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Release and establishment of the little decapitating fly Pseudacteon cultellatus (Diptera: Phoridae) on imported fire ants (Hymenoptera: Formicidae) in Florida.

Imported fire ants (Solenopsis spp.; Hymenoptera: Formicidae) in North America have population densities 5-10 times higher than those normally found in their South American homelands (Porter et al. 1992, 1997). Escape from numerous natural enemies left behind in South America is a likely reason for intercontinental differences in fire ant abundance (Porter et al. 1997).

The little decapitating fly (Pseudacteon cultellatus Borgmeier; Diptera: Phoridae; Fig. 1) was selected for release as a self-sustaining biological control agent for imported Solenopsis fire ants in the United States because this species parasitizes the smallest sizes of fire ant workers. Small fire ant workers are the most abundant size class in fire ant colonies, especially in young colonies (Porter & Tschinkel 1985a, 1985b) and in multiple-queen or "polygyne" colonies (Greenberg et al. 1985). Polygyne fire ant colonies are found in about 10-15% of the imported fire ant range in the United States, except in Texas where 50% of the state has multiple-queen fire ants (Porter et al. 1991; Porter 1992; Porter et al. 1992).

Like other Pseudacteon flies, when P. cultellatus maggots are ready to pupate, they decapitate their host workers and use the newly emptied host head capsules as pupal cases (Porter 1998). All Pseudacteon species that attack fire ants, including P. cultellatus, appear to be specific to Solenopsis fire ants (Porter & Gilbert 2004; SDP unpublished data).

The natural geographic range of P. cultellatus in South America is from Buenos Aires, Argentina, north through Corrientes, Argentina, then northeast into Brazil through the states of Parana and Sao Paulo to the states of Minas Gerais and Goias (Patrock et al. 2009). In South America, this species is known to attack both red and black fire ants (i.e.; Solenopsis invicta Buren and Solenopsis richteri Forel) and probably Solenopsis saevissima F. Smith as well. The most abundant populations were found in gaps in subtropical gallery forest habitats (Calcaterra et al. 2005). The climatic preferences of P. cultellatus flies are rather broad ranging from tropical to temperate; consequently, its potential range in the United States could be anywhere imported fire ants are found.

Five species of phorid decapitating flies have been released and established as fire ant biocontrol agents in the United States: Pseudacteon tricuspis Borgmeier (Callcott et al. 2011), Pseudacteon curvatus Borgmeier (Callcott et al. 2011), Pseudacteon litoralis Borgmeier (Porter et al. 2011), Pseudacteon obtusus Borgmeier, (Gilbert et al. 2008; Porter & Calcaterra 2013), and Pseudacteon nocens Borgmeier (Plowes et al. 2012).

The objective of this paper is to document the release of P. cultellatus, a sixth species of fire ant decapitating fly, at 5 sites in Florida and the establishment of this fly at 2 of these sites.

MATERIALS AND METHODS

The original P. cultellatus flies used in this study were collected in Dec 2006 from a marsh area along the Parana River (~2,700 flies) about 20 km NE of Corrientes, Argentina (S 27.371[degrees] W 58.687[degrees]) and from a picnic/camping area (~2,000 flies) about 20 km SE of Corrientes (S 27.559[degrees] W 58.725[degrees]). Most of the host ants at these sites were S. invicta, but local biotypes of S. invicta were probably not as similar to US biotypes as those found further north along the Paraguay River in the province of Formosa (Ascunce et al. 2011). Immature stages of P. cultellatus were carried to a USDA quarantine facility in Gainesville, Florida in parasitized hosts accompanied by an Argentine export permit (DFS No. 19517) and a USDA/ APHIS import permit (#64943).

Prior to field release in 2010, the flies were mass reared on red imported fire ants from the United States using large attack boxes (1 m wide x 2-2.5 m long x 0.7 m high) with either 8 or 14 smaller attack trays similar to those described by Vogt et al. (2003) except that humidity was controlled by a vaporizer in a chamber mounted under the attack chamber. Every morning, new flies emerged from pupation trays directly into the attack chamber where they could parasitize worker ants, which were trailing back and forth between 2 cups as they were automatically raised and lowered in each of the attack trays. Flies were allowed to parasitize workers for 3 or 4 days before the workers were removed. Photographs of attack boxes as well as notes and photographs about rearing procedures can be viewed in Supplementary Material for this article in Florida Entomologist 96(3) (2013) online at http://purl. fcla.edu/fcla/entomologist/browse, as well as at http://www.ars.usda.gov/saa/cmave/ifahi/cultellatus. Extensive care was necessary when rearing P. cultellatus to limit contamination from workers already parasitized with P. curvatus flies in the field (see Supplementary Material for details).

