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Nikos A. Kouloussis (corresponding author), Laboratory of Applied Zoology and Parasitology, School of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece. E-mail: email@example.com
Previous work has demonstrated that exposure of males of the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae), to the scent of sweet orange increases their mating success relative to non-exposed males. Here, we demonstrate that exposure to the commercial essential oils of bitter orange, mandarin orange, lemon and grapefruit confer to males a significant mating advantage as well. Likewise, exposure to peel-wounded mandarin oranges and lemons, and to peel-wounded sweet oranges of the Merlin, Newhole and Naveline varieties, also conferred a significant mating advantage. Exposure to peel-wounded grapefruits and bitter oranges as well as to sweet oranges of the local Greek variety Xino Artas enhanced mating success, however, not significantly. These results suggest that the chemicals responsible for mating enhancement in C. capitata are present in several citrus fruit species and varieties. In an experiment involving different doses of commercial sweet orange peel oil, males exposed to medium doses of 12.5 or 25 μl achieved significantly higher numbers of matings with virgin females than control males. Lower doses did not increase mating success, whereas higher doses killed a number of males. In another experiment we tested the effect of the seven most abundant individual chemical compounds of citrus oil (some of them also existing in the pheromone of males) and detected compounds with some potential for further trials. In a final experiment we created a mixture of those compounds (geraniol, α-pinene, limonene, β-myrcene and linalool) and found that wild or sterile males exposed to it achieved over 70% of all matings when competing with non-exposed males. Since the mixture did not contain α-copaene (an expensive compound that is often invoked as responsible for the phenomenon of mating enhancement) our data could lead in a more cost-effective and efficient implementation of sterile insect technique (SIT) programmes against C. capitata.
Early evidence regarding the influence of sweet oranges was provided by a study showing that in mixed orchards containing multiple hosts, including a small percentage of citrus, nearly 50% of Mediterranean fruit fly matings occurred on orange trees (Hendrichs and Hendrichs 1990). Other experiments conducted in field cages showed that C. capitata males were strongly attracted to and landed on sweet oranges and on other citrus fruits that were wounded in the flavedo region of the peel (Katsoyannos et al. 1997). Subsequent work conducted in the last decade has clearly demonstrated that male Mediterranean fruit flies exposed to wounded sweet orange peel or to commercial sweet orange essential oil obtained significantly more copulations when competing with males denied such an exposure (Papadopoulos et al. 2001, 2006; Shelly et al. 2004a,b; Shelly and Villalobos 2004).
This tight association between sweet orange and the mating system of this insect may be put into practical use for the control of C. capitata. During sterile insect technique (SIT) operations large numbers of mass-reared sterile males are released in on an area-wide basis in high sterile to wild over-flooding ratios (Dyck et al. 2005). Females that mate with sterile males lay unfertilized eggs that do not develop into larvae and thus the population declines. The success of the SIT depends on the ability of sterile males to compete with wild males in attracting, courting and mating with wild females in mating aggregations or leks (Prokopy and Hendrichs 1979). Large-scale, pre-release treatments of released males with suitable behaviour modifying chemicals, such as sweet orange, or ginger root oil as suggested by Shelly et al. (2007), may increase matings and thus enhance the effectiveness of SIT. An adequate understanding of this influence could lead to increased efficiency of the method.
Although several studies have dealt with the effect of the scent of sweet orange on the mating behaviour of C. capitata, it is not known which other citrus species or which sweet orange varieties enhance mating competitiveness. The only other citrus species for which an effect similar to that of orange has been observed is grapefruit (Citrus paradisi Macfad.) (Shelly 2009). The identity of the compound(s) responsible for this effect and its mode of action are also unknown. It has been suggested that the hydrocarbon sesquiterpene α-copaene, a powerful attractant to male Mediterranean fruit flies (Flath et al. 1994a,b), may play an important role, as males exposed to pure α-copaene had a higher mating frequency than non-exposed males (Shelly 2001). This chemical is not only present in ginger root oil, but also in sweet orange and other citrus fruits. However, there is no data showing if this compound is actually required for mating enhancement to occur, or if other citrus compounds can have a similar or even better effect in the absence of α-copaene.
Here, we describe a series of laboratory experiments that further explore the association between citrus oil and mating enhancement in male C. capitata. In particular, we present the results of experiments that examined mating success of males exposed to (i) chemicals found in different citrus species and sweet orange varieties, (ii) five different doses of sweet orange oil, (iii) seven different compounds contained in both citrus oils and male pheromones, and (iv) a mixture of five of these compounds.
