Study of Reproductive Behaviour of Cranberry Tipworm, Dasineura oxycoccana: Prelude to Identification of Pheromone for Monitoring This Insect Pest

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1 Report to BC Cranberry Marketing Commission Submitted November 23, 2009 Study of Reproductive Behaviour of Cranberry Tipworm, Dasineura oxycoccana: Prelude to Identification of Pheromone for Monitoring This Insect Pest Principal Investigator Dr. Sheila Fitzpatrick, Research Entomologist, Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre (AAFC-PARC), Agassiz, BC. Assistants Dan Peach (100% time; SFU Co-op student); Sneh Mathur (10% time; technician, AAFC- PARC), Ringa Kurniawan (10% time; UBC Co-op student), Melissa Cook (10% time; SFU Master of Pest Management student). Collaborators Dr. Gerhard Gries and Regine Gries, Department of Biological Sciences, Simon Fraser University; Dr. Bradley Sinclair, Canadian Food Inspection Agency, Ottawa; Drs. John Huber and Lubomir Masner, AAFC Eastern Cereals and Oilseeds Research Centre, Ottawa.

2 1 Study of Reproductive Behaviour of Cranberry Tipworm, Dasineura oxycoccana: Prelude to Identification of Pheromone for Monitoring This Insect Pest Report to BC Cranberry Marketing Commission Submitted November 23, 2009 Principal Investigator Dr. Sheila Fitzpatrick, Research Entomologist, Agriculture and Agri- Food Canada, Pacific Agri-Food Research Centre (AAFC-PARC), Agassiz, BC. Assistants Dan Peach (100% time; SFU Co-op student); Sneh Mathur (10% time; technician, AAFC-PARC), Ringa Kurniawan (10% time; UBC Co-op student), Melissa Cook (10% time; SFU Master of Pest Management student). Collaborators Dr. Gerhard Gries and Regine Gries, Department of Biological Sciences, Simon Fraser University; Dr. Bradley Sinclair, Canadian Food Inspection Agency, Ottawa; Drs. John Huber and Lubomir Masner, AAFC Eastern Cereals and Oilseeds Research Centre, Ottawa. Start and end date of project May 1, 2009, to November 30, 2009 Location of work Cranberry farms in Pitt Meadows; Pacific Agri-Food Research Centre in Agassiz; Simon Fraser University in Burnaby. Amount contributed by BCCMC $10,050 Other funding In-kind from AAFC: use of fleet vehicle; technical support; growth chambers and growth rooms; microscopes, vials and other supplies; computer, software, network access and computer support. In-kind from SFU: technical support, gas chromatograph, electroantennograph, and related supplies.

3 2 Executive Summary Pheromone-release behaviour, known as calling, was observed during the morning of the first and subsequent days of female midge life until. Calling females extended their ovipositors and remained motionless. Sometimes the ovipositor was only partially or slightly extended; this behaviour was termed subtle calling. Females on their first, second or third day after emergence called throughout the first 8 hours of the 16-hour photophase (light period). In the second 8 hours of the 16-hour photophase, calling was infrequently seen. During the photophase, females sometimes curved their ovipositors to the substrate then walked or flew dragging the ovipositor. A drop of fluid -- possibly a marking pheromone -- was left where the ovipositor first touched the substrate. This behaviour was called marking. Females were not watched during the 8-hour scotophase (dark period), so their behaviour during that time remains unrecorded. During three months of observing cranberry tipworm midges under controlled conditions, we saw male tipworm midges that were attracted to containers vacated by calling females, to the calling females themselves, and to drops of fluid deposited by marking females. We have yet to conduct controlled experiments to demonstrate that male tipworm midges are attracted to calling female in the lab and field. We began designing containers to hold live female tipworm midges in pheromone traps. We found that female (and male) tipworm midges are very dependent on humidity, and died very quickly in dry air. We made a number of attempts to wick water vapour into the tiny tube containers, but were not successful at maintaining the female midges for more than 12 hours. Further work on this objective is required. In July and August, 2009, R. Gries at SFU began investigating the chemical components in female tipworm midge pheromone. She immersed calling female midges in hexane to extract chemical components, then injected the hexane extract into a gas chromatograph. Effluent from the chromatograph was split to pass over a flame ionization detector and a male tipworm midge antenna connected to two electrodes. In this early work, Gries obtained several gas chromatograms of potential pheromone components and electroantennograms of male antennae responding to these compounds. G. and R. Gries have agreed to collaborate on this research in Additionally, D. Peach discovered two species of parasitoids emerging from cranberry tipworm, and M.P.M. student M. Cook obtained comprehensive and unique data on cranberry tipworm phenology on cranberry and blueberry.