Fire ant workers used in the attack boxes were obtained from field colonies because workers from laboratory colonies proved unsuitable for fly development if they had been infected by the SINV-3 virus (SDP--unpublished data), which at that time was a major problem in our rearing facility (Porter et al. 2013; Valles & Porter 2013). We used only the smallest fire ant workers for hosts because this improved production efficiency and limited contamination from other Pseudacteon species, which parasitize medium- and large-sized workers. A second major rearing problem was that fire ant workers frequently chewed out fire ant head capsules with newly pupated flies, thus killing the pupae and greatly reducing productivity of the fly culture. This problem was mitigated by collecting dead fire ant workers with pupating larvae 6 days a week, using larger holding boxes for parasitized workers, and providing darkened nests for the workers so fewer workers were active in the holding box (see Supplementary Material above).

Field releases were conducted at 5 sites in Florida beginning in the spring of 2010 (Table 1). Sites were selected which contained large populations of fire ants (probably 1500-3000 workers/ m2; Macom & Porter 1996) with small workers like those associated with polygyne colonies. The sites were all in open disturbed areas, but releases were conducted using mounds as close as possible to bushes, trees, and water sources to ensure refugia for the fire ants and flies in case of drought or hot weather (Plowes et al. 2011). The 4 releases conducted in 2010 (Table 1) used larvae in parasitized ants as was previously done with P. curvatus (for details see: Graham et al. 2003; Vazquez et al. 2006). The 2011 release (Table 1) was done by burying P. cultellatus pupae in an insolated box with an exit pipe in the top similar to the method described by Plowes et al. (2012). Our implementation of this method needed improvements because about one quarter of the 13,600 release flies were trapped in the crack between the cooler box and its lid. Also, cool ground temperatures (15-20[degrees]C) in the early spring delayed emergence of the flies longer than was desirable. In May 2010, 2 attempts were made to release adult flies over disturbed fire ant mounds as was done with P. tricuspis (Porter et al. 2004), but the P. cultellatus flies dispersed rapidly (within 5-15 min) without attacking many fire ants so further attempts were discontinued.

Sticky tri-stand traps in 150 x 25 mm Petri dishes were used to monitor the establishment of flies at release sites (Puckett et al. 2007; Porter & Calcaterra 2013). Petri dishes were coated on the inside with a Fluon-like suspension of fluoropolymer resin (Daikin Polyflon PTFE DX-9025; Daikin America, Inc., Orangeburg, New York) and placed in fire ant mounds so disturbed workers would fall into the dish and not be able to escape. Sometimes additional fire ant workers were added from lab colonies if ant numbers were too small to reliably attract phorid flies.

Voucher specimens of P. cultellatus and the other Pseudacteon species have been deposited in the Florida State Collection of Arthropods, Division of Plant Industries, Florida Department of Agriculture, Gainesville, Florida, and the Museo de Ciencias Naturales de La Plata, Buenos Aires, Argentina.

RESULTS

The little decapitating fly, P. cultellatus, was established at 2 of 5 release sites in Florida (Table 1). Flies were first released at the University of Florida Organic Gardens site in the spring of 2010 around the edge of a pond. Several first-generation field-reared flies were recovered in June 2010, but no additional P. cultellatus flies were found on 6 subsequent dates in Aug and Sep so an additional fall release was conducted. Several likely first-generation field-reared flies were recovered at the end of Oct 2010. Additional flies have been recovered on 11 of 26 different occasions from Mar 2011 to May 2013, more than 30 months after the fall 2010 release (Table 1).

By the end of the first yr (fall 2011), flies had expanded outward about 200 m from the release site in 3 of 4 directions (E, W, S). No additional expansion was observed at the end of the second yr (fall 2012), but a fly was found out 500 m in the fourth direction (N) in spring 2013. Flies were found on 3 of 8 trap dates in 2012 and 2 of 3 dates for the spring of 2013. The relative abundance of P. cultellatus has remained very low at the Organic Garden site from 2010-2013 (0.3-0.7%; Table 2).