Materials and Methods
Laboratory conditions and insects used
The experiments were conducted in the laboratory at 25 ± 2°C, 65 ± 5% RH, and a photoperiod of L14 : D10, with photophase starting at 06:00 h. In all experiments except one the flies used were retrieved from field-infested figs (Ficus carica L.) collected on Chios Island, Greece. Before being used the flies were reared in the laboratory for 1–6 generations on an artificial diet (Boller 1985). These flies were considered as ‘wild-like’. In a single test we also used mass-reared sterile males of the VIENNA-8 tsl genetic sexing strain, which is used around the world for C. capitata SIT programs. The flies were derived from the mother colony of this strain maintained at the Entomology Unit of the FAO/IAEA Agriculture and Biotechnology Laboratory, Seibersdorf, Austria. All adults were kept in cages provided with food [a mixture of yeast hydrolysate (ICN Biomedicals Inc., Irvine, CA) and sugar in a ratio of 1 : 4] and water. The experiments with wild-like males or females were conducted at adult age of 14–16 days and with sterile males at adult age of 7 days.
Effect of chemicals found in five different citrus fruits and four different sweet orange varieties
The first experiment involved wild-like male exposure to ripe fruits of five different citrus species. Sour oranges (Citrus aurantium L.) were picked from seedling trees, whereas the other four fruit species were of known varieties and were obtained from the local market. These were sweet oranges (C. sinensis) of the Merlin variety, mandarin oranges (Citrus reticulata Blanco) of the Clementine variety, lemons (Citrus limon L. Burm.) of the Eureka variety and grapefruits (Citrus paradisi Macfad.) of the Dunkan variety. Each fruit was wounded with a scalpel razor following the procedure described in Katsoyannos et al. (1997). For each exposure of flies we used one wounded sweet orange or grapefruit with five cuts each. As mandarin oranges, lemons and bitter oranges were smaller in size, we used two of them per cage, one with two wounds and one with three wounds.
Fifteen minutes after being wounded, the fruits were introduced into a group cage containing 100–120 males. The exposure of flies to the wounded fruits lasted 24 h and ended 12 h before the experiment. At about the middle of the exposure period the fruits were replaced with freshly wounded ones. Control males were exposed to a dummy fruit treated with water and kept in cages in a different room to avoid inadvertent side exposure. To distinguish between treated and control individuals, 1 day before exposure males of each category were marked with a small dot of a different colour of water paint on the notum of their thorax (FAO/IAEA/USDA 2003).
Following fruit exposure ten treated and ten control males were introduced into a Plexiglas cage (15 × 15 × 15 cm), with a round (ca. 10 cm diameter) nylon-mesh-screened window in one side. At 10:00 h of the next day, ten virgin females were introduced into the cage. Cages were observed at 5-min intervals until 14:00 h, and the pairs formed with either treated or control males were recorded and removed from the cage, together with a randomly chosen male of the opposite category. This was done so that the ratio of the three categories of flies in a cage (treated males : control males : virgin females) always remained constant. In this test we conducted 13 replicates.
The second part of this experiment involved male exposure to commercial oils from the five citrus species tested in the experiment described above. Bitter orange oil was from New Direction Aromatics Inc. (San Ramon, California), whereas the rest of the oils from Sigma–Aldrich GmbH (Steinheim, Germany). The method regarding exposure and testing was similar as described in the first experiment. Using a microcapillary pipette, 25 μl of oil was applied to a piece of white filter paper placed on the bottom of the cage. Control males were exposed likewise to 25 μl of water also applied to a piece of white filter paper. Exposure started at 14:00 h the day before the experiment and continued until 19:00 h. In this experiment we conducted 15 replicates.
Another experiment involved male exposure to peel-wounded sweet orange fruits of the varieties ‘Merlin’, ‘Newhole’, ‘Naveline’ and the local Greek variety ‘Xino Artas’. The group mating competitiveness method regarding exposure and testing was similar as described above for the wounded fruits of the five different citrus species. In this experiment we conducted 11 replicates.
Effect of different doses of sweet orange oil
This experiment involved wild-like male exposure to different doses of sweet orange oil. The exposure was performed inside transparent plastic drinking cups (400-ml volume) which had been transformed into small cages with food and water. Before exposure to oil, 25 males were placed in a cage through a small opening using an aspirator. With the help of a microcapillary pipette we then applied 1, 3, 6, 12.5, 25, 50 μl of the oil (or 25 μl of water as control) to a disk of white filter paper placed on the bottom of the cage. Exposure started at 15:00 h and continued until 19:00 h. During exposure the number of males walking, flying or staying immobile within the cage was monitored at 5 min intervals by an observer. Following exposure one treated and one control male were introduced into a new small cage. At 10:00 h of the next day, one virgin female was introduced into the cage. Cages were subsequently observed at 5-min intervals until 14:00 h, and the pairs formed with either treated or control males were recorded. Fifty to 70 replicates of the single mating competitiveness test were conducted for each of the six exposure treatments (doses) under comparison.