4 3 Introduction and Original Objectives Cranberry tipworm overwinters as a pupa in the trash layer or soil on the floor of the cranberry bed. As the cranberry plants come out of dormancy, the tiny adult midges begin emerging from the pupal stage. The adults mate and the female lays eggs in the tender buds at the tips of uprights. The larvae that hatch from the eggs are so tiny that they complete most of their development undetected. The cupped leaves indicating tipworm presence only become apparent when the larva within has almost finished its development, by which time the tip has been damaged or killed by the larva s rasping mouthparts. Monitoring for cranberry tipworm involves examining upright tips under a microscope to detect eggs and early stage larvae. This is an excellent, accurate way of monitoring eggs and larvae, but it is time-consuming. It would be helpful to have a way of monitoring the adult midges, so that there could be some foreshadowing of the appearance of eggs and larvae in the tips. The most specific way to monitor adult insects is to attract the males to a trap baited with a synthetic version of the female s sex pheromone. Three of the world s foremost experts in midge pheromone identification are located at Simon Fraser University. These three biologists Dr. Gerhard and Regine Gries and chemist Dr. Grigori Khaskin have previously collaborated on blackheaded fireworm and cranberry girdler pheromone studies and are willing to collaborate on the tipworm pheromone study. However, they are extremely busy with other cutting-edge research on insect communication. Therefore, before asking them to identify the sex pheromone chemical(s) and to synthesize pheromones for field testing, the basic biology of pheromone release and attractiveness needed to be well characterized. It is for this reason that the present study of tipworm reproductive behaviour was proposed. Objectives as written in the original proposal: 1. Determine how soon after emergence and at what time of day or night female cranberry tipworm midges show pheromone-release behaviour. 2. Confirm preliminary observations (by another researcher) that male tipworm midges are attracted to female tipworm midges that are calling (showing pheromone-release behaviour) in the lab and in the field. 3. Determine what pheromone trap type is best suited to monitoring cranberry tipworm. 4. Using results from objectives 1, 2 and 3, establish a collaborative research project for 2010 with pheromone experts G. and R. Gries and G. Khaskin at Simon Fraser University. Research carried out in April through September, 2009, met the majority of these objectives. In addition, new information on cranberry tipworm lifespan and parasitism was generated.

5 4 Methods Simon Fraser Co-op student Dan Peach was hired to work from May through August, 2009, on this project. With assistance and supervision from the principal investigator, Dan carried out most of the field and laboratory work described here. Beginning on May 26, cranberry uprights were collected weekly from three farms in Pitt Meadows, placed in a cooler and transported to PARC Agassiz. When it became obvious that one farm had few infested uprights, collection was reduced to two farms. At least 50 uprights per week were collected from each of the two farms until August 11, when collection ceased. At PARC Agassiz, the stem of each upright was immersed in water in a glass scintillation vial, and the top of the upright was covered with an inverted scintillation vial (Fig.1a). A layer of parafilm over the lower vial prevented emerged tipworms from drowning in the water. When adult tipworm midges emerged from pupation in the tips (Fig. 1b), each midge was placed individually in an inverted glass scintillation vial with moistened filter paper in the cap, but no upright. Figure 1a (left) Tipworm-infested cranberry uprights enclosed in scintillation vials. 1b (right) A female cranberry tipworm (midge) after emergence from the upright. Infested uprights and emerged midges were initially kept in the lab at 21 to 24 o C under natural photoperiod. However, when it was discovered that the lab remained partially illuminated at night, all material was kept in a rearing room or a growth chamber at a photoperiod of 16 hours light: 8 hours dark. In the rearing room, lights went on at 0900 hours and off at 0100 hours; temperature ranged from 20 to 22 o C when light and to 19 o C when dark. In the growth chamber, lights went on at 0100 hours and off at 00 hours; temperature was steady at 21, 22 or 23 o C when light and or 19 o C when dark. Temperature in each location was measured by a Hobologger. By staggering the light periods in the rearing room and growth chamber, we were able to observe female midge behaviour during the first 8 hours and the last 8 hours of photophase. From late June until late July, unmated female midges 1, 2 or 3 days old were observed every half hour during the 16-hour photphase, and their behaviour noted. A headset magnifier (Optivisor 7X) was often used to see the fine details.

