Revista de la Sociedad Entomológica Argentina ISSN: 0373-5680 [email protected] Sociedad Entomológica Argentina Argentina

HEIT, Guillermo E.; SARDOY, Pedro; COHEN, Graciela R.; MAREGGIANI, Graciela Locomotor activity of (Coleoptera: ) exposed to volatile semiochemicals and to direct contact with the odour source Revista de la Sociedad Entomológica Argentina, vol. 66, núm. 3-4, 2007, pp. 197-203 Sociedad Entomológica Argentina Buenos Aires, Argentina

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Locomotor activity of Cycloneda sanguinea (Coleoptera: Coccinellidae) exposed to volatile semiochemicals and to direct contact with the odour source

HEIT, Guillermo E.*, Pedro SARDOY*, Graciela R. COHEN** and Graciela MAREGGIANI*

*Cátedra Zoología Agrícola, Facultad de Agronomía, Universidad de Buenos Aires. Avda. San Martín 4453. (1417) Buenos Aires, Argentina; e-mail: [email protected], [email protected] **Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. Pabellon 2, Ciudad Universitaria. (C1428EHA) Buenos Aires, Argentina; e-mail: [email protected]

Actividad locomotriz de Cycloneda sanguinea (Coleoptera: Coccinellidae) expuesta a semioquímicos volátiles y al contacto directo con la fuente de olor

„ RESUMEN. Cycloneda sanguinea (Linnaeus) (Coleoptera: Coccinellidae) es un predador polífago oportunista y cuando su menú está dominado por áfidos, es esperable un comportamiento complejo de forrajeo. Un movimiento activo, caracterizado por alta actividad locomotriz, juega un rol importante en la búsqueda de esta presa. En este trabajo se analizó el comportamiento de adultos de C. sanguinea expuestos a los semioquímicos volátiles, emitidos por sustratos de Capsicum annuum Linnaeus (Solanaceae) sanos o infestados con Myzus persicae (Sulzer) (Hemiptera: Aphididae), o simplemente puestos en contacto directo con esos sustratos. Los tratamientos evaluados fueron: A) hojas de pimiento infestadas con alta densidad de áfidos, B) hojas de pimiento infestadas con baja densidad de áfidos, C) hojas de pimiento sanas y D) control. La actividad locomotriz no difirió significativamente entre los distintos tratamientos cuando C. sanguinea se expuso solamente a los volátiles. En cambio, cuando los coccinélidos se pusieron en contacto directo con los sustratos evaluados, se encontraron diferencias significativas entre los tratamientos. Estos resultados preliminares indicarían que la sola presencia de un estímulo de olor, no sería suficiente para modular un patrón locomotor diferente en C. sanguinea.

PALABRAS CLAVE. Comportamiento de forrajeo. Capsicum annuum. Cycloneda sanguinea. Myzus persicae. Actómetro.

„ ABSTRACT. Cycloneda sanguinea (Linnaeus) (Coleoptera: Coccinellidae) is an opportunistic polyphagous predator and when aphids dominate its menu, a complex foraging behaviour can be expected. An active movement characterized by a high locomotor activity plays an important role in the search of this prey. The behaviour of C. sanguinea adults exposed to volatile semiochemicals emitted by Capsicum annuum

Recibido: 29-V-2007; aceptado: 26-IX-2007 198 Rev. Soc. Entomol. Argent. 66 (3-4): 197-203, 2007

Linnaeus (Solanaceae) substrates, healthy or infested with Myzus persicae (Sulzer) (Hemiptera: Aphididae), or in direct contact with these substrates was here analyzed. The treatments evaluated were: A) high aphid-infested pepper leaves, B) low aphid-infested pepper leaves, C) healthy pepper leaves and D) control. Locomotor activity was not significantly different among treatments when C. sanguinea was exposed only to the volatiles. However, when adults were placed in direct contact with the evaluated substrates, statistically significant differences were found among treatments. These preliminary results could indicate that the sole presence of an olfactory stimulus could not be sufficient to modulate a different locomotor pattern in C. sanguinea.

KEY WORDS. Foraging behaviour. Capsicum annuum. Cycloneda sanguinea. Myzus persicae. Actometer.