Pseudacteon cultellatus was also established at a highly disturbed former mulch site adjacent to a major canal in the Everglades restoration area southwest of Miami, Florida (Table 1). The first P. cultellatus fly recovered at this site was in Aug 2011, 8 months after the releases concluded. Extensive trapping in and around this release site on 10 Jan 2012 failed to find either P. cultellatus or evidence of its expansion. However, 4 flies were found in Apr 2012 and 6 more flies were found in May 2013 (28 months after the releases). As with the previous site, P. cultellatus females only accounted for a small fraction of the total female Pseudacteon flies collected (0.2-1.0%; Table 2). We did not attempt to monitor fly expansion out of this release site in 2013 because of logistical constraints.

Releases of P. cultellatus appear to have failed at 3 additional sites near Gainesville, Florida (Table 1). Several field-reared flies were trapped at each site 1-5 months after release; however, no additional flies have been found since, despite 13, 9, and 7 additional trapping dates over periods of 29, 21, and 30 months for the Waldo Ballpark, Waldo Storage, and Hawthorn sites, respectively. We were not able to discern any critical habitat differences between the 2 successful and 3 unsuccessful sites. Weather conditions during the releases and monitoring were not unusually extreme.

Trap data from the 4 release sites in the Gainesville area (Table 2) show P. curvatus was the most abundant species, accounting for about 91% of total female decapitating flies (range: 83100% by date) followed by P. obtusus with about 8% (range: 0-17%). Like P. cultellatus, P. tricuspis was rarely captured at the Gainesville release sites. At the release site near Miami, P. tricuspis accounted for about 20% (range 0.6-27%) of total female flies with P. curvatus making up most of the remainder (Table 2).

Females accounted for 52% (1,344/2,565) of total P. obtusus flies trapped in the Gainesville area while females accounted for only 36% (609/1,674) of total P. tricuspis flies trapped at the site near Miami. More than 99% of P. curvatus flies trapped were females and all of the P. cultellatus flies were females because males of both species do not mate with females while they are ovipositing in fire ant workers.

DISCUSSION

The little decapitating fly P. cultellatus appears to be established in Florida because populations have persisted for more than 2 yr at each of 2 release sites (Table 1). The P. cultellatus biotype that we released clearly finds S. invicta from the United States a suitable host, unlike the P. curvatus biotype from black fire ants, which was released 7 times on red fire ants in Florida without any of the releases establishing (Graham et al. 2003). Nevertheless, P. cultellatus populations remain very low (Table 2) and expansion rates, so far, appear to be limited to a few hundred m. About 91% of the female flies collected in this study were P. curvatus, similar to the results of Porter & Calcaterra (2013) in 2009-2010, while almost all of the remaining females were P. obtusus. The once abundant P. tricuspis continued to be trapped throughout the study, but only at extremely low levels (Table 2). Curiously, P. tricuspis accounted for about 20% of females trapped at the release site near Miami (Table 2), thus demonstrating that, while P. tricuspis generally does poorly in competition with P. curvatus (LeBrun et al. 2009; Porter & Calcaterra 2013), in some areas and seasons this is not true.

Expansion rates for other Pseudacteon species released in the United States have been quite variable with some fly populations showing little or no expansion for 1-3 yr while other populations have expanded several kilometers in the first yr and up to 50 km/yr in subsequent years (Porter et al. 2004; Henne et al. 2007; LeBrun et al. 2008; Porter 2010; Porter et al. 2011). Slow spread of P. cultellatus is probably a result of low population densities and perhaps a low natural rate of dispersal. Competition with the very abundant P. curvatus is likely a major factor affecting population densities because P. curvatus depressed P. tricuspis populations in large parts of Texas and Florida (LeBrun et al. 2009; Porter & Calcaterra 2013). Competition with P. curvatus and perhaps P. obtusus is also a likely reason why we were only able to establish flies at 2 of 5 release sites (Table 1). Plowes et al. (2011) report that success rates of establishing P. obtusus in Texas were reduced by the presence of P. curvatus and P. tricuspis at their release sites.

