Behavioral responses of parasitoids in the genus Melittobia (Hymenoptera: Eulophidae) to volatiles emitted by natural and factitious hosts. Dakota Camino 1, Antonino Cusumano 2, and Jorge M. Gonzalez 1 1Department of Plant Science, California State University, Fresno, CA, USA. 2Department of Agricultural and Forest Sciences, Universita` degli Studi di Palermo, Palermo, Italy ABSTRACT: Responses of macropterous females of seven species belonging METHODOLOGY: RESULTS AND DISCUSSION: to four species-groups of the ectoparasitoid genus Melittobia (Hymenoptera: Laboratory stock cultures of seven Melittobia species (M. acasta, M. assemi, M. Eulophidae) to direct cues emitted by some natural as well as laboratory hosts • After testing the seven Melittobia species on three different hosts (a australica, M. clavicornis, M. digitata, M. hawaiiensis and M. sosui ) have been were investigated using a Y-tube olfactometer. To locate the different hosts the pollinating bee (Megachile rotundata ), a muddauber wasp (Sceliphron wasps exploit volatile kairomones. These parasitoids did not respond in the maintained in continuous culture at 25 °C and 75% RH either on Trypoxylon spp. caementarium ) and a flesh fly (Sarcophaga bullata ), results indicated a overall same way to the volatiles emitted by all the offered hosts. Our results indicate (Hymenoptera: Crabronidae) prepupae or the factitious host Sarcophaga bullata preference towards the natural hosts (the bee and the wasp) than to the that odors emitted by the hymenopterans tested elicit a stronger response by the (Sarcophagidae) pupae. For comparison purposes, the Melittobia species were factitious host (the fly). different species of Melittobia. However, a limited attraction was found to the analyzed and arranged phylogenetically based on Tanner et al. (2011) (Figs 7-9) dipteran host, suggesting that parasitism of these hosts may be incidental, due to the broad host plasticity of Melittobia wasps. Our preliminary results are part Long Winged females (the dispersive group of any Melittobia species population) of our efforts to better understand the evolutionary trends that have led to the 1 – 5 days old, presumably mated, were collected from the stock cultures and formation of the species-groups and their interrelationships. separated in gelatin capsules prior to each trial. INTRODUCTION: Melittobia parasitoid wasps (Hymenoptera: Eulophidae) are Prepupae/pupae of several known hosts were tested in terms of response toward small (1 mm) gregarious idiobionts that parasitize many species of pollinating volatiles by Melittobia spp. All prepupae/pupae used were inside their cocoons. bees, as well as wasps (Hymenoptera) (Fig. 1), and even some flies (Diptera). Hosts used were: Alfalfa leaf-cutter bee (Megachile rotundata ), muddauber All species are sexually dimorphic (Figs. 2-3). They are cosmopolitan and 12 (Sceliphron caementarium ) and Flesh Flies (Sarcophaga bullata ). The two first are species have been described worldwide (Dahms 1984). Nine of them are natural hosts while the third one is a factitious one. present in North America, and they belong to four “species-groups” (species that share similar traits or closely related behaviors). Fifty different females of every Melittobia species were tested for each different host. Their response to volatile cues from the tested hosts were investigated using a Y-tube olfactometer made up of polycarbonate (stem: 90 mm long; arms: 80 mm long, with an angle of 130° in between; inner diameter: 15 mm) sandwiched between two glass sheets (Fig. 5). A 10-mm diameter hole was drilled through the device into the end of each arm to allow the connection with air tubes and the introduction of the test wasp (Fig. 5). Purified (by a charcoal filter) and Figs. 7 - 9. Melittobia wasps responses to host kairomones. Figs 7 – 8 show the responses to natural hosts (Leaf-cutter bee and mud-dauber wasp) of the seven tested Melittobia species humidified (by bubbling into distilled water jars) air was passed through the tubes tested. Fig. 9 shows the responses of the Mellitobia wasps to a factitious host. and the air flow was regulated by flowmeters at 35 ml per min. Two chambers (one containing the tested host, the other empty) were located at the end of the • All Melittobia wasps showed a preference to the host Megachile rotundata , a Fig. 1. Honey bee prepupae covered with Larvae and some adult, mainly females, of the ectpoparasitoid bee which is a very efficient