Effect of citrus peel substances on male Mediterranean fruit fly behaviour

Proceedings of 6th International Fruit Fly Symposium 6 10 May 2002, Stellenbosch, South Africa pp. 13 17 Effect of citrus peel substances on male Mediterranean fruit fly behaviour Byron I. Katsoyannos 1, Nikos T. Papadopoulos 1 *, Nikos A. Kouloussis 1 & Jorge Hendrichs 2 1 Laboratory of Applied Zoology and Parasitology, Department of Agriculture, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece 2 Insect Pest Control Section, Joint FAO / IAEA Division, Vienna, Austria The response of wild Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) males to chemicals from the peel of citrus fruits was quantified with choice tests in large field cages housing naturally planted orange trees. Males were strongly attracted to and arrested on oranges (and to a lesser extent other citrus fruits) on which superficial cuts were made in the oily region of the peel (flavedo). Response was observed throughout the day and increased as the number of cuts per fruit increased. Feeding on the peel wounds,and homosexual activity on or near them,was frequently observed.no similar response was observed for females and it is possible that male-specific response might be related to the mating behaviour of the fly. In a second set of experiments, the possible effect of orange peel chemicals on increasing mating competitiveness of males exposed to them was further studied in the laboratory. Males were also attracted to, landed and arrested on, and fed on superficial wounds made on the peel of ripe oranges. Exposure to wounded oranges conferred to males a mating advantage over unexposed males. In flies of 1 10 days of age, this advantage was independent of the age of the flies during exposure and lasted at least 10 days following exposure. Twenty-four hours of exposure ensured this effect. Exposure to intact oranges, or to wounded oranges covered with a wire-screen which allowed olfactory response and landing on the screen but not direct contact with the fruit, did not confer any mating advantage. It is possible that the acquisition of certain substances in the flavedo, most probably through their ingestion, is responsible for the observed phenomenon. Enhancement in mating competitiveness of released sterile males exposed to peel chemicals may improve the effectiveness of sterile insect technique programmes. INTRODUCTION Resource-finding behaviour in insects is the product of responses to an array of sensorial information from the environment, which interact simultaneously or sequentially. Tephritid fruit flies use both olfactory and visual cues to seek and assess habitat, food, ovipositional and mating resources (Prokopy & Roitberg 1984; Prokopy 1986; Katsoyannos 1989). In some species, plant chemicals are sequestered and used as sex pheromones or pheromone precursors. In others, pheromone is released in response to host plant cues, while a synergistic effect of plant compounds with sex pheromones has also been reported (Landolt & Phillips 1997). The Mediterranean fruit fly (Medfly), Ceratitis capitata (Wiedemann), is a polyphagous tephritid that attacks the ripening fruit of numerous commercially important fruit crops (Liquido et al. 1991).There are several studies aimed at explaining the mating behaviour of this species, where there are still many unclarified aspects.for example,little information exists about the role that host plant chemicals might play in mating behaviour. Teranishi et al.(1987) observed male attraction to odour from oranges and suggested -copaene as *To whom correspondence should be addressed. Present address: Department of Agriculture Crop Production and Rural Environment, University of Thessaly, Fytokou str. N. Ionia 384 46 Magnisia Greece. E-mail: nikopap@agr.uth.gr the active compound without, however, supporting these observations with quantitative data. Papaj et al.(1989) showed that Medfly females were more likely to land on and attempt to oviposit into oranges that were artificially wounded (by piercing the fruit peel and pulp and causing the release of juice).in another study,levinson et al.(1990) tested the response of a laboratory strain of Medflies to certain fruits,including Citrus spp.,and inferred that fruit volatiles were not essential for the location of those fruits by the flies.however, in the same study electrophysiological recordings from olfactory sensilla on the antennal funiculi revealed that essential oils of orange and lemon fruits provide more olfactory stimuli to females than to males. Nonetheless,there are no comparative field studies on the response of of male and female Medflies to substances emanated by citrus fruits and on the importance of this response for the fitness of the two sexes, especially for males. Existing information suggests that females use olfactory stimuli to locate a suitable host-fruit for oviposition.however, it is not known why males respond to plant compounds. In other tephritids such as Bactrocera dorsalis (Hendel), it is known that males acquire plant-originated substances, use them as pheromone precursors (Landolt & Phillips 1997), and males ingesting them gain an advantage in mating

