Effects of a Fipronil Spot Treatment on Field Colonies of Coptotermes formosanus (Isoptera: Rhinotermitidae)

Transcription

1 HOUSEHOLD AND STRUCTURAL INSECTS Effects of a Fipronil Spot Treatment on Field Colonies of Coptotermes formosanus (Isoptera: Rhinotermitidae) W.L.A. OSBRINK, 1,2 M. L. CORNELIUS, 3 A. T. SHOWLER, 1 AND J. M. POUND 1 J. Econ. Entomol. 107(2): 727Ð740 (2014); DOI: ABSTRACT This Þeld study investigated the colony effect of a Þpronil spot treatment applied to active infestations of Formosan subterranean termite, Coptotermes formosanus Shiraki. Spot treatments were applied to a single active independent monitor from each of four colonies in which multiple independent monitors were established. All treated monitors were abandoned, and the contents of the treated monitors were replaced with untreated wood at the 30-d posttreatment inspection. All colonies survived treatment and only one colony exhibited long-term effects, which included significant reductions in termite collections and increased worker size. The affected colony was treated within 1mofitsprimary nest. Two colonies exhibited a correlation between monitor termite production and distance from treatment. Distance appears to be a factor limiting ÞpronilÕs colony effects. The Formosan subterranean termite may not be a good candidate for the exterior perimeter and localized interior treatment label option because of the large range and size of the colony. KEY WORDS Formosan subterranean termite, colony effect, nonrepellent Economic loss from termites in the United States causes US$11 billion per year (Su 2002). The Formosan subterranean termite, Coptotermes formosanus Shiraki, native to Asia (Bouillon 1970), has become an economically important pest in the southern United States (Su and Tamashiro 1987). In addition to structural infestations, C. formosanus infestations of living trees are common in the New Orleans, LA, area (Osbrink et al. 1999, Osbrink and Lax 2003). It has been suggested that partial postconstruction treatments with slow-acting, nonrepellent insecticides might result in elimination of termite colonies (Thorne and Breisch 2001, Potter and Hillery 2002, Vargo and Parman 2012). Such an approach reduces labor, cost, and environmental hazard compared with a traditional postconstruction barrier treatment. One such termiticide is Þpronil (Termidor SC; BASF, Research Triangle Park, NC), which disrupts nerve impulse transmission by antagonism of gamma-aminobutyric acid-gated chloride channels (Cole et al. 1993). Past research has shown Þpronil can suppress termite populations (Potter and Hillery 2002, Shelton 2003, Wagner 2003, Ripa et al. 2007). However, preliminary data suggest that a Þpronil treatment of an infested structure was not effective in eliminating an offsite C. This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or recommendation by the USDA for its use. USDA is an equal opportunity provider and employer. 1 USDA-ARS-SPA Knipling-Bushland U.S. Livestock Insects Research Laboratory, 2700 Fredericksburg Rd., Kerrville, TX Corresponding author, weste.osbrink@ars.usda.gov. 3 USDA-ARS-BARC, Baltimore Ave., Bldg., 007, Rm., 313, Beltsville, MD formosanus colony. The objective of this research was to determine C. formosanus colony effects of a Þpronil spot treatment applied to actively infested wood located away from the primary nest. Materials and Methods Independent Monitors. Termite-infested (2 by 4 by 30 cm [h by w by l]) pine stakes that had been hammered 22 cm into the soil and checked monthly were replaced with independent bucket trap termite monitors (monitors) active with C. formosanus (Su and Scheffrahn 1986). Each monitor was a 3.8-liter plastic bucket with a removable lid, the bottom removed, buried in the ground such that the lid was 1 cm below the soil surface. Monitors were Þlled with 50 slats (4 by 0.5 by 8 cm [h by w by l]) of spruce, Picea spp. Foraging ranges of Þve C. formosanus colonies were delineated using markðreleaseðrecapture detailed below. Colonies were labeled as follows: 1) SRRC service building yard (SRRC), 2) University of New Orleans Liberal Arts Building atrium (LAB), 3) central City ParkÕs Diagonal Drive (DIAG), 4) southern City ParkÕs magnolia (Magnolia grandiflora L., Magnoliaceae) stand adjacent to the north bank of Bayou Metairie (MAG), and 5) untreated central City Park Diagonal Drive quadruplex oak tree (QO; Figs. 1 and 2; Table 1). Individual monitors for each treated colony were labeled with Þpronil treated monitors followed by an F as follows. SRRC: S350F, S351, S354, S364, and S365; LAB: U50, U316, U317, U318, U319, U320, and U322F; DIAG: C15, C17, C330, C331, and C353; MAG: C56, C302, C304F, C305, and C360; QO: C300, C326, C332, and C333 (Fig. 2).

