conventional markers, too.”

As one example, a study published in March 2021—led by Roy Faiman, Ph.D., at the National Institutes of Health, with colleagues at NIH and the University of Sciences, Techniques and Technologies in Mali—tested SmartWater for marking Anopheles gambiae mosquitoes, a species that spreads malaria. They found that SmartWater markings could be detected in field-caught mosquitoes with a handheld UV flashlight, without causing death of the mosquitoes.

The SmartWater fluorescent dye method has clear applications for ecological research, for studies informing control of crop pests and natural enemies of crop pests, and for the study of arthropods that vector disease. “This work is preliminary,” Hagler says. “We plan on conducting follow-up studies in the next field season. If true, this will be a game-changer on the methods used to mark insects for mark-release-recapture research.”

John P. Roche, Ph.D., is an author, biologist, and educator dedicated to making rigorous science clear and accessible. Director of Science View Productions and Adjunct Professor at the College of the Holy Cross, Dr. Roche has published over 200 articles and has written and taught extensively about science. For more information, visit https://authorjohnproche.com/.

Update, December 22, 2021: An earlier version of this post misidentified Roy Faiman’s affiliation. He works at the National Institutes of Health. The post has been updated accordingly.