Aracveness of Solitary Bee Nesng Cues to a Cleptoparasite Samuel Malan1 and Theresa Pis-Singer2 1Utah State University Department of Biology 2USDA-ARS Pollinang Insects Research Unit

Background Experimental Design Results Megachile rotundata F. (Hymenoptera: Megachilidae), the Bioassay: To test the aracveness of olfactory cues on In the first trial (blank vs. odor cue 1), 15 wasps chose the alfalfa leafcung bee, is a solitary bee commonly used in the Sapgya pumila, diapausing wasps were found among nest cells blank and 22 chose odor cue one. In the second trial (blank vs northwestern United States (including Utah and Idaho) for of M. rotundata. They were reared in an incubator at 29 °C. S. whole cocoon), 4 wasps chose the blank and 17 chose the whole pollinaon of alfalfa for seed producon. Managing solitary bees pumila. Emerged wasps were placed in a benchtop Plexiglas cocoon odor plume (Fig 3). presents challenges, including low seasonal return of cage so that males and females were allowed three days to Stascal analyses reveal that olfactory cues derived from commercial populaons and low rates of offspring survival. One mate and ovary maturaon could occur before being females the whole cells of M. rotundata are significantly aracve to S. major factor causing the low return rate is parasism (Pis- were tested. During this waing period, the S. pumila were pumila, while odor cue 1 is not. Further analysis will allow Singer 2007; James and Pis-Singer 2013). maintained at 29°C and fed a mixture of 90% water and 10 % researchers to isolate the organic compound contained in the Megachile rotundata is not a nave species to the United local honey. This was done to ensure that females were cocoon that is most aracve to S. pumila. States, and is heavily affected by parasism of the nave mature and acvely seeking a host. On the third day aer cleptoparasic wasp Sapyga pumila (Cresson, Hymenoptera: emergence, cocoon extract was applied to filter paper and Discussion Sapygidae). S. pumila exhibit a high rate of parasism, and a allowed to dry before use on the same day. For trials, each relavely small populaon can parasize a high number of M. wasp was introduced to a Y tube apparatus (Fig. 3) within Further research is necessary to isolate the compounds that rotundata offspring. The high rate of parasism is partly due to which it was allowed to walk about freely. Each apparatus was are most aracve to S. pumila from the M. rotundata cocoon. the long life span of the Sapgya wasp, which gives it the ability enclosed by a large cardboard box equipped with a full Separaon of the components that make up the cocoon odor to parasize nests for the enre flight period of the alfalfa spectrum light to exclude external visual cues that may have will allow for comparing the aracveness each component. S. leafcung bee. Because a small populaon is capable of a biased wasp movement. The Y-tube apparatus itself consisted pumila may be aracted to volale compounds released by the parasizing many M. rotundata, it is difficult for bee managers of an air pump that drew air through a charcoal filter, which leaves of the alfalfa that surround the cocoon of M. rotundata, to trap and kill enough S. pumila to significantly reduce rates of purified the air of other potenal cues in the environment. The or to compounds found within the cocoon itself. parasism. Effecve traps are essenal in order to achieve an air was passed over the cues (in vesbules) and then through M. rotundata is not a species that is nave to the United effcve reducon of parasism (Torchio 1974). the arms of the Y-tube before reaching the wasp. The wasp States, and we speculate that S. pumila is not aracted to an was then observed for up to 30 minutes to see which cue it odor contained in the cocoon of M. rotundata that is unique to followed. The first choice of cue was recorded. A choice was M. rotundata. Instead, we speculate that S. pumila is aracted Figure 1. Megachile rotundata female and to volale compounds released by freshly cut leaf pieces, as this cocoon wrapped in leaf pieces. constuted by the insect touching the extremity of the Y-tube nearest one cue or the other. Significant differences between is a more reliable indicator of a potenal host (Torchio 1974). choices were determined using a Chi Square analysis. Once individual aracve components are revealed, it may be possible to improve the efficacy of traps to reduce Chemical Extracon: Extracts were made using 100 empty populaons this parasite where M. rotundata are used as cocoons and associated leaf pieces. The cocoons were placed