Effect of different citrus oil compounds
This experiment involved wild-like male exposure to limonene, linalool, β-myrcene and α-pinene (which are all present in abundance in citrus oils), to geraniol and farnesene (present in small quantities) and to indole (present in abundance in citrus flowers) (Attaway et al. 1968). Limonene, linalol, β-myrcene, farnesene and indole were included for the additional reason that they are constituents of the pheromone of male C. capitata (Howse and Knapp 1996). Indole, farnesene and linalool were purchased from Sigma–Aldrich GmbH; geraniol, α-pinene and limonene from Alfa Aesar (Kalsruhe, Germany); and β-myrcene from Fluka (Lyon, France). The purity of β-myrcene was 90% and of the rest of the compounds 97–98%. The method regarding exposure and testing was similar as described in the group experiments involving different citrus species and varieties. We used 5 μl of each compound and for indole 1 g of crystals diluted in 1 ml of ethyl alcohol. For each compound we conducted 15 replicates.
Effect of a mixture of citrus compounds
This experiment involved sterile and wild-like male exposure to a mixture consisting of equal quantities (1 : 1 : 1 : 1 : 1) of geraniol, α-pinene, limonene, β-myrcene and linalool. To decrease evaporation rate we applied 25 μl of the mixture (or water for the control) to a disk of white filter paper placed on the bottom of a 5.5-cm diameter plastic Petri dish. The cover of the dish bore a 5-cm diameter hole in which a plastic, hollow, red hemispheric dome of the same diameter was fitted. The dome had 100 equidistant holes (<0.5 mm diameter) through which the mixture evaporated. Two such domes were placed inside a holding cage (20 × 20 × 20 cm) containing 50 wild-like or sterile males at 08:00 h 2 days previous to the experiment. At 10:00 h on the next day each dome was replenished with an additional 25 μl of the mixture; the domes remained inside the cage until 18:00 h. Each replicate consisted of one treated male competing with one untreated male for a virgin female. We ran 60 replicates with wild-like males and 85 with sterile males.
Means in group mating competitiveness tests were separated by paired t-test. We also estimated the proportion of mating increase in treated males over control non-exposed males and we submit these data to one way analysis of variance and mean separation by Tukey’s HSD test (Sokal and Rohlf 1995). In single-mating competitiveness tests involving different doses of sweet orange oil we used chi-square test. The proportion of matings achieved by treated males was regressed against the five different doses. In the single-mating competitiveness tests involving the mixture of five citrus oil compounds the deviation from random mating (P = 0.5) was tested using a normal approximation of the binomial test (Z-test) corrected for continuity (Snedecor and Cochran 1989).
Effect of chemicals found in five different citrus fruits and four different sweet orange varieties
Exposure to wounded sweet oranges, mandarin oranges and lemons conferred to males a significant mating advantage over control males (t12 > 3.6, P < 0.01), whereas exposure to grapefruit and bitter orange did not confer a significant advantage (t12 = 1.4 and 1.5 respectively; in both P > 0.05) (fig. 1). It must be noted, however, that exposure to any fruit resulted in an increase in matings in treated males. The highest percentage of matings relative to the respective control was observed in sweet orange (79.4% of all matings), followed by mandarin orange (78.4%), lemon (63.9%), bitter orange (59.6%) grapefruit (59.3%). Further analysis of the results by anova (on the proportion of mating increase of treated males over control non-treated males) confirmed the significant effect of the different fruit on mating competitiveness (F5,72 = 3.75, P = 0.004) allowing direct comparisons among effects of different citrus species. With the exception of grapefruit, all fruit resulted in an increase in matings relative to the control (Tukey’s HSD test, P < 0.05). There were no differences between sweet orange, mandarin orange, lemon and bitter orange; and grapefruit was not significantly different from lemon and bitter orange (Tukey’s HSD test, P > 0.05).
When commercial oils rather than wounded fruits were used, all of them (including bitter orange and grapefruit) conferred to males a significant mating advantage over control males exposed to water [t14 = 2.2–4.4; P < 0.05 (lemon) or P < 0.01 (all other fruit)]. The order of effectiveness of the different fruit (judging based on the percentage of matings observed relative to the control) was somehow different that in the previous test. Highest was the percentage of matings achieved by males exposed to grapefruit (80.9% of all matings), followed by bitter orange (76.5%), mandarin orange (75.0%), sweet orange (74.4%) and lemon (62.8%). anova confirmed that competitiveness was significantly influenced by exposure to the oils (F5,79 = 3.28, P = 0.01). Lemon, however, was not different from the control, but also not different from sweet orange or mandarin orange (Tukey’s HSD test, P > 0.05).
