Microsoft Word - rjs-vol-ii-wickramasekara-new-1.2.doc © 2007 Faculty of Science University of Ruhuna RUHUNA JOURNAL OF SCIENCE Vol. 2, September 2007, pp. 89-95 http://www.ruh.ac.lk/rjs/rjs.html ISSN 1800-279X 89 Abstract. Aphidius rhopalosiphi is a parasitoid, which parasitizes the aphid Rhopalosiphum padi on wheat and is a potential candidate in Biological Control Programmes. The effect of honeydew excreted by host aphids A. rhopalosiphi and Metapolophium dirhodum, and honeydew extracted by non-host aphids, Mizus percicae and Brevicoryne brassicae on searching and walking time of A. rhopalosiphi was tested. Water was used as the control. Walking time of A. rhopalosiphi on honeydew treated area was also investigated. When parasitoids were released, the time taken to reach the honeydew treated discs was significantly shorter than the time taken, to reach the control discs (P ≤ 0.05). The time taken by parasitoids to reach areas treated with honeydew of different aphid species did not differ significantly among the species (P ≥ 0.05). The time spent on different honeydew treated discs was significantly higher than controls (P ≤ 0.05). The time spent on non-host honeydew was significantly shorter than the time spent on the host honeydew (P ≤ 0.05). Speed of movements of A. rhopalosiphi on treated area was significantly higher than non- treated area (P ≤ 0.05). These results revealed that honeydew of both hosts and non -hosts act as an attractant. Distinct behaviour pattern suggests that host honeydew act as a stimulant as well. Keywords: Aphidius rhopalosiphi, searching behaviour, host honeydew, non-host honeydew 1. Introduction Aphids (Hemiptera: Aphidae) are one of the major groups of insect pests of wheat (Emden, 1972). Aphidius rhopalosiphi L. (Hymenoptera: Aphidiidae) is a well-known parasitoid of the aphid Rhopalosiphum padi (Hemiptera: Aphidae) which feeds on wheat. Searching behaviour of the aphid parasitoid Aphidius rhopalosiphi (Hymenoptera : Aphidiidae) in response to honeydew excreted by aphids on different host plants M.G.V. Wickremasinghe Department of Zoology, Faculty of Science, University of Ruhuna, Matara, Sri Lanka Correspondence: vineetha@zoo.ruh.ac.lk 90 Wickramasinghe:Searching behaviour of the aphid… R u h u n a J o u r n a l o f S c i e n c e 2 , p p . 89-95 ( 2 0 0 7 ) Biological control has drawn the attention of scientists as well as policy makers (Martin, et al., 2003) as an alternative to chemical control that brings about harmful effects on the environment as well as on human health. Therefore, it is very important to investigate the searching behaviour of a parasitoid to find the host. Searching time of a parasitoid is one of the indicators of its performances (Vinson, 1978). The olfactometer studies revealed (Wickremasinghe and Van Emden, 1992) that several species of aphid parasitoids have been attracted to the odour of the plant which is host of their aphids, more than to the odour of host aphid or their honeydew, in the absence of the host plant. However, a combination of host plant and host aphid odour proved the most attractive stimulus, of those tested. Although not tested directly the magnitude of the responses suggests that honeydew was a stronger attractant than host aphids. Honeydew is excess sap excreted by aphids following feeding on the host plant phloem sap. The behaviour of parasitoids on honeydew contaminated plants suggested that honeydew could be an arrestant as well as a searching stimulant. An arrestant may be defined as a chemical that congregates insects as a result of undirected kinetic reactions, either the slowing down or stopping locomotion (Orthokinesis) (Shorey, 1977). To investigate this further, effect of honeydew from host and non-host aphid species on searching and walking times of the parasitoids were investigated under the laboratory conditions. 2. Materials and Methods 2.1 Aphids and host plants The four different aphid species and their host plants selected for the study were Metapolophium dirhodum and Rhopalosiphum padi found on wheat, Brevicoryne brassicae found on Brussels sprouts and Myzus persicae found on tomato plants. Each aphid species was reared on the relevant host plant. Plants were kept in culture cages (25×35×60 cm) covered in muslin. Wheat, brussels sprouts and tomato were grown in separate pots (12 cm diameter) and were infested with the relevant aphid species. 2.2 Collection of honeydew To collect honeydew 10 aphids from each species were caged separately on leaves of its host plant in para film lined clip cages for 24 hours. Honeydew excreted by each aphid species was collected on para film and was removed using capillary tubes. Immediately after collection 2µl of honeydew was applied on to para film discs (3.5 cm diameter) and placed inside a Petri dish. A hole of 1.5 diameter was made in the center of the lid of each Petri dish and a perspex tube (1.5 cm wide and 6 cm long) was fixed to make a passage for introducing parasitoids in to each test chamber. 2.3 Rearing of Aphidius rhopalosiphi Aphidius rhopalosiphi was reared on R. padi infesting the wheat variety Hobbit. Wheat was grown in two separate cages (25×35×60 cm). Parasitized aphids were collected using a wet paint brush and placed in specimen tubes lined with cotton wool to prevent desiccation of the parasitized aphids. Parasitoids, 24-48 h after emergence, were used in this experiment. Wickramasinghe:Searching behaviour of the aphid… 9 1 R u h u n a J o u r n a l o f S c i e n c e 2 , p p . 