The ultimate fate of the P. cultellatus releases in Florida is unknown. Drought or other extreme weather conditions combined with strong competition from pre-existing species may eventually lead to extinction of what are currently small and localized populations. If P. cultellatus is able to disperse from our release sites, it may remain as a rare species in most areas with some locally abundant populations as is currently the case for P. litoralis in Alabama (Porter et al. 2011). After passing through a latent phase (Henne et al. 2007), P. cultellatus may eventually become a more abundant fly once it adapts to local conditions and fly populations become large enough to ensure easy mating and dispersal. Populations of a P. curvatus biotype from red fire ants remained so low at 2 release sites in Florida that none were recovered for almost a yr; however, eventually their numbers increased dramatically and rapid expansion followed (Vazquez et al. 2006; Porter 2010). Similar results have also been reported for P. tricuspis in Louisiana (Henne et al. 2007). The prediction was that P. cultellatus would do especially well with polygyne fire ant hosts because polygyne colonies have much smaller workers on average than monogyne colonies (Greenberg et al. 1985). Current data is not sufficient to evaluate this prediction; however, the presence of colonies with small workers at each of the release sites did not ensure successful establishment. The release site near Miami contains what appear to be mostly polygyne colonies with a predominance of small workers, but the Organic Garden site in Gainesville has become almost entirely monogyne since the original releases.

Around Corrientes, Argentina, P. cultellatus is only locally abundant where it was originally collected. Differences in habitat, hosts, or the presence of small polygyne workers are possible explanations. However, the relative abundance of Pseudacteon species in South America is not correlated well with the abundance of Pseudacteon species, which have been established in the United States (Porter & Calcaterra 2013). Pseudacteon cultellatus is currently being reared by USDA/APHIS for release in additional states in hopes that it may do better in other habitats or climates. Nevertheless, only a few more releases are probably justified unless at least some of these releases result in vigorous localized P. cultellatus populations.

To date, 6 species of Pseudacteon decapitating flies have been released against the red imported fire ant (S. invicta) in the United States and each have shown different levels of establishment and dispersal. While additional candidate species remain in South America (Patrock et al. 2009), P. cultellatus will probably be the last Pseudacteon species our group will release against S. invicta. Most of the remaining species are either too rare to be reliably collected, too difficult to rear in quarantine, and/or of questionable host association with the US biotype of S. invicta. Several small Pseudacteon species, which attack fire ants along trails, may be useful if other research groups can find effective ways to rear and/ or release them in the United States. We are still contemplating releasing 1-2 Pseudacteon species against black imported fire ants (S. richteri) and hybrid fire ants in Tennessee and the northern parts of Mississippi, Alabama, and Georgia, but our research efforts are beginning to shift to other potential groups of agents, especially viruses and microsporidian pathogens (Poinar et al. 2007; Oi et al. 2010; Valles 2012; Porter et al. 2013).

Caption: Fig. 1. Male and female Pseudacteon cultellatus decapitating flies (left). A posterior view of the ovipositor (right) by which female flies are easily identified (Porter & Pesquero 2001). The male can be distinguished in the United States by its small size, triangular anal tube, and a dark-brown hairy antenna with a narrow curved apical extension. The gray scale line is 0.5 mm (left) and 0.2 mm (right).

Supplementary Appendix: Porter S.D., Kumar, V., Calcaterra L.A, Briano, J.A., and Seal D.R. 2013. Release and establishment of the little decapitating fly Pseudacteon cultellatus (Diptera: Phoridae) on imported fire ants (Hymenoptera: Formicidae) in Florida. Florida Entomologist 96(4):

Rearing Pseudacteon cultellatus: Notes and Photos

Sanford Porter

USDA-ARS, Center for Medical, Agricultural and Veterinary Entomology, Gainesville, FL, USA 32608

June 2013

Changes to Minimize Chewed-out Heads

1. Collect dead workers 6 days a week.

2. Use larger trays (#3 Rubbermaid TakeAlongs).

3. Use a stainless steel cup (2.5 oz., ~$0.50 each) over plaster block with foil patch on tray bottom (Beaty Restaurant Supply, 352 376-5939).

Changes to Minimize P. curvatus Contamination

1. Hold workers from field for 10-21 days in lab at 21-24[degrees]C to allow as many P. curvatus as possible to pupate before use. Don't hold workers longer than 3 weeks to avoid possibility that they become infected with SINV-3 virus.

2. Sieve workers to remove larger ants which P. curvatus prefers.

--Collect colonies with a predominance of small workers.