14 Proceedings of the 6th International Fruit Fly Symposium success over deprived males (Nishida et al. 1997). This study summarizes results on quantitatively assessed behavioural responses of both sexes of wild Medflies to citrus fruit peel and pulp chemicals, under semi-natural conditions, and in a subsequent laboratory experiment we investigated the biological significance of orange peel chemicals on male sexual behaviour, especially on the mating competitiveness of males. MATERIALS AND METHODS Field response to citrus chemicals Field-cage experiments were conducted on the island of Chios, Greece, in a citrus orchard. Two cylindrical (2 m high, 2.6 m diam.), plastic-screen field cages were used, each housing a 20-year-old sweet orange tree (Citrus sinensis Osbeck) of 2.5 m canopy diameter. Adult food (a mixture of yeast hydrolysate and sugar) and water were provided ad libitum. Adult Medflies were obtained from naturally infested sour oranges (Citrus aurantium L.) collected from orchards in the area. They were kept at ambient conditions in groups of 200 300 individuals of both sexes,in 40 40 60 cm organdy gauze net cages with wire frames and provided with water and food. All flies used in the experiments were 1 12 days old. To conduct a test, 200 mated or unmated flies (sexed at eclosion) of a given age and sex were introduced into each field cage the evening before the initiation of the test. During an experimental day, the same flies were subjected to up to eight replicates of various treatments. To assess response to chemicals from the outermost oily region of the peel (flavedo), uninfested, ripening sour oranges, sweet oranges and lemons (Citrus limon L.) were wounded by removing a c. 0.5 mm deep disk of peel tissue at five different positions on each fruit with a sharp razor. Each removed disk left a fruit surface area of approximately 3 cm 2, which contained severed oil glands, resulting in the release of peel oil components. For each test, fruits were introduced into the field cages immediately after they were treated. Fruits were suspended from their stems by 10-cm long wires in shadowed areas on the tree branches.four fruits were suspended on each tree,of which one or two were untreated (controls). Fifteen minutes after the introduction of the fruits into the cages, the number of insects on each fruit were counted. Counts of responding flies were supplemented by direct observations of fly behaviour made by an observer inside each cage. All tests were conducted between 09:00 and 18:00. To evaluate essential oils, a piece of cotton wool was treated with 0.5 ml of essential oil (purchased locally) from either mandarin (Citrus reticulata Blanco), orange or lemon fruit.the cotton was then gently rubbed onto the surface of a yellow, plastic, 7.0-cm diameter hollow sphere and then attached to the upper part of the sphere. Two such spheres and two untreated controls were each suspended from a caged tree by a 10-cm long wire.the experimental procedure which followed is as described above. Mating competitiveness laboratory experiments The experiments were conducted in the laboratory at 25 ± 2 C, 65 ± 5% R.H. and a photoperiod of L14:D10, with photophase starting at 06:00. The flies used were retrieved from field-infested apples collected in autumn and reared in the laboratory for one generation on sour oranges. One day after emergence they were separated by sex and placed in wire-screened, wood-framed holding cages (30 30 30 cm), provided with food and water. All oranges used in this study were mature, Navel variety, purchased from the local market. The wounded oranges used had five superficial cuts in the flavedo region. The main treatment consisted of confining male flies with ripe oranges that were superficially wounded on the peel as described above. Before testing,males were kept for various periods in holding cages containing a wounded orange, in groups of 50 70 per cage. A newly wounded orange was provided daily when exposure lasted more than one day.control males were kept in the same room and type of cage as the treated ones (but without an orange), at a distance of 4 5 m from the treatment cages. The experimental procedure consisted of competitiveness tests where one treated male (which was in most cases a male exposed to wounded oranges) and one untreated control male of the same age competed for a virgin female of the same age and origin. Tests were conducted after adult day 11 when the flies were sexually mature (Papadopoulos et al. 1998). On the day before testing, one treated and one control male were introduced into a 15 15 15 cm perspex cage. At 10:00 the next day, one virgin female was introduced into the cage.cages were observed at 5 min intervals until 14:00, and the pairs formed with either treated or control males were recorded. Before testing, both treated and control males

Katsoyannos et al.: Effect of citrus peel substances on male Mediterranean fruit fly behaviour 15 were marked with a dot of different colour using correction fluid (Tipp-Ex ). In another experiment we tested the mating competitiveness of the following categories of treated males against control males: (a) males that were freely exposed to a wounded orange, (b) males that were exposed to a wounded orange that had been placed inside a small wire-screen cage that prevented direct contact of males with the orange, and (c) males that were exposed to an unwounded orange. Treated males were exposed to oranges from emergence up to adult day 10, and mating competitiveness tests were conducted on day 11. To study the persistence of mating advantage, males were exposed to wounded oranges from adult days 1 to 10. Afterwards they were denied access to wounded oranges and their mating competitiveness against control males was subsequently tested 1,3,6,10 and 12 days after exposure. To study the effect of fly age on exposure,in a final experiment males of different ages (1, 5 and 9 days old) were exposed to a wounded orange for 24 hours. Mating competitiveness tests were conducted on adult day 11. RESULTS Field response to citrus chemicals To test whether flies respond to peel chemicals from the flavedo of oranges, males or females were given a choice of oranges with or without five peel cuts. Males were sighted often on treated oranges and very rarely on controls, exhibiting a statistically significant (paired t-test, P < 0.05) positive response to chemicals from the cuts (Fig. 1). Males were frequently observed lowering their head and contacting the sap from the cuts with their mouthparts. Male homosexual activity (mating attempts) on or near the cuts was also commonly observed. Direct observations revealed none to few males on the control fruits throughout the 15-min test period.after landing on treated fruits,most individuals remained there throughout the 15-min period. These observations strongly suggest that flies were attracted from a distance to properties of the treated fruit, and thereafter arrested on it. By contrast, virgin or mated mature females visited the treated and the control oranges at equal rates, displaying no heightened response to peel chemicals (Fig. 1). Direct observations showed that none of the few females recorded on fruits was exploiting the peel cuts for oviposition or other purposes. These results demonstrate a marked Fig. 1. Field-cage response of Ceratitis capitata to peel chemicals from ripening oranges bearing five superficial disk cuts in the flavedo (200 males or females were used per replicate; the duration of the bioassay was 15 min). disparity between males and females in their responses to peel chemicals. When given a choice of orange, sour orange or lemon, all with peel cuts, sexually immature, unmated males responded only to chemicals of orange, whereas mature, mated males were also sensitive to chemicals of sour orange (Fig. 2). By contrast, immature, mated and unmated females were not responsive to peel chemicals from any of the fruits. When given a choice of spheres treated with commercial essential peel oils of orange,mandarin, or lemon, the degree of male attraction differed significantly. Males were particularly responsive to orange oil, less sensitive to mandarin oil and unresponsive to lemon oil (Fig. 3).Virgin and mated mature females displayed very low responses (approximately zero) to all three oils, even lower than the response to control spheres. Fig. 2. Field-cage choice response of Ceratitis capitata to peel chemicals from different ripening citrus fruits bearing five superficial wounds in the flavedo (200 males or females were used per replicate; the duration of the bioassay was 15 min).

