Attraction of released male Mediterranean fruit flies to trimedlure and an α-copaene-containing natural oil: effects of lure age and distance

Tephritid fruit flies (Diptera: Tephritidae) pose a threat to global agriculture owing to increased movement of people and commodities as well as global warming that is allowing range expansion of pest species (Papadopoulos et al. 2024). Many countries operate monitoring programs to detect incursions of pest tephritids (Kean et al. 2024). These programs rely on trapping networks based primarily on so-called male lures, which are synthetic or natural compounds attractive to males only. The most commonly used male lures are: cue-lure (for certain Bactrocera and Zeugodacus species), methyl eugenol (for certain Bactrocera species), and trimedlure (for certain Ceratitis species) (Tan et al. 2014). Of these, methyl eugenol is considered the most potent lure and trimedlure perhaps the weakest (McInnis et al. 2017). Attempts to improve detection of Ceratitis males involve modifications of trimedlure (e.g., ceralure [Warthen et al. 1994] and capilure [Leonhardt et al. 1984]) or complete replacement with natural oil-based lures.

The attractiveness of particular natural oils to males of the Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann), derives largely from the presence of the compound α-copaene (Guiotto et al. 1972), and studies in sub-Saharan Africa (Manrakhan et al. 2017) reported that enriched ginger root oil (so named, because the natural concentration of α-copaene was increased via fractional distillation) was more attractive to certain Ceratitis spp. than trimedlure. More recently, data from Tunisia, Costa Rica, and Hawaii showed that a natural oil, with enhanced α-copaene content, consistently attracted more C. capitata males than trimedlure (Kharrat et al. 2024; Shelly et al. 2023).

The present study provides additional data regarding the relative attractiveness of trimedlure and the aforementioned natural oil via release-recapture trials of male medflies. Specifically, we compared the two lures based on captures of males released near or far (15 or 30 m) from fresh or aged (weathered < 2 weeks or 10–12 weeks, respectively) lures. Shelly et al. (2023) found that, when trimedlure and natural oil baits were weathered for the same duration, the natural oil lure consistently attracted more wild male medflies than trimedlure. Consequently, we predicted that, for each release distance, fresh and aged natural oil would attract more males than comparably aged trimedlure.

Trials were conducted in a macadamia nut orchard (Macadamia integrifolia Maiden & Betche, Proteaceae; 19.6428611 °N, 155.0800361 °W) near Hilo, Hawaii. Macadamia is neither an ovipositional nor adult food source, thus eliminating these parameters as potentially influencing fly movement. Trees were uniform in size (4–5 m high) and planted in a grid, with rows 8–9 m apart and trees within a row separated by 4–5 m. Releases were performed during July 2024 to February 2025. Average daily minimum and maximum temperatures on release days (N = 32) were 18.1 °C (range: 15.9–19.7 °C) and 26.8 °C (range: 24.2–27.8 °C), respectively. Rainfall was < 4 mm on all release days except for one instance (16.2 mm on 27 November 2024).

Flies were captured using Jackson traps, which are widely used in fruit fly monitoring programs (FAO/IAEA 2018; Scentry Biologicals Inc., Billings, Montana, USA). Jackson traps are white, ‘delta’ traps made of thick waxed paper (12.7 L × 9.5 W × 8.4 H cm), with a removable, sticky floor panel. Trimedlure plugs (Scentry Biologicals Inc.) and natural oil bulbs (ChemTica Intl.; Heredia, Costa Rica) both loaded with 4 g of active ingredient) were placed inside perforated, plastic baskets suspended above the sticky floor of the trap. Operationally, fresh lures were those weathered for < 2 weeks, while aged lures were weathered for 10–12 weeks. Weathering occurred outdoors on a covered porch at the United States Department of Agriculture, Agricultural Research Service (USDA-ARS) laboratory in Hilo under environmental conditions similar to the field site. Use of these intervals was based on earlier results (Shelly et al. 2023) that revealed (i) neither trimedlure nor natural oil showed significant decline in attractiveness over the first 2 weeks of weathering and (ii) both trimedlure and natural oil weathered > 10 weeks were significantly less attractive than their fresh counterparts. Traps were suspended 2.0–2.5 m above ground from branches in the tree canopy.