commercial pollinators. However, because both whole cell and in a glass bole, and 25ml of a 2:1 chloroform:methanol leaf piece extracts also are aracve to M. rotundata (Pis- mixture was added (25ml is the minimum volume needed to Singer 2007), trap designs using odors of these substrates must cover all cocoons). The glass bole was then agitated by hand be developed to prevent accidental capture of M. rotundata. Approach and Objecves for 5 minutes. Aer agitaon, large pieces of cocoon were The purpose of this experiment is to isolate organic filtered from the extract via rough glass fiber filtraon. compounds that can be used as aractants in traps specific to S. Extracts were stored in glass scinllaon vials. pumila. Olfactory cues derived from the cocoons of another For dosage of whole cocoon extracts, a 1 cocoon equivalent managed solitary bee species, Osmia lignaria (Megachilidae) have (CE) was used for Y-tubes. 250ul of extract was applied to the proven to be an effecve aractant of its parasite, S. elegans filter paper. Acknowledgements (Pis-Singer 2007; Pis-Singer et al. 2014; Malan and Pis-Singer, Figure 3. unpublished). We predict that S. pumila will also be aracted to We thank Ellen Klinger, the same compounds that were aracve to S. elegans (odor cue Ellen Klomps, and 1). If S. pumila are not aracted to the same compounds as S. Derek Artz for their elegans, we predict that S. pumila will be aracted to olfactory assistance conducng cues derived from the cocoon of M. rotundata (whole cocoon). this study. We tested the aracveness of odor cue 1 and the whole cocoon extract in two different trials. Figure 4. Adult Sapyga female Whole cocoon olfactory cues were created using a methanol chloroform extracon of 100 M. rotundata whole cells from which bees had already emerged (see extracon protocol for References cocoon extract). This cue was tested for aracveness against a blank in a Y tube apparatus (Fig 2). Allsopp M.H., de Lange, W.J., Veldtman, R. 2008. Valuing insect pollinaon services with cost of replacement. PLoS ONE 3(9): e3128. doi:10.1371/journal.pone.0003128 Figure 2. Le: Sam Malan observing wasp during Y tube p > 0.1 James, R.R. and Pis-Singer, T.L. 2013. Health status of alfalfa bioassay. Right: Diagram of Y tube apparatus. leafcung bee larvae (Hymenoptera: Megachilidae) in United States alfalfa seed fields. Environmental Entomology 42(6): 1166-1173. Pis-Singer, T.L., Kemp, W.P., Moreland, D., Peterson, S., Buckner, J.S., Hagen, M. Patent Applicaon, Docket No.:143.12 Pis- Singer. Bee Aractants. Filed March 7, 2014. Pis-Singer, T.L. 2007. Olfactory response of megachilid bees, Osmia lignaria, Megachile rotundata, and M. pugnata, to individual cues from old nest cavies. Environ. Entomol. 36: 402-408 Pis-Singer, T.L. and Cane, J.H. 2011. The alfalfa leafcung bee, Megachile rotundata: the world’s most intensively managed solitary bee. Annu. Rev. Entomol. 56: 221-237. Torchio, P. 1974 Biology and Control of Sapyga pumila, a Parasite p < 0.005 of the Alfalfa Leafcung Bee.Logan, Utah notes

Figure 5. Sapyga trap and nesng site for M. Rotundata • Ongoing research to improve the efficacy of the pollinators known commonly as the blue orchard bee (Osmia lignaria) and the alfalfa leafcuer bee (Megachile Rotundata) is necessary to render both species a viable opon for large-scale agricultural pollinaon. • Megachile rotundata is responsible for two-thirds of the pollinaon of alfalfa in the United States, and Osmia lignaria is used as a commercial pollinator of tree fruit. • However, certain obstacles must be overcome in order to achieve effecve large-scale pollinaon using O. lignaria and M. rotundata. One of these obstacles is parasisim. • Both bees are parasized by wasps of the genus Sapyga, leading to reduced numbers of bee offspring. This decreases the populaon size and the rate of pollinaon. • In a previous study, olfactory cues derived from O. lignaria cocoons were isolated and tested for efficacy in aracng the natural enemy of O. lignaria known as Sapyga elegans. Two cues were tested extensively and determined to be aracve to Sapyga elegans. However, research must sll be done to isolate compounds that are aracve to Sapyga pumula, the natural enemy of the alfalfa leaf cuer bee. The purpose of this research is to find aractants of Sapyga sp. That can be used in traps near nesng sites of M. rotundata. • Tesng started with two olfactory cues that were previously shown to be aracvie to Sapyga elegans. These olfactory cues did not aract Sapyga pumula, and researchers moved on to test odors extracted from the cocoons of M. rotundata.