2 728 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 2 Fig. 1. Extended map of Southern Regional Research Center, New Orleans, LA, indicating locations of Formosan termite colonies spot treated with 0.125% Þpronil. Mark Release Recapture Colony Delineation. Foraging ranges of C. formosanus colonies were regularly delineated by dyeing termites for markðreleaseðrecapture procedure (Messenger and Su 2005). Termites collected from selected monitors in the Þeld were fed Whatman #1 Þlter paper dyed with either Nile Blue A (0.1%, wt dye:wt paper) or Neutral Red (0.5%, wt:wt; Sigma-Aldrich, St. Louis, MO) for 1wk in the laboratory until termites became blue or red, respectively. Dyed termites were released back into the same monitor they were collected from. Termites from all monitors in the area were collected monthly and examined for dyed individuals in the laboratory. Monitors or structures containing dyed termites were considered to be part of the same colony as the station in which the dyed termites were released. This dye procedure was repeated at least every 4 mo. Visual inspections of a southern live oak tree (Quercus virginiana Miller, Fagaceae) harboring a C. formosanus primary nest (SRRC colony) and two sheds adjacent to and infested with the SRRC colony were conducted at least monthly to monitor termite activity, presence of dyed termites, and presence of alates. Fipronil Spot Treatment. Colonies used in this study had stable and robust monitor collection histories (Table 2). Fipronil (Termidor SC) was applied at the higher label rate of 0.125% active ingredient. The lid of one active monitor for each colony was removed and the wood slats and termites within were sprayed to run off and the lid replaced. Treatments were applied 1 wk before the scheduled monthly inspection date. Amount of Þpronil delivered was determined by weight (Table 1). Time (d) to Þrst posttreatment inspection varied by colony depending on amount of wood present at time of treatment, after which collections were monthly. Termites had abandoned all spot-treated monitors by the Þrst posttreatment inspection. At the 1 mo posttreatment collection, wood from the abandoned spot-treated monitors were replaced with untreated wood. The number of termites collected from each monitor was calculated by weight, with worker numbers estimated by subtracting the total weight of soldiers and brachypterous nymphs from the total weight of termites collected (Osbrink et al. 2008). Individual weights were calculated by weighing four groups of 10 workers, soldiers, and brachypterous nymphs. Total number of soldiers and brachypterous nymphs were counted. Collection factors were subjected to analysis of variance (ANOVA) as detailed below, and when signiþcantly different (P 0.05), separated by FisherÕs

3 April 2014 OSBRINK ET AL.: EFFECTS OF FIPRONIL ON C. formosanus COLONIES 729 Fig. 2. Map of monitor locations of Formosan subterranean termite colonies spot treated with Þpronil. F indicates location of spot-treated monitor. (A) MAG, (B) DIAG, (C) SRRC, (D) LAB. Individual monitors for each colony were labeled as follows: SRRC: S350F, S351, S354, S364, and S365; LAB: U50, U316, U317, U318, U319, U320, and U322F; DIAG: C15, C17, C330, C331, and C353; MAG: C56, C302, C304F, C305, and C360. (Online Þgure in color.) least-signiþcant difference (LSD), unless otherwise indicated (Systat Software 2008). Factors being evaluated were as follows: Table 2, total number of termites collected by colony among years; Tables 3, 7, 8, and 9, total number of termites collected pretreatment among monitors, total number of termites collected posttreatment among monitors, total number of termites collected for each individual monitor among pre- and posttreatment; Tables 4 and 10, total number of termites collected by colony among posttreatment collections; Tables 5, 11, 12, and 13, worker weights by collection date pre- and posttreatment among monitors, worker weights by individual monitor among preand posttreatment dates; Table 6, number of alates collected among years, number of alates collected each year among sexes. Square roots of mean proportion (%) of soldiers were arcsine transformed and subjected to ANOVA, and when signiþcantly different (P 0.05), separated by LSD (Systat Software 2008). Transformed data were converted back to mean percentage ( SE) for reporting. Pearson correlation coefþcient (r) was computed between number of termites collected per monitor and the distance from the treatment monitor (Systat Software 2008). Acoustical Emission Detector (AED). An AED acoustical emissions detector (Acoustical Emissions Consulting, Fair Oaks, CA) was used to quantify termite activity within a southern live oak tree. A spot-treated C. formosanus colonyõs remote primary nest in SRRC oak S354 located 5.6 m from the treatment was acoustically monitored during the study. Two wave guides in the form of lag bolts (150 by 9 mm) were screwed into predrilled holes in the oakõs trunk 20 cm above the ground facing north and south. C. formosanus colony acoustical emissions were detected using a sensor (model SP-1L) probe and a model DMH-30 high force magnetic accessory attachment (Acoustic Emission Consulting, Fair Oaks, CA). Table 1. Parameters of spot-treated (fipronil 0.125%) Formosan subterranean termite colonies Parameter Colony SRRC LAB DIAG MAG QO No. monitors 3Ð Date treated 15 June May May May 2011 None Treatment amt 95.0 ml ml 14.1 ml 9.3 ml None Treated monitor S350 U322 C15 C304 None First post-treat collection 07/12/10 (27 d) 05/25/11 (7 d) 06/01/11 (6 d) 05/27/11 (1 d) Ð AED monitor Yes No No No No Location (lat.) Location (long.) Ð, not applicable.