pollinator of alfalfa, carrots and other vegetables. Melittobia . Fig. 2: Female of Melittobia parasitoid wasp on host. branch, close to the air source (Fig. 5). To minimize cues from the location holding the Y-tube olfactometer, the room has no windows, no direct exposure to light, However the most primitive Melittobia wasps (assemi and sosui ) show a significant preference for the bee (Fig 7). Insect parasitoids, like Melittobia wasps, rely mainly on semiochemicals for and was equipped with 40-w fluorescent bulbs covered with red tube guards • In the case of the Muddauber, frequently found parasitized by Melittobia wasps location and recognition of hosts, and several studies have been published on completely lighting it in red (Fig. 6). Since Melittobia wasps can not see under red light they would be guided solely by smells. Wasps movements inside the in the field (González et al. 2004), it is surprising that some Melittobia species the role played by various chemical cues in the host selection behavior of (assemi , sosui and clavicornis ) actually avoid the host (Fig. 8). hymenopteran parasitoids. Also, parasitoids, at the initial steps of the host olfactometer was followed by a camera connected to a computer to better follow • When the flesh fly is tested, the most primitive Melittobia wasps tend to avoid location process, exploit mainly volatile cues since they can be perceived at their movements (Fig. 6). Each wasp was followed up to 15 min. If a choice was not made by that time, that particular trial was discarded. it, while the most evolved ones (acasta & digitata ) have a tendency to prefer long range distance, whereas slightly volatile cues whereas contact cues and use this factitious host. become progressively more important when parasitoids are in the close range. A Chi square test was performed to analyze the wasps’ first choice in both arms of • Our tests suggest that some pollinating bees seem to be the natural hosts of the Y-tube olfactometer. Melittobia wasps, but the parasitoid plasticity is allowing them to adapt in the Chemical cues are definitively relevant in communication of Melittobia . The field to other insect host groups. antennae of Melittobia males and females possess numerous mechano- and • We are still in the process of testing other hosts to clarify our current views on chemoreceptors (Figs. 3-4), suggesting that chemical cues play a key role in this parasitoid –host relationship in an evolutionary perspective. their biology. However, little is known about the role played by semiochemicals during the host location process of Melittobia. The purpose of this research is ACKNOWLEDGEMENTS: Funding for this study was provided by the California State University Agricultural Research Initiative Seed Grant (Study of chemically mediated to study the response of several Melittobia species to volatiles emitted by behaviors of bee associated Polyphagous Parasitoid Wasps) to professor Jorge M. natural and factitious hosts. González. Thanks to Jun Abe (Kanagawa University) for helping us with the SEM of Melittobia wasps antennae (figs 3-4). Thanks also to Marlene Miyasaki (Dept. Plant Science, Fresno State) for her unconditional help at various stages of this investigation. REFERENCES: Dahms, E. C. 1984. Revision of the genus Melittobia (Chalcidoidea; Eulophidae) with the description of seven new species. Memoirs of the Queensland Museum 21: 271-336. González, J.M., J.A. Genaro & R.W. Matthews. 2004. Species of Melittobia (Hymenoptera: Eulophidae) established in Bahamas, Costa Rica, Cuba, Hispaniola, Puerto Rico, and Trinidad. Fig. 5. Plexiglass olfactometer covered by glass and showing chambers (red arrows; one of Florida Entomologist 87(4):619-620. Fig. 3 – 4. Scanning Electron Microscope pictures of male and female antennae of Melittobia acasta and the chambers containing a host, the other empty) inside the branches. Yellow arrows Tanner, D.A., J.M. González, R.W. Matthews, S.B. Vinson & J.P. Pitts. 2011. Evolution of the M. australica respectivelly. Notice all the setae and sensillae covering both antennae. The presence and indicate the direction of the air flow. Figure 6. Olfactometer was set inside a room courtship display of Melittobia digitata (Hymenoptera: Eulophidae). Molecular complexity of sensilla and setae indicates how prone to semiochemicals detection these wasps are. illuminated with red lights to avoid influence of the lights on the wasps tested. Phylogenetics and Evolution 60: 219 – 227..