89-95 ( 2 0 0 7 ) Behaviour of parasitoids was observed in an observation chamber. A Petri dish containing a honeydew-treated para film disc was placed inside the observation chamber. The top and the four sides of this chamber were covered with opaque oil paper to prevent entry light which could disturb the behaviour of parasitoids. Experiment was started by releasing a single parasitoid at the top of the tube leading to Petri dish in the test chamber. A parasitoid was released by inverting the specimen tube containing the parasitoid over the opening of the central tube. Time taken by parasitoids to reach the honey dew treated disc and the time spent for searching inside the honeydew treated disc was recorded. Ten parasitoids were allowed to search on each honeydew treated para film disc. Eight replicates were used for each treatment. Discs treated with 20 µl of water were used as controls. In another experiment, whether the honeydew acts as a searching stimulant was tested. The movement of A. rhopalosiphi was observed in the treated para film discs. Twenty microliters of honeydew solution was applied to a 4 cm diameter spot in 0.5µl droplets. Treated discs were air dried before use. Eight such discs were prepared. At the beginning of the experiment a Petri dish with a honeydew-treated filter paper was placed inside the observation chamber and a single parasitoid was released into the arena, and the behaviour was observed. The movement of the parasitoid was measured by tracing the path followed by the parasitoid on the filter paper. A transparent paper was placed above the top of the Petri dish and trace of the path was marked directly on the transparent paper. The time spent outside and inside honeydew treated area by each parasitoid was measured and the distance travelled was measured using the trace. Ten parasitoids were allowed to search on treated discs. Eight treated discs were used in each experiment. 3. Results The time taken since the release of the parasitoid into the tube above the arena to reach the treated part of the test disc was significantly shorter with honeydew treated than the control (P ≤ 0.05) To water treated discs 44% of the parasitoids tested failed to react at all. The time taken by parasitoids to reach areas treated with honeydew produced by different aphid speciesdid not differ significantly (P ≤ 0.05) (Table 1). Table 1 : Time spent on searching on host/non host honeydew by A. rhopalosiphi Honeydew producing spp. Time spent on searching X ± SE(s) Time taken to reach the test disc X ± SE(s) % parasitoids reached the test chamber R. padi on wheat 39.342 ± 2.14c 42.93 ± 7.65a 100 92 Wickramasinghe:Searching behaviour of the aphid… R u h u n a J o u r n a l o f S c i e n c e 2 , p p . 89-95 ( 2 0 0 7 ) M. dirhodum on wheat 36.56 ± 2.15c 44.65 ± 7.47a 100 M. persicae on tomato 14.03 ± 1.24b 51.87 ± 6.95a 100 B. brassicae on brussels sprouts 15.34 ± 1.43b 48.84 ± 5.57a 100 Control (water) 9.22 ± 0.76a 181.81 ± 28.3b 56 Means with different supper script letters in each column are significantly different (One way ANOVA using log transformed data). Means separated by LSD at P ≤ 0.05. Searching time of a parasitoid is a good indicator of its performances. However, the time spent for searching on honeydew of R. padi and M. persicae fed on wheat were similar and significantly shorter than the time spent searching on the honeydew of B. brassicae fed on Brussels sprouts and M. persicae on tomato (P ≤ 0.05). The speed of movement of A. rhopalosiphi inside the honeydew treated area was significantly lower than the out side (P ≤ 0.001) (Table 2). Table 2 : Rate of movement of A. rhopalosiphi in the presence of honeydew Location Movement (mm/s) a) Outside the treated area 14.14 ± 0.31a b) Inside the treated area 3.24 ± 0.47b Means followed by different supper script letters are significantly different at P ≤ 0.05. When a parasitoid was introduced in to the tube leading to the arena it did not immediately orient itself to the arena containing honeydew but took some time to walk in to the arena. Parasitoids walking towards the area appeared excited, probing the substrate with the antennae as they walked. The time taken by the parasitoid to reach the honeydew treated disc and their behavior inside the inverted tube was similar regardless of the type of honeydew. In the control, parasitoids were not active; many of them remained on the top of the inverted specimen tube without moving out. Only 56% walked inside the tube, but even those took about four times longer duration to reach the water treated compared to honeydew treated discs. All parasitoids responded positively to the honeydew treated discs, indicating the presence of an attractant. The parasitoids searched for discs treated with honeydew of their hosts (R. padi and M. dirhodum feeding on wheat) took about half a minute. Abdominal protractions and frequent antennation were observed. Some of the parasitoids tried to oviposit on the honeydew droplets. When parasitoids searched discs with the honeydew of M. persicae and B. brassicae fed on tomatoes and Brussels sprouts respectively, frequent antennation could be observed but abdominal protractions were rarely observed. This suggests that the host honeydew could be a stimulant most probably for oviposting. Wickramasinghe:Searching behaviour of the aphid… 9 3 R u h u n a J o u r n a l o f S c i e n c e 2 , p p . 