6 5 Results Objective 1. Determine how soon after emergence and at what time of day or night female cranberry tipworm midges show pheromone-release behaviour. Pheromone-release behaviour, known colloquially as calling, was observed during the morning of the first day of female midge life, and during the following mornings until death. Calling females extended their ovipositors and remained motionless on the side of the vial or, if a cranberry upright was present, on a leaf (Fig. 2a). When fully extended, the ovipositor was nearly as long as the female, an estimated 1.5 mm. Sometimes the ovipositor was only partially or slightly extended; this behaviour was termed subtle calling (Fig. 2b). We are not sure if pheromone is released during subtle calling but there is a possibility that residual pheromone remains on the ovipositor. Figure 2a (left) Pheromone-release behaviour calling by female cranberry tipworm midge. Note extended ovipositor, which is very fine and almost as long as the female. 2b (right) A female cranberry tipworm midge in subtle calling posture. Females on their first, second or third day after emergence called throughout the first 8 hours of the 16-hour photophase (Figs 3, 4 and 5; upper graphs). A small percentage (<25%) of females called within minutes of lights-on. Within 1.5 to 2.5 hours after lights-on, the percentage of females calling increased to 65% (day 1), 80% (day 2) and 90% (day 3). The majority of females called continuously from 1 hour to 6 or 6.5 hours after lights-on. Females not calling in the first 8 hours of the photophase were usually in subtle calling posture, particularly at the beginning and end of the 8-hour period (Figs 3, 4 and 5; upper graphs).

7 % of Females % of Females 6 Day 1 Females - First 8 hours after Lights On Rearing Room 9G Call Subtle Call Oviposit Mark :00 5:30 6:00 6:30 7:00 7:30 8:00 8:30 9:00 9:30 10:00 10:30 11:00 11:30 12:00 12:30 13:00 Time (24-hour) Day 1 Females - 8 hours before Lights Off Growth chamber 7A Call Subtle Call Oviposit Mark :00 13:30 14:00 14:30 :00 :30 16:00 16:30 :00 :30 18:00 18:30 19:00 19:30 20:00 20:30 21:00 Time (24-hour) Figure 3. Day 1 Females. Periodicity of calling, subtle calling, ovipositing and marking behaviour among unmated female cranberry tipworm midges during the first 8 hours (top) or last 8 hours (bottom) of a 16-hour photophase. Number of females observed each half hour is shown above the bars.

8 % of Females % of Females 7 Day 2 Females - First 8 hours after Lights on Rearing Room 9G Call Subtle Call Oviposit Mark :00 5:30 6:00 6:30 7:00 7:30 8:00 8:30 9:00 9:30 10:00 10:30 11:00 11:30 12:00 12:30 13:00 Time (24-hour) Day 2 Females - 8 hours before Lights off Growth Chamber 7A Call Subtle Call Oviposit Mark :00 13:30 14:00 14:30 :00 :30 16:00 16:30 :00 :30 18:00 18:30 19:00 19:30 20:00 20:30 21:00 Time (24-hour) Figure 4. Day 2 Females. Periodicity of calling, subtle calling, ovipositing and marking behaviour among unmated female cranberry tipworm midges during the first 8 hours (top) or last 8 hours (bottom) of a 16-hour photophase. Number of females observed each half hour is shown above the bars.