INTRODUCTION circumscribed search, passing over long straight paths quickly and making just a few Food sources are distributed along the stops (Ferren & Dixon, 1993; Carter et al., environment, both in time and space. To 1984; Bond, 1980). In this way, they forage successfully, predators and parasitoids minimize the waste of time on unprofitable must face a trade-off between the most proper areas, avoiding unnecessary energy moments to look for their food and the expenditure (Gendron & Staddon, 1983). ACS amount of resources present in a certain foraging behaviour would match to some volume of space. extent the distribution of resources in a Coccinellids are opportunistic continuous patchy environment. polyphagous predators, but their essential Some authors consider that coccinellids food requirements are very specific (Soares look randomly for their preys and can detect et al., 2004). In Argentina, Cycloneda them only when direct contact is achieved sanguinea (Linnaeus) (Coleoptera: (Ferran et al., 1994). This foraging behaviour Coccinellidae) is considered an important has great advantage when the prey lives aphids predator, but its impact in biological gregariously, such as aphids. Perhaps this is control is very variable, particularly due to the reason why coccinellid ovipositions are its polyphagy. When aphids dominate their mostly located in aphid-infested places or menu, a complex foraging behaviour near them (Nakamuta, 1982), favoring representative of a huge range of generalist successful prey foraging when ladybirds predators, can be expected (Dicke, 1999; larvae hatch. On the other hand, newly Ninkovic et al., 2001). emerged adult coccinellids must look for new According to the Area Concentrated patches, because usually all the preys around Search (ACS) behaviour model, if predators them have been consumed during juvenile such as coccinellids detect their preys, which development (Obata, 1986). are usually distributed in patches throughout Other authors (Dicke, 1999; Al Abassi et the environment, after feeding, they will al., 2000; Ninkovic et al., 2001; Acar et al., explore the prey-patch intensely (Wiens, 2001) propose that a wide diversity of 1976). They will do this in order to delimit a generalist predators such as ladybirds use concentrated area to search for more different semiochemicals or infochemicals resources. This area will be slowly swept emitted by plants and to mediate in a following sinuous ways and making series of key processes during foraging numerous stops in order to raise the behaviour. probability of finding more preys. On the An active movement of predators, other hand, if predators do not find preys, characterized by a high locomotor activity, they will undertake a more extensive or less plays an important role in the search for preys HEIT, G. et al. Locomotor activity of Cycloneda sanguinea 199

(Bell, 1990). However, little research on the squares (1.5 cm side) of a squared grid crossed effect of volatile semiochemicals in by the during its movement for 60 coccinellid locomotor activity has been done. seconds (Tourniaire et al., 1999). Data were The aim of the present investigation was analyzed with three way ANOVA and Tukey to evaluate the locomotor activity of the multiple comparison test (α=0.05). Square predator C. sanguinea, exposed to volatile root transformations were used to fulfil the semiochemicals emitted by Capsicum assumptions of the test (Sokal & Rohlf, 1995; annuum Linnaeus (Solanaceae) plants, Zar, 1999). Data were analyzed using InfoStat healthy or infested with Myzus persicae program. (Hemiptera: Aphididae), or in direct contact with these materials. Two experimental series were done: MATERIAL AND METHODS First series: exposure to volatile Rearing of predator and prey. Culture of semiochemicals. Locomotor activity of plant-host Cycloneda sanguinea was evaluated with an actometer (modified from Manrique et al., Adults of Cycloneda sanguinea, collected 2006). In each test, a male or a female of C. in the campus of FAUBA (Facultad de sanguinea, chosen randomly from a Agronomía, Universidad de Buenos Aires), randomized sample of 24 h starved were reared in the laboratory. Ladybirds were individuals, was put in the experimental fed on nymphs and adults of the aphid M. arena. The actometer (Fig. 1) consisted of an persicae (Hemiptera: Aphididae). Every two acrylic cylindrical box 12 cm diameter, days, C. sanguinea eggs were collected and composed by two horizontal compartments put in a Petri dish until larval emergence. of the same height (2 cm), separated by a wire Neonate larvae from each cohort were reared mesh to allow the diffusion of odour stimulus separately in glass flasks closed with gauze. to avoid direct contact between the insect and The adults obtained were used for the the volatiles source. The upper compartment, bioassays. the arena, where the insects were placed, had Myzus persicae colonies were multiplied a removable and transparent cover on the top, in the laboratory on pepper plants, Capsicum with a squared grid drawn on its surface. The annuum CV. California Wonder, cultivated in lower compartment, the diffusion volatile 350 cc polyethylene pots. C. annuum was chamber, was connected by means of a selected as plant host due to its economic perforated plate in the centre of its floor, to a importance as horticultural crop and to its stimulus delivery chamber, where the odour high susceptibility to M. persicae sources were placed. After the introduction colonization. Predators, prey and host were of the insect into the arena, 4 minutes maintained in standardized conditions (24º acclimations were spent until the initiation C ± 2º C, 65 ± 10% RH, photoperiod: 16:8 h of the experiment, to allow the uniform light-darkness). diffusion of the volatile chemicals. The volatile sources evaluated were: A) Bioassays high aphid-infested pepper leaves (80 aphids/ replica) on filter paper, infested with Myzus Standardized conditions were used for the persicae for one month before the bioassays (24ºC±2ºC, 65±10% RH and light experiments, B) low aphid-infested pepper 60 watts). Every test was performed in the leaves (20 aphids/replica) on filter paper, morning (9-12 h) and in the evening (13-16 infested with M. persicae for one week before h). Cycloneda sanguinea individuals from the the experiments, C) healthy pepper leaves on same cohort, which were food deprived for filter paper, and D) control: clean filter paper. 24 hours, were tested. The behavioural Nine independent replicates resulting response, evaluated as the locomotor activity, from the combination of sex, moment of the was estimated according to the number of day (morning and afternoon) and volatiles 200 Rev. Soc. Entomol. Argent. 66 (3-4): 197-203, 2007