The results of the two experiments combined reveal that all five fruit species contain chemicals responsible for the phenomenon of mating enhancement in C. capitata. These chemicals, however, might not be released at equal rates from the wounds of the fruit and/or may be contained in different quantities or ratios in the oils used in the tests.
Exposure to wounded sweet oranges of all four varieties conferred to males a mating advantage (fig. 2). The difference from the control were significant in the varieties Merlin, Newhole and Naveline (t10 = 2.6–5.2; P < 0.001, P < 0.01 and P < 0.05 respectively), but not in the variety Xino Artas (t10 = 1.5, P > 0.05). The percentage of matings observed relative to the control was highest in Newhole sweet oranges (75.0%), followed by Merlin (72.0%), Naveline (64.9%) and Xino Artas (60.5%). Further analysis with anova suggested no differences among the four different varieties (F3,40 = 0.13, P = 0.94).
Effect of different doses of sweet orange oil
Treated males achieved greater numbers of matings compared to the control males in all doses of sweet orange peel oil (fig. 3). Nevertheless, only the doses of 12.5 μl and 25.0 μl conferred to males a significant mating advantage (64.5% and 62.8% of all matings respectively) over control males (chi-square test, P < 0.05). Effectiveness increased as the dose increased from 1.0–3.0 μl to 12.5 μl of oil and then decreased again at 25.0 μl of oil. There was no significant relationship between dose and effectiveness (linear regression, F1,3 = 7.4, P =0.07; quadric regression, F1,3 = 10.3, P = 0.08).
Observations performed during exposure of males to the oils (prior to the mating competitiveness test) revealed that as soon as the oil was introduced into a cage males became immobile, especially so in the higher doses. A few minutes later, as the oil evaporated, males tended to resume crawling, flying, feeding and other activities in the lower doses (1, and 3 μl), but not in the higher doses (6, 12.5 and especially 25.0 μl). In the dose of 25.0 μl several males never resumed normal activities and 4–8% of them died before the exposure ended. In the highest dose of 50.0 μl all males died. In the lower doses males were often observed lowering their head and touching the oil-treated filter paper with their proboscides in an apparent attempt to feed.
Effect of different citrus oil compounds
None of the seven compounds tested conferred to males a significant mating advantage over control males exposed to water (t14 < 1.5, P > 0.05; data not shown). Nevertheless, a substantial tendency towards more matings was observed by males treated with geraniol (58.2% of total matings) and limonene (56.9%). A small advantage was also conferred to males treated with β-myrcene, linalool, α-pinene and indole.
Exposure to a mixture of citrus compounds
Exposure to the 1 : 1 : 1 : 1 : 1 mixture of geraniol, α-pinene, limonene, β-myrcene and linalool conferred to wild-like males a significant advantage over the control males, with treated males achieving 73.3% of total matings (Z = 4.9, P < 0.05) (fig. 4). Likewise, sterile males of the VIENNA-8 tsl strain achieved 70.8% of total matings with wild-like females (Z = 3.7, P < 0.05). These data suggest that mixtures made from pure chemicals of citrus oils may confer a high mating advantage to males.
Our data demonstrate a strong effect of several citrus species and sweet orange varieties on enhancing mating performance of male C. capitata. This effect stems from chemical constituents of the flavedo region that seem to be associated with the sexual behaviour of this species. These ingredients are present in several citrus fruits and varieties, although in differing proportions. The variability observed in our experiments in the mating enhancing properties between wounded fruit of different species or different sweet orange varieties is probably the result of qualitative differences in the chemical composition of the flavedo area between species and varieties (Lota et al. 2002; Moufida and Marzouk 2003) as well as quantitative differences stemming from the density of oil glands and the amount of essential oils in the flavedo area in the various fruits (Papachristos and Papadopoulos 2009). Exposure to wounded bitter orange and grapefruit did not confer a significant advantage. However, exposure to the commercial essential oils of these fruits (as well as of all other fruits tested) enhanced male mating competitiveness. The effect of grapefruit and grapefruit oil was also shown in another recent study (Shelly 2009). The chemical composition of the commercial oils depends on the method of extraction, the variety used and several other factors, and may therefore be responsible for the above discrepancy in effects between wounded fruits and the respective commercial oils.