9 % of Females % of Females 8 Day 3 Females - First 8 hours after Lights on Rearing Room 9G Call Subtle Call Oviposit Mark :00 5:30 6:00 6:30 7:00 7:30 8:00 8:30 9:00 9:30 10:00 10:30 11:00 11:30 12:00 12:30 13:00 Time (24-hour) Day 3 Females - 8 hours before Lights off Growth Chamber 7A Call Subtle Call Oviposit Mark :00 13:30 14:00 14:30 :00 :30 16:00 16:30 :00 :30 18:00 18:30 19:00 19:30 20:00 20:30 21:00 Time (24-hour) Figure 5. Day 3 Females. Periodicity of calling, subtle calling, ovipositing and marking behaviour among unmated female cranberry tipworm midges during the first 8 hours (top) or last 8 hours (bottom) of a 16-hour photophase. Number of females observed each half hour is shown above the bars.

10 9 In the second 8 hours of the 16-hour photophase, calling was infrequently seen. A small percentage of females were in subtle calling posture, and that percentage increased as females aged (Figs 3, 4 and 5; lower graphs). The few 3-day-old females that called during the second 8 hours of photophase died that same afternoon. Females sometimes curved their ovipositors to the substrate then walked or flew dragging the ovipositor. A drop of fluid -- possibly a marking pheromone -- was left where the ovipositor first touched the substrate. This behaviour was called marking (Fig. 6). If the ovipositor was curved to the substrate and the female remained stationery or released eggs, the behaviour was called ovipositing. In hindsight, it is recognized that ovipositing and marking behaviour may be a continuum of linked behaviours. These behaviours were infrequently seen during the first 9 hours of photophase (Figs 3 upper; 4 upper; and 5 upper and lower graphs). Females were not observed during the 8-hour scotophase (dark period), so their behaviour during that time remains unrecorded. Figure 6. Female cranberry tipworm midge marking the inner surface of glass vial. Note drops of fluid that may be a marking pheromone.

11 10 Objective 2. Confirm preliminary observations (by another researcher) that male tipworm midges are attracted to female tipworm midges that are calling (showing pheromonerelease behaviour) in the lab and in the field. During three months of observing cranberry tipworm midges under controlled conditions, we saw male tipworm midges that were attracted to containers vacated by calling females, to the calling females themselves, and to drops of fluid deposited by marking females. We have yet to conduct controlled experiments to demonstrate that male tipworm midges are attracted to calling female in the lab and field. Objective 3. Determine what pheromone trap type is best suited to monitoring cranberry tipworm. Work on this objective was initiated but not completed. We began by designing containers to hold live female tipworm midges in pheromone traps. We found that female (and male) tipworm midges are very dependent on humidity, and died very quickly in dry air. We made a number of attempts to wick water vapour into the tiny tube containers, but were not successful at maintaining the female midges for more than 12 hours. Further research is required on this objective. Objective 4. Using results from objectives 1, 2 and 3, establish a collaborative research project for 2010 with pheromone experts G. and R. Gries and G. Khaskin at Simon Fraser University. In July and August, 2009, R. Gries began investigating the chemical components in female tipworm midge pheromone. She immersed calling female midges in hexane to extract chemical components, then injected the hexane extract into a gas chromatograph. Effluent from the chromatograph was split to pass over a flame ionization detector and a male tipworm midge antenna connected to two electrodes. In this early work, Gries obtained several gas chromatograms of potential pheromone components and electroantennograms of male antennae responding to these compounds. G. and R. Gries have agreed to collaborate on this research in Additional progress Cranberry tipworm midge lifespan. Most of the unmated male and female cranberry tipworm midges kept in glass vials under the controlled conditions in this study lived for one to four days, with a very few living as long as 9 or 10 days (Fig. 7). The longest-lived midges were in the rearing room, where temperatures were a little cooler than in the other sites. Average lifespan for males was 3.4 ± 0.2 days; for females, 3.2 ± 0.2 days (ANOVA: F 1,202 = 0.47, P = 0.5)

12 11 Figure 7. Lifespan of 93 unmated male and 111 unmated female cranberry tipworm midges kept under the controlled conditions of this study.