4 730 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 2 Table 2. Annual total number of Formosan subterranean termite collected from fipronil spot-treated colonies (mean SE) Year Colony SRRC LAB DIAG MAG QO (untreated) 2007 ND 6, a 3, bc 10, ,349.6a 3, a 2008 ND 6, a 1, bd 3, b 6, ,168.3a , ,650.2a 5, a 7, a 4, b 5, a , ,94.6bc 3, a 3, bc Combined with , a 2010 PT 1, c NA NA NA 4, (PT)a a , b 4, ab 4, ,036.7c 4, b a 2011 NA PT b PT d PT 4, b 4, (PT)a b F F F F F df 3, 66 df 5, 397 df 5, 217 df 9, 167 df 9, 439 P P P P P ND, no data; NA, not applicable; PT, post treatment; means within a column followed by the same letter are not signiþcantly different, LSD (P 0.05). a Analysis consistent with SRRC. b Analysis consistent with LAB, DIAG, and MAG. Acoustical emissions were recorded by a laptop personal computer using Acoustical Emissions Consulting software to convert termite sounds to counts per second. Acoustical output for each attachment was captured for 60 s (Osbrink and Cornelius 2013a,b). Ten consecutive count values represented termite activity associated with each unique attachment. Both bolts were used to quantify acoustical output and the most active chosen to represent termite activity. Readings were taken monthly and increased to daily concurrent with monitor treatment, including a pretreatment reading on treatment day and posttreatment readings 1, 2, and 3 d posttreatment. Acoustical data were subjected to ANOVA, and when signiþcant (P 0.05), separated by LSD (Systat Software 2008). Alate Trapping. C. formosanus alates were trapped annually from 2009 through 2011 using a universal blacklight trap with 12-watt U -shaped blacklight tube (BioQuip Products, Rancho Dominguez, CA). The light trap was located outdoors 20 m south of the oak tree colonized by the Þpronil spot-treated SRRC C. formosanus colony. In 2011, another light trap was placed inside an adjacent infested shed. Light traps were checked every morning during April, May, and June, when local C. formosanus populations swarm. Annual mean alate catch and sex were analyzed using ANOVA, and if signiþcantly different (P 0.05), LSD separated means (Systat Software 2008). Results Overall, Þpronil spot-treated colonies initially showed signiþcant reductions in termites collected from monitors in two of four C. formosanus colonies (SRRC and DIAG) and a numerical reduction in LAB. Fipronil spot treatments did not eliminate remotely connected C. formosanus colonies as indicated by continued termite activity in monitors and AED surveillance. All four spot-treated monitors had no termites 1 mo posttreatment, which motivated the replacement of treated wood with untreated wood. Two (50%) treated monitors remained termite free for the rest of the season. SRRC Service Building Yard. There was a numerical reduction ( 2,000) in C. formosanus mean numbers of termites collected posttreatment in 2010 compared with pretreatment in 2010, and a signiþcant rebound in 2011 ( 3,000), with no comparable changes in the untreated colony (Table 2). Posttreatment 2010 collections were signiþcantly reduced from a combined pretreatment 2010 and 2009, followed by a signiþcant increase in 2011 (Fig. 3; F 4.242; df 2, 69; P 0.019). Table 3. Total number of Formosan subterranean termite collected by monitor from colonies receiving fipronil spot treatments (mean SE) Monitor 2009Ð2010 Pretreat 2010 Posttreat 2011 SRRC S350 a 4, ,175.7Aa Bb 6, ,026.7Aa H b df 2 P S351 6, ,423.2Aa 6, Bb 2, Ab F df 2, 17 P S354 6, ,851.1Aa 2, Aa 4, ,747.3Aa F df 2, 16 P S364 ND ND 3, ,198.2 S365 ND ND 3, ,081.5 F F F df 2, 16 df 2, 14 df 4, 30 P P P ND, no data. Means within a column followed by the same capital letter are not signiþcantly different, LSD (P 0.05). Means in a row followed by the same small letter are not signiþcantly different, protected LSD Test (P 0.05). a Treated monitor. b KruskalÐWallis ANOVA on ranks (P 0.050), means separated with DunnÕs method (P 0.050).