--Rapid pre-sieve using #12 U.S. Standard sieve (1.7 mm diameter opening) and heavily etherized workers, especially if sex brood and sexuals are common (optional).

--Remove brood with sorting sheets.

--Slow sieve the remaining workers using a #25 sieve (0.71 mm opening). Use workers passing through this sieve. We used 1 g workers & 1.5 g brood. (The brood was needed to keep workers running when flies attacked.)

3. Discard all dead in boxes with parasitized workers at 12 days (oldest date) when holding parasitized workers at 27.5 [+ or -] 0.5[degrees]C. We tag all boxes with the date at which the oldest parasitized ants turn 12 days (or 11 days if 12 is on a Sunday). Collect and plate dead workers and pupating maggots beginning at 13 days. Note: This works because most P. curvatus pupate between 10 and 12 days at 27[degrees]C.

4. Hold pupal trays in reach-in boxes at 23-25[degrees]C until day before P. cultellatus begin emerging. Usually this is 21-24 days depending on temperature. Best to use two boxes: one for oldest date and another for the next 4-5 dates for flies that emerge early. Note: This works because most P. curvatus flies emerge from the pupae 1-3 days before the P. cultellatus pupae do. The exact time depends on temperature.

5. Monitor species of flies emerging in reach-in boxes to adjust timing and abundance of curvatus.

6. Monitor random samples of 30-40 pupae 1-2 times a week to warn of buildup of curvatus.

7. If all else fails, be prepared to check all emerging flies to remove curvatus females. This is best done under a microscope using C[O.sub.2] to knock them down and a cold table (9-12[degrees]C) to keep them immobile while they are inspected. If caught early enough, this can be done to half of the colony at a time.

8. Keep P. curvatus and P. cultellatus colonies in different rooms.

Changes to Minimize Mites and Trash Phorids

1. Use tight seals on all holding boxes.

2. Move to new holding box after about 5 days so adult mites or adult flies do not have time to emerge and re-infest new trays.

3. Put holding boxes in rooms where they will be separated from trash phorid flies (especially, with new trays).

For further information about Pseudacteon fly rearing see:

Vogt, J. T., S. D. Porter, D. A. Nordlund, and R. Smith. 2003. A modified rearing system for production of Pseudacteon curvatus (Diptera: Phoridae), a parasitoid of imported fire ants. Biol. Control 28: 346-353.

Caption: 1. Parasitized ants. They are kept for 1 month or 28 days at 27-28[degrees]C.

Caption: 2. Vented 1 gal RubberMaid TakeAlong box with parasitized ants. Ants like to place dead workers in the corner depressions.

Caption: 3. Test tube for water. Castone (dental plaster) cap keeps ants from digging into cotton and cotton fibers getting mixed with dead ants which makes it hard to count the pupae. Two Castone moisture blocks were used to maintain the high humidity required by the pupating fly larvae. Metal cup with 2 small notches cut in lip inverted over a third Castone moisture block. Sugar wick on plastic lid. Foil tape is affixed under bottom of tray to blackout inside of metal cup and help keep negatively phototactic ants inside.

Caption: 4. Castone block under cup.

Caption: 5. Large Kimwipes used to make sugar wicks with 2 M sugar solution.

Caption: 6. Moisture blocks are made of a very soft mixture of Castone so they absorb a lot of water quickly.

Caption: 7. At 12 days old, we throw away all dead workers and then begin collecting the dead on day 13. This helps to reduce numbers of P. curvatus getting through. If 12 days falls on a Sunday, we pull on day 11 instead. Timing may need adjusting if temperature varies.

Caption: 8. Orphan box: used for live workers accidently collected with dead workers. It is replaced monthly.

Caption: 9. Plaster tray for holding dead workers. This tray is partly moistened before putting dead workers on and then again after they are evenly spread across the tray. The 11 lines creating 12 strips are used for conducting 1/2- or 1/4-count estimates of pupal production.

Caption: 10. Trays with dead parasitized ants in Holding Box. Workers are spread evenly over trays. Several bank trays are added and moistened to make sure humidity is in the 85-95% range. High humidity is critical for Box 1 and good for box 2. Holding boxes may need smaller vents or watering more often if room is drier.

