Being some of the staples of the terpene kingdom, limonene, beta-myrcene, and linalool are known for their all-around soothing properties, especially from an anxiolytic and analgesic standpoint. This is why a group of scientists investigated if these effects translate when the terpenes are administered to zebrafish.  Scientists often use zebrafish in studies of vertebrate development and gene function. These fish have 70% similarity to the human genome  as well as many organs and physiological features that require genes that are conserved between the fish and us. Thus, any disease that can occur in these body parts in us can be modeled in the fish.
The zebrafish were separated into four groups – a control group and three others, as defined by the administered terpene. Since this was the first study on terpenes that used zebrafish as an animal model, the scientists performed careful pilot testing to gauge the appropriate terpene concentrations. During that preliminary step, the zebrafish displayed a high sensitivity to linalool, reminiscent of extreme sedation, so the linalool dose was significantly reduced and was overall much lower than the concentrations evaluated in previous studies on the terpenoid. The concentrations of terpenes used were: limonene 0.25, 0.5, 0.75%; β-myrcene 0.001, 0.01, 0.1%; and 0.0001, 0.001, 0.00125% linalool.
The study involved two major parts: acute and repeated administration, with the latter being a seven-day period.
The terpenes’ influence on zebrafish was assessed based on the following parameters: anxiety-like behavior, boldness, and locomotion.
Anxiety-like behavior and locomotion were gauged through an open field test. Anxiety-like behavior was “quantifed as time spent in the various ‘virtual’ zones of the arena (thigmotaxis, transition, and center zone), with more time being spent near the walls of the arena in the thigmotaxis zone being indicative of anxiety-like behavior.” Thigmotaxis simply refers to the motion or orientation of the zebrafish in response to being touched.
Locomotion, respectively, was defined by the distance moved, immobility, high mobility, and meandering.
Boldness, on the other hand, was measured through a novel object approach test, and can overlap to an extent with anxiety-like behavior, or rather the lack thereof. A novel object – a LEGO figurine – was introduced in the natural habitat after the open test trial. The zebrafish’s willingness to interact with the object was defined by their movement patterns, using the same virtual zones as baselines.
Limonene and beta-myrcene were found to significantly decrease the anxiety-like behavior and mobility, and respectively, an increase in boldness. Linalool, however, didn’t seem to produce noteworthy effects on either count, with the most noticeable alterations being observed in locomotion. Perhaps this can be attributed to the low concentration of linalool, since these findings aren’t in line with previous studies and the pilot testing.
It’s important to note that limonene’s and beta-myrcene’s effects faded drastically upon repeated administration, which indicates that the zebrafish developed a tolerance toward the terpenes.
“In conclusion, our study provides support for an anxiolytic and sedative effect in zebrafish in response to acute limonene and β-myrcene exposure that is no longer present after one week of repeated exposure.”
1- Szaszkiewicz J, Leigh S, Hamilton TJ. Robust behavioural effects in response to acute, but not repeated, terpene administration in Zebrafish (Danio rerio). Sci Rep. 2021;11(1):19214. Impact Factor = 4.379
2- Howe K, Clark M, Torroja C. et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature. 2013; 496:498–503.
Image Credits: Wikimedia Commons