13 12 Additional progress Parasitoids of cranberry tipworm. In early June, parasitoids were observed emerging from infested cranberry shoots collected from the farms Pitt Meadows. Parasitoids are parasitic insects (tiny wasps, in this case) that lay eggs in host insects. Parasitoid larvae kill host larvae (tipworms, in this case) and are thus potential biological control agents. Parasitoid emergence increased throughout the summer (Fig. 10). Parasitoids were particularly numerous on the farm that did not apply insecticide after July 10. Photographs and specimens of all parasitoids were sent to systematists John Huber and Lubomir Masner (AAFC Ottawa), and are presently being identified. From the photographs, Huber determined that the earlier emerging parasitoids probably belong to the genus Aprostocetus (family Eulophidae) (Fig. 8), and that the later emerging parasitods are probably in the genus Platygaster (family Platygastridae) (fig. 9). Figure 8. Male adult parasitoid in the genus Aprostocetus. Adult parasitoids in this genus ranged from 1.0 to 1.8 mm long. Figure 9. Male adult parasitoid in the genus Platygaster. Adult parasitoids in this genus were about 1.0 mm long.

14 % 80% 60% 40% 20% 0% 60* May 26th June 2nd May 26th June 2nd Number of tipworms and parasitoids emerged 100* 54 June 9th June 16th Diazinon May June 23rd June 30th Diazinon June 12 0 July 7th July 14th Diazinon July 10 June 9th June 16th June 23rd June 30th July 7th July 14th July 21st July 28th August 4th August 11th Tipworms and parasitoids as percent of total emerged July 21st July 28th August 4th August 11th Parasitoids Tipworms Tipworms Parasitoids Figure 10. Number (upper graph) and percent (lower graph) of cranberry tipworm midges and parasitoids that emerged from infested tips collected weekly from one farm in Pitt Meadows. In the upper graph, the number of tips collected is shown above the bars No tips collected June 30 Additional progress Related study on monitoring of cranberry tipworm in cranberry and blueberry. Melissa Cook (Master of Pest Management student, SFU) conducted a study of phenology of cranberry tipworm on three cranberry and three blueberry farms in Pitt Meadows in Her research has generated a comprehensive data set on the similarities and differences in tipworm occurrence on the two host plant species. She has sent voucher specimens of tipworms from all sites to Brad Sinclair (CFIA, Ottawa) for identification. Cook hopes to determine if cranberry tipworm on cranberry is a different host race or a different species than on blueberry. Preliminary work by Sneh Mathur (AAFC, Agassiz) suggests that mitochondrial DNA of midges reared from cranberry differs from that of midges reared from blueberry.

15 14 Discussion, Deliverables, and Future Work This study is the first to describe the pheromone-release (calling) behaviour and calling periodicity by cranberry tipworm, D. oxycoccana. These results build on observations by Gagné (1989) that newly emerged females of many species of gall midge often exert and wave their ovipositor, frequently with a drop of fluid, presumably pheromone, at the tip. Voss (1996), working in Wisconsin, also observed that cranberry tipworm females extend their ovipositor before mating. Calling and mating behaviour of other species of cecidomyiid midges has been reviewed by Harris and Foster (1999). Females of orange wheat blossom midge call in scotophase (Pivnick and Labbé, 1992). Further study is needed to find out if cranberry tipworm females call during scotophase, or if they release pheromone during subtle calling posture. It would also be interesting to discover if females use a host-marking pheromone, and if it differs from the sex pheromone. This study is also the first to determine longevity of cranberry tipworm midges under controlled conditions, and to note the rapid death of midges in dry containers. In the field, cranberry tipworm midges probably seek humid locations within and around plants. Future work on attraction of males to calling females in the lab and in pheromone traps in the field must maintain adequate humidity in those locations. Encouraging results have been obtained from initial attempts to extract and detect sex pheromone produced by cranberry tipworm females. Collaborative research with Regine and Gerhard Gries and Grigori Khaskin at Simon Fraser University is planned for This team has a strong track record of discovering midge pheromones (Gries et al. 2000, 2002, 2005). The discovery of two species of parasitoids emerging from cranberry tipworm in BC is new, unexpected, and encouraging. Voss (1996) reported parasitoids in the same two families as ours Eulophidae and Platygastridae emerging from cranberry tipworms in Wisconsin. Sampson et al. (2006) discovered several species of parasitoids, including Aprostocetus sp. and Playgaster sp., in D. oxycoccana and a related midge in blueberry in Florida. César Rodriguez at Rutgers University has also discovered parasitoids emerging from D. oxycoccana on blueberry. In BC, it is possible the parasitoid activity can be conserved and enhanced by minimizing insecticide sprays, particularly in late July and August. The following posters and presentations were generated by this research. Pheromone-release behaviour of female cranberry tipworm, Dasineura oxycoccana, (Diptera: Cecidomyiidae). Authors S.M. Fitzpatrick and D.A.H. Peach. Poster at BCCMC Cranberry Field Day, August 2009; Oral Presentation at Entomological Society of BC Meeting, October, Hymenopteran parasitoids from cranberry tipworm, Dasineura oxycoccana, collected from a cranberry farm in BC. Authors D.A.H. Peach and S.M. Fitzpatrick. Poster at BCCMC Cranberry Field Day, August 2009; Oral Presentation at Entomological Society of BC Meeting, October, 2009.