Caption: 11. Pupae Holding Box 1. Pupae are placed here immediately after collecting dead. Blank trays are watered twice a week with regular water and pupae trays are watered on day four or five. After collection, it is best not to water pupae again until they have extended their respiratory horns at 3-4 days. All water must soak in so plaster in trays is not shiny. We leave the trays in Holding Box 1 for 4-5 days and then move them to Holding Box 2.

Caption: 12. Pupae Holding Box 2. Transferring trays to box 2 at five days keeps mites and trash phorids from completing development inside the box and contaminating new trays. Trays in Holding Box 2 are watered with 1% bleach solution twice a week to keep humidity high. Trays are transferred from this holding box to Emergence Box B and then to Emergence Box A.

Caption: 13. 4-5 days of trays are held in Emergence Box B along with several additional trays for moisture. This box is backup in case our aim with Box A is too long.

Caption: 14. Emergence Box A with Emergence Box B behind. We hold pupae trays in Box A until just before the P. cultellatus emerge. The trays are moved out into the attack box at about 18 days but when the room is cooler it can be as much as day 20 or 21. This is done to make sure the P. curvatus have a chance to emerge first. If more than a dozen or so P. cultellatus flies begin emerging, the waiting period is too long (unless we are still getting a lot of P. curvatus).

Caption: 15. Only one date is held in Box A. Other trays are to ensure moisture remains high. Water is added daily to trays except on Sunday.

Caption: 16. Copper tubing port in center of photo used to attach Allen aspirator to a tube inside the box which is used with hands through the two side access ports to extract flies in the box.

Caption: 17. Black strip in corner of Box A. Flies are attracted to strip so they are easier to collect and the strip may aid in mating.

Caption: 18. Vaporizer attached to a humidistat used in winter to keep room from getting too dry (<35%).

Caption: 19. Room humidistat.

Caption: 20. Preferred room temp and humidity.

Caption: 21. Cold table. Note embroidery hoop with sheer cloth for holding flies. We used several squares of plastic needlework canvas from a craft store below the hoop to maintain temperature at 10-12[degrees]C. More of the plastic squares are added if the hoop gets too cold or removed if it gets too warm.

Caption: 22. Data book for recording numbers of female P. cultellatus and P. curvatus emerging in Box A and B. Might want to keep track of total males too.

Caption: 23. Embroidery hoop with guidelines drawn on sheer material with thermocouple to monitor temperature.

Caption: 24. CO2 box for knocking flies down prior to putting on cold table. Screened vials are inserted in place of the black stopper so the CO2 can diffuse into vial.

Caption: 25. Double-chambered Allen-type aspirator fitted to handle vials with 60-mesh screen caps that can be fitted to the CO2 container to anesthetize the flies prior to sorting on cold table.

Caption: 26. Attack box. The temperature in the box is controlled by the halogen lights during the day and the room temperature during the night. Humidity is controlled by a vaporizer placed in a box under the attack box. A fan continually circulates moist air up to and back from the attack chamber. A bank of fluorescent lights illuminate the attack tray during the day. The emergence box on the right with the big "C" is where pupae trays are put just prior to the flies emerging.

Caption: 27. Attack trays and "lifter lids". The ants run back and forth to between the two lids in each tray as the lids are raised or lowered by the PVC rod above. Note that each tray has a vial of sugar water and a vial of tap water.

Caption: 28. "Lifter Lids" used in large attack box. Workers use chains workers to escape when lid is raised. The brass clip glued to bottom lip of the black spray paint can lid shims it up thus avoiding crushing ants when it is lowered. A wooden dowel is screwed to the top of the black cap and a fluoned PVC pipe is slipped over the dowel. Two inverted cups are attached to the top of the dowel. The inside of the outer cup and both sides of the inner cup are fluoned to ensure that ants do not escape up the lifter screen. The black lid is filled to within 7-10 mm of the lip with plaster and the plaster is coated with paraffin wax to discourage ants from simply riding up and down under the lid.

Caption: 29. Pupae trays in attack box with pupae ready to emerge. Trays are transferred to the attack box from Emergence Box A. Seven trays is usually enough to ensure all flies emerge before removal. About 85% emergence rates indicate good conditions. Trays are watered each afternoon.

Caption: 30. Preferred temp in attack box is 27[degrees]C during the day and about 23-24[degrees]C at night with and 80-85% humidity maintained 24 hours a day.