Fig. 1. Actometer scheme. Lateral view shows the disposition and dimensions of the compartments. Upper view shows the removable and transparent cover with a squared grid drawn on its surface, a wire mesh as the arena’s floor, and below this, the connection of the diffusion volatile chamber with the stimulus delivery chamber by means of a plate with holes, which allowed the diffusion of volatiles to the arena.

source were made for each treatment. The substrates, its locomotor activity was not experimental unit consisted of a male or a significantly different among treatments female of Cycloneda sanguinea. (F3,113,0.05=1.366, p=0.257). No differences Second series: Direct contact of predator were found between the mean locomotor with the odour source. The experimental activity in either female or male adults of this arena consisted of a Petri dish, 9 cm diameter, predator measured during the morning and with a squared grid drawn in the upper cover. afternoon (F1,113,0.05=3.683, p=0.058) (Fig. 2). In each test, a male or a female of Cycloneda However, when adults of the coccinellid sanguinea, chosen randomly from a were placed in direct contact with the randomized sample of 24 h starved evaluated substrates, statistically significant individuals, was put in the experimental differences were found among treatments arena, which allowed the contact with the (F3,112,0.05=73,62, p<0,0001) and between both substrate evaluated. moments of the day (F1,112,0.05=9.54, Treatments evaluated were the same as in p=0.0025) (Fig. 3). the first series, but in this case the aphids were The mean locomotor activity of removed from the infested leaves with a Cycloneda sanguinea measured in contact brush, to avoid fortuitous stops of predators with pepper leaves, with either high or low to eat, which could affect the locomotor density of infestation by Myzus persicae, was activity. Eight independent replicates resulting significantly lower than that recorded in from the same combinations as in the first contact with uninfested pepper leaves or series of experiments were performed. In the clean filter paper. Furthermore, C. sanguinea second series, the experimental unit consisted was significantly more active on filter paper of a male or a female of Cycloneda sanguinea, than on healthy pepper leaves (p<0.05). as well. In both experimental series, 3 gr of On the other hand, the mean locomotor pepper leaves were used as the odour source activity non discriminating among treatments for each bioassay. neither between sexes resulted statistically lower (p=0.0025) in the morning RESULTS (mean=3.87;S.E.=0.24) than in the afternoon (mean=4.44; S.E.=0.19). When Cycloneda sanguinea was exposed No significant differences between sexes only to the volatiles of the evaluated were observed neither during the volatile- HEIT, G. et al. Locomotor activity of Cycloneda sanguinea 201

Fig. 2. Locomotor activity of Cycloneda sanguinea adults (male and female), exposed to several substrate volatiles, evaluated during morning or afternoon hours. Locomotor activity was estimated according to the number of squares crossed by the insect during 60 seconds. Substrates: A) High-infested pepper leaves, B) Low-infested pepper leaves, C) Healthy pepper leaves, D) Control. Mean locomotor activity and standard error are shown.