16 Proceedings of the 6th International Fruit Fly Symposium Mating competitiveness in the laboratory The results of the experiment that included the treatment with the screen cover showed that free exposure to orange wound exudates conferred to males a mating advantage (74% of all mating was achieved by treated males and only 26% by control males) over control males (paired t-test, P < 0.05). Exposure to wire-screen-covered,wounded oranges or uncovered, unwounded oranges did not confer any mating advantage (51 58% of all mating for treated males and 49 42% for control males) over control males (P > 0.05). These results strongly suggest that the mating advantage observed in those males confined with wounded oranges is dependent on direct contact of males with the wounded oranges, and most probably on the ingestion of substances from the severed flavedo tissues. Males exposed for 10 days to wounded oranges retained a mating advantage over the untreated males for at least 10 days after exposure (P < 0.05). This mating advantage gradually decreased as the number of days since exposure increased. Treated males achieved 88% and 65% of all matings 1 and 10 days after exposure respectively. However, 12 days after exposure there was no significant difference in mating success between treated ( 60% of matings) and control males ( 40% of the matings; P > 0.05). Since the age of the flies increased in each subsequent test so that those flies tested 12 days after exposure were 22 days old, age differences may to a certain degree have affected the results obtained. Males exposed to wounded oranges for 24 h on adult day 1, 5 and 9 and tested on day 11, all gained a strong mating advantage (70 91% of all matings) over control males (P < 0.05). Since the tests were conducted 10, 5 and 1 days after exposure, respectively, the differences in the strength of mating advantage of the three treatments is presumably attributed to the number of days between exposure day and test day. The results show that irrespective of the day of exposure (including the first day after emergence), 24 hours of exposure is enough to ensure a high mating advantage of treated over control males for at least 10 days after exposure. DISCUSSION This paper integrates the results of two recent studies (Katsoyannos et al. 1997; Papadopoulos et al. 2001). It demonstrates, first, a strong field response of male Medflies (and a lack of response of females) to chemicals emanating from citrus Fig. 3. Field-cage response of Ceratitis capitata to 7-cm diameter spheres treated with 0.5 ml of peel oil from different citrus fruits (200 males or females were used per replicate; the duration of the bioassay was 15 min). peels, and second, the acquisition of a significant mating advantage of males coming into direct contact with orange peel substances over unexposed males. The marked disparity in response between the two sexes in the field cages possibly reflects differences in ecological needs for hostfruit recognition between males and females. Males gained a mating advantage only after landing on a wounded orange and bringing their proboscis in contact with chemicals exuding from the wounds. This is a strong indication that ingestion of flavedo chemicals may be the factor responsible for the subsequent enhancement of mating competitiveness. It is not unlikely that C. capitata males use these same chemicals in the biosynthesis of sex pheromone. -Copaene might be an active compound responsible for C. capitata male attraction to orange peel substances and the enhancement of mating competitiveness. This substance is known to be very attractive to males and occurs in the essential oils of many plant species (Teranishi et al. 1987). The monoterpene limonene, which is the principal component of orange oil, may also be involved in the observed enhancement in male mating competitiveness (Stashenko et al. 1996). Enhancement of mating competitiveness of mass-reared sterile males after exposure to orange peel chemicals may considerably improve their mating ability. Our finding that the mating advantage of males exposed to orange peel substances is long-lasting (at least 10 days after exposure) and independent of the age of males at exposure (up to the tenth day of age) may be very important in sterile insect technique programmes. If young sterile male Medflies are exposed to orange peel substances before being released, a substantial

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