Caption: 31. Black strips in attack box held on with Velcro. Mating appears to occur around the black strips. P. cultellatus males do not mate with female whiles they oviposit on the ants so they are not collected in the field like P. tricuspis or P. obtusus males.

Caption: 32. Black strips in attack box held on with Velcro.

Caption: 33. Pneumatic motor used to drive the large central PVC pipe which raises and lowers the lifter lids.

Caption: 34. Solenoid which controls the direction the pneumatic motor turns. The bleeder valves on the bottom adjust the rate of turn so that the cups rise and fall slowly.

Caption: 35. Timers for halogens (on at 10 am and off at 6 pm), fluorescent lights (on at 9 am and off at 6:30 pm) and lifter motor (on at 12 noon and off at 6 pm).

Caption: 36. Room light timer (on at 7:00 am and off at 7:00 pm).

Caption: 37. Dusk light on for 30 min before and after room light (6:30am-8:00pm). 40 watts.

Caption: 38. Second dusk timer on for another 30 min in morning and evening (6:00am-8:30pm). 7.5 watts.

Caption: 39. Custom made lifter timer: about 11 minutes one way and 13 the other. Controls pneumatic motor from noon to 6 pm.

Caption: 40. Attack box humidistat. Dental tape used to raise and lower cups. Swivel fishing clip used to attach and remove cups for cleaning and repair.

Caption: 41. Safety off thermostat. Senses when water in vaporizer gets too hot because humidistat is stuck on. Turns vaporizer off. Set to about 90[degrees]F. Without this safety, the vaporizer would occasionally keep pumping vapor into the air in the box until it condensed on the plexiglas top and rained into the trays filling them with water and drowning many of the ants. Many of the flies would also die when they were trapped in the condensation.

Caption: 42. Vaporizer for controlling humidity in attack box. A vaporizer is much better than a ultrasonic humidifier because the vaporizer does not leave calcium dust everywhere. Water was added to tray daily during the week. Water was drained and replaced monthly to avoid accumulations of salts which may have caused the vaporizer to run too hot so that the relay in the humidistat would be stuck on. Holes in the vaporizer reservoir allowed water in from the larger tray.

Caption: 43. Using an aspirator to collect pupae for shipping or study. Ant heads with pupae are sucked into double-chambered Allen-type aspirator (BioQuip) so that they are not wind-tunneled as would happen in a single-chamber aspirator. We connected the aspirator to a vacuum line and used a small flexible tube with a trimmed pipette tip to suck up the pupae.

ACKNOWLEDGMENTS

Darrell Hall (USDA-ARS, Gainesville) is thanked for his assistance with ant rearing, field releases, and monitoring efforts. Cynthia Vann (USDA-ARS, Gainesville) is thanked for managing fly rearing efforts. Field release of P. cultellatus would not have been possible if Charlie Brown (USDA-APHIS, PPQ, Riverdale, Maryland) had not personally shepherded a FONSI (Finding of No Significant Impact) through regulatory channels. Orlando Coy (South Florida Water Management District) and Dallas Hazelton (Miami-Dade County Park and Recreation Dept.) guided us in finding potential release sites in the Miami area. Jason Smith (South Florida Water Management District) is thanked for his persistent efforts in obtaining approval for fly releases on Water Management District land. DeWayne Shoemaker (USDA-ARS, Gainesville), Kelly Loftin, Sim Barrow (both Univ. of Arkansas), and two anonymous reviewers read a draft of this paper and provided valuable comments. A Porter family fund covered unexpected international travel and shipping expenses for the flies. Funding provided by USDA-APHIS, PPQ assisted substantially with rearing and release efforts.

REFERENCES CITED

ASCUNCE, M. S., YANG, C. C., OAKEY, J., CALCATERRA, L., WU, W. J., SHIH, C. J., GOUDET, T., J., ROSS, K. G., AND SHOEMAKER, D. 2011. Global invasion history of the fire Ant Solenopsis invicta. Science 331: 1066-1068.