16 Cranberry Tipworm, Dasineura oxycoccana (Johnson) (Diptera: Cecidomyiidae) and the potential for host race formation in cranberry and blueberry fields. Authors M. Cook, S. Fitzpatrick and B. Roitberg. Poster at BCCMC Cranberry Field Day, August 2009; Oral Presentation at Entomological Society of BC Meeting, October, It is anticipated that this work will also be presented at the 2010 Cranberry Congress, and will be published in the scientific literature. Anticipated funding request in 2010 Dan Peach, the Co-op student hired in 2009, has a strong interest in pursuing research on cranberry tipworm pheromone and parasitoids, and wants to return to work on this project in Melissa Cook will continue her M.P.M. project on cranberry tipworm phenology in cranberry and blueberry, and would benefit from a half-time student assistant. Collaborators R. and G. Gries at SFU will require some funding for materials related to gas chromatography, electrophysiology and pheromone synthesis. I thank the BC Cranberry Marketing Commission and BC Cranberry Growers Association for funding this research in References Gagné, R.J The plant-feeding gall midges of North America. Cornell University Press, Ithaca, NY. Gries, R., Gries, G., Khaskin, G., King, S., Olfert, O., and Kaminski, L.-A Sex pheromone of orange blossom wheat midge, Sitodiplosis mosellana. Naturwissenschaften 87: Gries, R., Khaskin, G., Gries, G., Bennett, R.G., King, G.G.S., Morewood, P., Slessor, K., and Morewood, W.D (Z,Z)-4, 7-tridecadien-(S)-2-yl acetate: sex pheromone of Douglas-fir cone gall midge, Contarinia oregonensis. Journal of Chemical Ecology 28: Gries, R., Khaskin, G., Bennett, R.G., Miroshnychenko, A., Burden, K., and Gries, G (S,S)-2, 12-, (S,S)-2, 13-, and (S,S)-2, 14-diacetoxyheptadecanes: sex pheromone components of red cedar cone midge, Mayetiola thujae. Journal of Chemical Ecology 31: Harris, M.O. and Foster, S.P Gall midges. pp in J. Hardie and A.K. Minks (eds.) Pheromones of Non-Lepidopteran Insects Associated with Agricultural Plants. CABI Publishing, CAB International, Oxford, UK. Pivnick, K.A. and Labbé, E Emergence and calling rhythms, and mating behaviour of the orange wheat blossom midge, Sitodiplosis mosellana (Gehin) (Diptera: Cecidomyiidae). Canadian Entomologist 124: Sampson, B.J., Rinehart, T.A., Liburd, O.E., Stringer S.J. and Spiers, J.M Biology of parasitoids (Hymenoptera) attacking Dasineura oxycoccana and Prodiplosis vacinii

17 16 (Diptera: Cecidomyiidae) in cultivated blueberries. Annals of the Entomological Society of America 99: Voss, K.K Studies on the cranberry tipworm (Dasineura oxycoccana (Johnson)) and a predator, Toxomerus marginatus (Say) in Wisconsin. M. Sc. Thesis, University of Wisconsin, Madison, WI.