Males for release were obtained from the California Department of Food and Agriculture (CDFA) Medfly Rearing Facility, Waimanalo, Hawaii, which rears a tsl genetic sexing strain for a sterile insect technique program operated in California. Non-dyed, non-irradiated male pupae were shipped overnight in chilled, insulated boxes and placed in cubical (30 cm per side) screen-mesh cages (excepting one side that was clear plexiglass; 10 mL or approximately 600 pupae per cage). Non-dyed flies were used, as wild medflies were absent at the study site. Based on CDFA quality control measurements, which follow international guidelines (FAO/IAEA/USDA 2019), adults emerged successfully from 85 % of the pupae, and 78 % of these adults were flight-capable (i.e., 100 pupae yielded 66 flight-capable adults; values based on averages of measurements obtained for daily batches [N = 32] of flies used in the experiment). Emerged flies were maintained at the USDA-ARS laboratory at 22.5 ± °C and 55 ± 3 % relative humidity under a 14:10 (L:D) photoperiod and were provided food (a 3:1 mixture of sugar and protein hydrolysate) and water ad libitum.

On a given test day, one trap containing trimedlure or natural oil was placed in each of two focal trees (separated by > 400 m). A focal tree (with trap) was located at the center of a circle with fly releases made at four points along the circumference corresponding to N, E, S, or W directions from the focal tree. The same combination of variables (i.e., new or old lures with near or far releases) was tested at both focal trees used on a given test day. For the releases, which occurred between 7 and 8 AM, cages were placed on the ground beneath the release trees, the sliding plexiglass side of the cage was gently lifted, and flies exited on their own volition. After 30 min, cages were tapped to induce flight among remaining males. For releases at 15 m, approximately 1,600 males was released (one cage or approximately 400 flight-capable males per release site), and for releases at 30 m, approximately 3,200 males were released (two cages or approximately 800 flight-capable males per release site). Traps were collected 2 days later and returned to the laboratory for counting. The release distances and male numbers were selected to insure sufficiently large recaptures for statistical analysis.

Tests were performed weekly and used alternate pairs of release trees, i.e., four different focal trees were used over the study (week 1: trees A + B; week 2: trees C + D; week 3: trees A + B and so on). This protocol resulted in a 2-week interval between successive releases around a given focal tree and thereby maximized the likelihood that captured flies derived from the most recent release. Also, over successive test days for a given pair of focal trees, the positions of the trimedlure and natural oil dispensers were rotated between trees. Releases were performed at each of two focal trees (one with trimedlure, the other with natural oil) on 8 days for each of the four combinations of variables (new or old lures each with near or far releases; 8 days × two releases (one per focal tree) per day × four treatment combinations = 64 total releases over 32 days).

For each combination of release distance and lure age, captures between trimedlure and natural oil lures were compared using a paired t-test (df = 7 in all cases), as (i) same-day releases were made at two focal trees containing a trimedlure- or natural oil-baited trap, respectively, and (ii) raw or log10 transformed data met the parametric assumptions in all cases. For a given release distance, captures for fresh and aged trimedlure plugs or natural oil bulbs were compared with an unpaired t test (df = 14 in all cases).

For the 15 m releases, fresh trimedlure and natural oil attracted similar numbers of male medflies, whereas traps baited with aged natural oil captured significantly more medflies than traps baited with aged trimedlure (Figure 1). For the 30 m releases, both fresh and aged natural oil bulbs attracted significantly more male medflies than weathered trimedlure plugs (Figure 1). Surprisingly, captures for 15 m releases were significantly higher for aged than fresh lures for both trimedlure and natural oil (P < 0.05 in both cases). This finding appears to suggest that male attraction increased as the release rate of the lures decreased with weathering. For trimedlure at least, this interpretation does not appear to be correct, as male attraction to new and aged trimedlure plugs were found to be similar in several studies (Dean et al. 2018; Shelly and Kurashima 2020). Comparable data are not available for the natural oil lure. Again, somewhat unexpectedly, captures for 30 m releases were not significantly different between fresh and aged baits for either lure (P > 0.05 in both cases).

Figure 1:

Captures of Ceratitis capitata males released at 15 or 30 m from Jackson traps baited with fresh or aged trimedlure or α-copaene enriched natural oil in a macadamia nut orchard in Hawaii. Bar heights represent average number (±1 SE) per trap (N = 8 replicates). Statistical results: ns – not significantly different; *P < 0.05; **P < 0.01.

Overall, these results confirm the greater attractancy of natural oil over trimedlure. Aside from 15 m releases with fresh lures, greater numbers of male C. capitata were captured in traps baited with natural oil than those baited with trimedlure. Recapture for 30 m releases (data from fresh and aged lures combined) were 11.3 % and 14.4 % for trimedlure and natural oil traps, respectively. Other studies performed releases 30–46 m from a central trimedlure-baited trap and reported recapture of 1 % (Meats and Smallridge 2007), 3 % (Lance and Gates 1994), 6 % (Shelly et al. 2014), and 36 % (Cunningham and Couey 1986). This latter study (like the present one) was conducted in a Hawaiian macadamia nut orchard, and the relatively high recapture values observed in this habitat suggest that the uniform, unbroken distribution of trees resulted in higher recapture than observed in residential areas.