Experiments involving application of different doses of commercial sweet orange peel oil revealed a significant effect of dose-exposure regimes on male mating behaviour. Over a range of doses (1.0–12.5 μl) it seems that there is a positive relationship between the amount of oil and its effect on mating success. However, dose increase beyond the level of 12.5 μl failed to further enhance mating competitiveness and, most importantly, it caused direct toxicant effects during exposure. These findings suggest the existence of an optimum exposure dose in citrus oils where the induction of male mating success is high and mortality low. The fact that flies became immobile or died in the presence of oil is probably due to the highly toxic and insecticidal effects of the compound limonene that is contained in large amounts in orange oil (Karr and Coats 1988; Siskos et al. 2009). Shelly et al. (2004b) used different doses of ginger root oil in large scale exposure schedules (plastic adult rearing containers) and found no apparent effects on sterile male survivorship; however the maximum dose tested in those experiments had a negative impact on the mating success of treated males. These data along with ours proves the necessity for detailed studies concerning the most effective dose regime to treat sterile males.
The chemical basis for the association between C. capitata male exposure to the scent of different citrus fruits and the increase in mating performance is unknown. Citrus peel essential oils are a rich mixture of several components belonging to different chemical classes such as hydrocarbons, aldehydes and alcohols (Mitiku et al. 2000; Verzera et al. 2004). A compound suggested as responsible for C. capitata male enhancement of mating competitiveness is the sesquiterpene hydrocarbon α-copaene (Shelly 2001; Shelly et al. 2004b). This compound is a known attractant of male Mediterranean fruit flies (Flath et al. 1994a,b) and occurs in a wide variety of plants including citrus fruits and leaves (Nishida et al. 2000); and references therein). Though male C. capitata exposure to pure α-copaene as well as to α-copaene containing oils (angelica seed oil and ginger root oil) enhances mating success, this does not constitute a proof that it is the only ingredient responsible for the observed phenomenon. Evidence for this is provided from our experiments involving male exposure to the mixture of pure compounds, which did not contain α-copaene but was still very powerful in enhancing the mating competitiveness of exposed males relative to unexposed control males. This is a strong indication that α-copaene is not a necessary ingredient for mating enhancement to occur.
Furthermore, C. capitata males do not appear to use the chemical in synthesizing their sex pheromone as far as neither α-copaene nor structurally similar compounds are present in the chemical profile of the pheromone (Nishida et al. 2000; Papadopoulos et al. 2006). On the contrary, the essential oils of citrus contain several compounds in common with components of the male sex pheromone (Howse and Knapp 1996; Light and Jang 1996) and it is therefore possible that males might use some of them to produce more effective pheromone emissions. Using a wind tunnel, Papadopoulos et al. (2006) found that female Mediterranean fruit flies showed greater arrestment on spheres emitting pheromone from sweet orange oil exposed males than on spheres emitting pheromone from non-exposed males. They suggested that this behavioural tendency by females may account for the greater mating success of males exposed to sweet orange oil due to the fact that such males would presumably have more opportunities to attract and successfully court females.
Based on the mating success rates reported in the literature, it is conceivable that citrus oils, and in particular the mixture of compounds that we used in our final experiment, may have an advantage over other oils of plant origin, such as ginger root oil and angelica seed oil, in enhancing the mating performance of released, sterile C. capitata males in the wild. This could be tested experimentally by directly comparing the various oils. Citrus oils are less expensive compounds than α-copaene, and thus their use in sterile male emergence and release centres could lead to a cost-effective and efficient implementation of SIT programmes against C. capitata.
Future experiments will aim to find out the active ingredients in citrus oils and their ratio in an optimized mixture that would have a maximum effect in enhancing mating competitiveness and could be used in SITs operations in a cost-effective way. The final evaluation of the mixture should be conducted in field cages keeping all conditions as close as possible to the ones found in natural settings. The appropriate combination of an optimum, pro-biotic bacteria and protein-containing pre-release adult diet, together with an optimized mixture of the main citrus oil compounds, might increase substantially the performance of released sterile males, and therefore, the efficacy of sterile insect release programs against the Mediterranean fruit fly (Gavriel et al. 2009; Maor et al. 2004; Yuval et al. 2002).
This work was supported by FAO/IAEA Grant 12857/RO to N.A.K. We thank A. Jessup for facilitating the shipping of sterile flies from the FAO/IAEA Agriculture and Biotechnology Laboratory in Seibersdorf, Austria, and G. Spyrou, K. Thalassinou, C. Damaskinou and N. Koufali for technical support. N.A.K. acknowledges the Fulbright Foundation in Greece for the award of a Fulbright Grant.