Fig. 3. Locomotor activity of Cycloneda sanguinea adults (male and female), in contact with several substrates, evaluated during morning or afternoon hours. Locomotor activity was estimated according to the number of squares crossed by the insect during 60 seconds. Substrates: A) High-infested pepper leaves (aphids removed), B) Low-infested pepper leaves (aphids removed), C) Healthy pepper leaves, D) Control. Mean locomotor activity and standard error are shown. only-exposure series of bioassays different visual and olfactory cues for orientation (Nakamuta, 1980; Ferren & (F1,113,0.05=0.0004, p=0.983) nor during those of direct contact with the odour source Dixon, 1993; Lambin et al., 1996). Olfactory cues such as volatile semiochemicals emitted (F1,112,0.05=1.025, p=0.313). by the aphids, the host or their interaction DISCUSSION are chemical messages essential for survival of these species. Coccinellids are important natural Some authors (Dicke, 1999; Al Abassi et enemies of aphids and have often been used al., 2000; Ninkovic et al., 2001; Acar et al., as a tool for biological control of several 2001; Heit et al., 2005) consider that plagues (Obrycki & Kring, 1998). These coccinellids would be able to use olfactory predators are constantly monitoring the and/ or visual cues to optimise their foraging environment in search for food by means of behaviour. In this case, a specific stimulus 202 Rev. Soc. Entomol. Argent. 66 (3-4): 197-203, 2007

linked to the presence of the prey or pepper exposed to volatiles and measured during the leaves could trigger a distinctive locomotor morning and afternoon is very close to the activity pattern in the predator, although there limit of significance (α=0.05), which suggests may not be any contact with the aphids or that additional replicates should be added in the host plant. However, according to our order to obtain more conclusive results. results (Fig. 3), once Cycloneda sanguinea Instead, this activity in males and females put was in contact with a patch-prey on pepper on direct contact with the substrates was leaves infested with Myzus persicae significantly higher during the afternoon than (regardless of prey density), it followed a during the morning hours (p=0.0025). These behaviour similar to the one described by the results agree with Elliott et al (2000), who Area Concentrated Search model (ACS) and found that searching activity of Hippodamia moved slowly scanning exhaustively the area convergens Guerin-Meneville, Hippodamia around. These results agree with those of tredecimpunctata tibialis (Say), and Ferran et al. (1994), who pointed out that Coleomegilla maculata (De Geer) coccinellids could only detect their preys (Coleoptera: Coccinellidae) adults is when they become in contact with them. On influenced positively as time of day and the other hand, when C. sanguinea did not temperature increase in spring cereal fields. detect a feeding resource, i.e. in contact with The above mentioned could point out to uninfested pepper leaves or clean filter paper the presence of a rhythm in the foraging (control), it moved significantly faster, activity of Cycloneda sanguinea (Mishra & monitoring very superficially the area devoid Omkar, 2004). Foraging behaviour is one of of food. the main adaptive functions which allow These results could have also been predator insects to find their preys. To be affected by the presence of honeydew, a successful, the search for food must be done sugar-rich sticky substance released by aphids when and where the preys are more as they feed, which could act as an additional available. In this sense, volatile patch cue in a similar way as other sugars semiochemicals and/or tactile cues from that are phagostimulants for phytophagous phytosuccivorous preys, the host plant or their insects do (Mitchell & Gregory, 1979; interaction, must be necessary. The increase Mitchell & Harrison 1984; Blaney et al., of the locomotor activity in the afternoon 1990; Nagnan-Le Meillour et al., 2000). It suggests an adaptive behavioural response of has been reported that honeydew could this predator. influence the behaviour of syrphids. The prey searching behaviour did not Budenberg & Powell (1992) point out that differ between sexes in both experimental females of Episyrphus balteatus (De Geer) series during morning and afternoon. (Diptera: Syrphidae) landed more frequently Probably, foraging in this predator is the main on ears contaminated with honeydew than diurnal activity in both sexes. Later, in the on clean ears, suggesting a response to evening or night, foraging would stop and honeydew volatiles. Additional experiments sexual activities would start, and then, male could be useful to evaluate the effect of and female locomotor activity could differ. honeydew on foraging activities of Cycloneda Mishra and Omkar (2004) observed that the sanguinea adults. major foraging activity and field presence Our results indicate that the sole presence levels of Propylea dissecta (Mulsant) of an olfactory stimulus could not be sufficient (Coleoptera: Coccinellidae) occur in the to modulate a different locomotor pattern in photophase while peaks of rhythm of mating, Cycloneda sanguinea. Perhaps the lack of oviposition, hatching and moult occur in response to volatiles could be due to a short scotophase, then supposing that the acclimation time to the odour sources (4 min). concentration of life events other than New assays with longer acclimation periods foraging in the same moments could be a would be useful. survival strategy of evolutionary significance. The p value 0.058 for the mean locomotor activity of Cycloneda sanguinea (both sexes) HEIT, G. et al. Locomotor activity of Cycloneda sanguinea 203

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