89-95 ( 2 0 0 7 ) 4. Discussion From the observations and responses measured, it is apparent that A. rhopalosiphi responds to odour of non-host and host honeydew and could discriminate host from non host honeydew upon contact. The time spent searching discs with honeydew of R. padi and M. dirhodum was about 2.5 times greater than the time spent searching with honeydew of M. persicae and B. brassicae. In the second experiment, parasitoids walking across the untreated area showed distinct behaviourial changes upon contact with the honeydew contaminated area. When parasitoids were released on to the test disc, firstly they walked rapidly; did not show any abdominal movements or antennation and they rarely turned back. However, when it entered the honeydew treated area, frequent abdominal protractions and zig- zag movements (angle turns) increased. Parasitoids stopped very frequently and touched the substrate with the antennae. Parasitoids moving out of honeydew treated area touched the filter paper with antennae and quickly turned back towards the area. Apparently the overall retention of parasitoids on honeydew is reflected through several different mechanisms shown by the parasitoids as arrest, decreased walking speed (orthokinesis) and increased turnings (klinokinesis). Honeydew therefore appears to be an arrestant, which keeps the parasitoid in the honeydew contaminated area, increasing the chance of contact of host aphids. The attraction of A. nigripes toward both non-host and hosts aphids has been reported by Bouchard and Cloutier (1984). A. nigrips responded positively to the odour of the honeydew of the preferred host Macrosiphum euphobiae but also that of less preferred hosts such as Myzus perssicae, Apis nasturtii and a non host Rhopalasiphum maidis. Hagen et al. (1976) reported that Chrysopa carnea, a predator of Theiroaphis trifolii was attracted to the odour of the host honeydew; van Emden and Hagen (1976) subsequently found that a break down product of the tryptophan in the honeydew was the attractant, which Hagen et al. (1976) reported as sucrose the arrestant. Behavior of A. rhopalosiphi suggestes that attractant may be present in both host and non-host honeydew, but the stimulant is present only in host honeydew. Frequent abdominal protractions by A. rhopalosiphi at host honeydew indicated that an oviposition stimulant may be present in the honeydew and this response would cause the parasitoid to increase its searching time. That abdominal protractions were rare on non- host honeydew indicated that the oviposition stimulant was either absent or most probably present in smaller amounts. Such differences between host and non-host honeydew might be expected to arise from diversity of chemicals present in the honeydew of aphids feeding on different plants (Auclair, 1958, 1963; Sidhu and Patton, 1970). Similar changes in the locomotory behaviour following contact with kairomones have been found for other parasitoids (Chiri and Leghan, 1982; Strand and Vinson (1982); Vet and van der Hoven, 1984; Waage, 1978). Strand and Vinson (1982) reported increased walking speed of Cardiochiles nigriceps. A behavioural response similar to that shown by A. rhopalosiphi in this study, has been reported by Waage (1978) for locomotory response of the ichneumoid Nemeritis canescence to the mandibular gland secretion of its host Plodia interpucntella. In response to the presentation of the host chemical on a surface, a walking Nemeritis exhibited a complex orthokinetic responses involving stopping, walking at a 94 Wickramasinghe:Searching behaviour of the aphid… R u h u n a J o u r n a l o f S c i e n c e 2 , p p . 89-95 ( 2 0 0 7 ) reduced speed and probing with the ovipositor. The wasps loosing contact with the treated area exhibited a klinokinetic response and returned back to the treated area. Two Leptopilina species such as L. fembriata and L. heterotoma, which attacks Drosophila spp. reduce their walking speed and increase their probing frequency (Chiri and Lengner, 1982). A significant increase in the number of both oviposition reactions (host stinging) and egg laying has been observed in Aphidius eivi when the dummies of aphids are coated with conical secretion (Larocca, 2007). However this enhancement has not been observed when the aphid dummies contained distilled water. Budenburg & Powell (1997) has reported that Episyrphus balteatus (Deg) landed more frequently on wheat ears contaminated with host aphid honeydew than on clean ears suggesting a response to honeydew volatiles. The number of eggs laid by E. baltetus has been increased with increasing honeydew concentration. Honeydew of host aphids attracts the parasitoids from a distance (Bouchard & Cloutier, 1984) then arrests the parasitoid in the contaminated area. Slowing down the increased antennae examination would increase the possibility of the parasitoid encountering the host. 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