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SANFORD D. PORTER (1) *, VIVEK KUMAR (2,4), LUIS A. CALCATERRA (3), JUAN A. BRIANO (3) AND DAKSHINA R. SEAL (2)

(1) Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, 1600 SW 23rd Drive, Gainesville, FL 32608, USA

(2) Tropical Research and Education Center, University of Florida, 18905 SW 280th St, Homestead, FL 33031, USA

(3) Fundacion para el Estudio de Especies Invasivas, Bolivar 1559 (B1686EFA) Hurlingham, Buenos Aires, Argentina

(4) Current Address: Mid-Florida Research & Education Center, University of Florida, 2725, S. Binion Road, Apopka, FL 32703, USA

* Corresponding Author; E-mail: sanford.porter@ars.usda.gov

Supplementary material for this article in Florida Entomologist 96(4) (2013) is online at http://purl.fcla.edu/fcla/entomologist/browse

TABLE 1. DATA FOR THE FIELD RELEASE OF THE FIRE ANT DECAPITATING
FLY PSEUDACTEON CULTELLATUS AT FOUR SITES NEAR GAINESVILLE,
FLORIDA AND ONE SITE IN MIAMI-DADE COUNTY NEAR MIAMI, FLORIDA.

Site                          Coordinates

UF Organic        29.6279[degrees]N 82.3584[degrees]W
  Gardens
NW of Hawthorne   29.6330[degrees]N 82.1492[degrees]W
Waldo Ballpark    29.7950[degrees]N 82.1703[degrees]W
Miami-Dade Co.    25.5971[degrees]N 80.5265[degrees]W
Waldo Storage     29.8198[degrees]N 82.1691[degrees]W

                                         #Flies
Site                 Release Dates      Released         Results

UF Organic        19 Apr-2 May 2010      14,000    1st Release Failed?
  Gardens         17-30 Sept 2010         7,400    Present [greater
                                                     than or equal
                                                     to] 2.5 years
NW of Hawthorne   10-27 May 2010         10,000    Several field-reared
                                                     flies at 5 months
Waldo Ballpark    1-18 Oct 2010          12,000    1st generation flies
                                                     at 5-7 weeks
Miami-Dade Co.    23 Nov-13 Dec 2010     12,500    Present > 2 years
Waldo Storage     Mar to mid-Apr 2011     8,500    One field-reared
                                                     fly, ~4 months

TABLE 2. PERCENT SPECIES COMPOSITION OF PSEUDACTEON FEMALES
TRAPPED DURING MONITORING AT P. CULTELLATUS RELEASE SITES
SHOWN BY YEAR.

                     Sample     Total
Release Site          Dates    Females
Year                 (Traps)   Trapped

UF Organic Gardens
2010                  7 (13)    262
2011                 11 (63)   3581
2012                  8 (53)   1835
2013                  3 (26)   1987

NW of Hawthorne
2010                  9 (28)    759
2011                  2 (3)      27
2012                  2 (12)    535
2013                  3 (24)   1834

Waldo Ballpark
2010                  4 (8)     419
2011                  8 (22)   1633
2012                  1 (3)     144
2013                  3 (15)    248

Miami-Dade Co.
2011                  6 (21)    389
2012                  2 (48)   2004
2013                  1 (18)    619

Waldo Storage
2011                  9 (61)   2324
2012                  1 (6)     305
2013                  3 (19)    205

                        % of Total Females by Pseudacteon Species
Release Site
Year                 curvatus   obtusus    tricuspis   cultellatus

UF Organic Gardens
2010                  92.0       7.3        0          0.7
2011                  91.0       8.5        0          0.5
2012                  82.5      17.2        0          0.3
2013                  95.8       3.9        0          0.3

NW of Hawthorne
2010                  98.0       0.8        0.9        0.3
2011                 100         0.0        0          0
2012                  96.8       2.8        0.4        0
2013                  97.4       2.6        0          0

Waldo Ballpark
2010                  95.0       4.5        0          0.5
2011                  96.5       3.5        0          0
2012                  88.2      11.8        0          0
2013                  85.1      14.9        0          0

Miami-Dade Co.
2011                  82.8       0.0 (a)   17.0        0.2
2012                  72.9       0.0 (a)   26.9        0.2
2013                  98.4       0.0 (a)    0.6        1.0

Waldo Storage
2011                  85.2      14.7        0.05       0.05
2012                  88.5      11.5        0          0
2013                  82.7      17.3        0          0

(a) P. obtusus is not yet established in South Florida


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Author:Porter, Sanford D.; Kumar, Vivek; Calcaterra, Luis A.; Briano, Juan A.; Seal, Dakshina R.
Publication:Florida Entomologist
Article Type:Report
Geographic Code:1USA
Date:Dec 1, 2013
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