5 April 2014 OSBRINK ET AL.: EFFECTS OF FIPRONIL ON C. formosanus COLONIES 731 Table 4. SE) Detailed posttreatment total number of Formosan subterranean termite colonies receiving fipronil spot treatments (mean Posttreatment (d) SRRC SRRC notes LAB LAB adjusted a LAB notes 7 Ð Ð 2, ,429.6a 2, ,989.7a 67 intox. workers a Ð Ð Ð Ð 52 1, ,044.7a 8 LBN 1, a 2, a 175 small workers 99 1, LBN 3, a 4, a 1,859 LBN; 250 small workers 129 1, LBN Ð Ð Ð 171 1, ,899.7a 16 LBN 3, a 4, a 273 LBN 221 6, ,945.0a S350 Mud tubes F F , ,653.8a 162 LBN df 3, 39 df 3, , ,038.2a Ð P P , a Ð 394 2, ,563.5a Ð 444 5, ,807.3a 250 small workers 50 small soldiers 457 4, a Ð F df 11, 45 P LBN, large brachypterous nymphs; intox. workers, intoxicated workers. Means within a column followed by the same letter are not signiþcantly different. a Abandoned monitors removed from analysis. Ð no data. SigniÞcant differences occurred between mean number of termites collected from individual SRRC monitors posttreatment in 2010 but not in 2011, the latter included newly established monitors S364 and S365 (Table 3). The Þpronil treated monitor (S350) had a signiþcant reduction with no termites collected posttreatment in 2010 followed by complete recovery in 2011 (Table 3). SigniÞcantly fewer termites were also collected from monitor S351 posttreatment in 2010 and 2011, with a numerical increase in 2011 (Table 3). No signiþcant posttreatment termite reduction occurred in monitor S354 (Table 3). The Þrst evidence of posttreatment termite activity in the SRRC-treated monitor (S350) was mud tubes at 221 d posttreatment (Table 4). Large brachypterous nymphs (LBN) were present in posttreatment monitors (Table 4). A cohort of obviously smaller workers and soldiers, 250 and 50, respectively, were a part of collections from monitors S350, S351, and S365 at 444 d posttreatment (Table 4). There was no signiþcant correlation between posttreatment termite numbers in individual SRRC monitors and monitor distance from the treatment (r 0.312; P 0.798; N 3). No signiþcant worker weight differences occurred between individual monitors from the same collection dates either pre- or posttreatment in 2010, but did occur in 2011 (Table 5). Five of six monitors had signiþcant worker weight differences from the same monitor at different collection dates, with no obvious pattern of increased or decreased change (Table 5). There was a numerical posttreatment increase in mean ( SE) soldier proportions with , , and %, pretreatment in 2009Ð2010, and posttreatment in 2010 and 2011, respectively (F 1.077; df 2, 33; P 0.353). Soldier proportions from the untreated QO during the same time intervals were , , and %, with only QOÕs 2.8% signiþcantly different from SRRCÕs 13.3% (F 2.397; df 5, 61; P ). SRRC Service Building Yard Acoustical Emission Detector (AED). Mean number of AED acoustical counts of oak S354 were signiþcantly reduced at 1 and 2 d posttreatment followed by increase and decrease Table 5. Formosan subterranean termite worker weight (mg) from fipronil spot-treated colonies by monitor (mean SE) Monitor Pretreatment Posttreatment 5/2010 9/2010 4/2011 8/2011 SRRC S350 a Aa ND Bb Aa F df 2, 11 P S Abc Ac Aa ABab F df 3, 15 P S A A AB AC F df 3, 15 P S364 ND ND ABa Cb F df 1, 7 P S365 ND ND Cb BCa F df 1, 7 P F F F F df 2, 11 df 1, 6 df 4, 19 df 4, 19 P P P P Means within a column followed by the same capital letter are not signiþcantly different, LSD (P 0.05). Means in a row followed by the same small letter are not signiþcantly different, LSD (P 0.05). a Treated monitor.

6 732 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 2 Table 6. Formosan subterranean termite alates trapped per night adjacent to fipronil spot-treated SRRC colony (mean SE) (in shed) Total 1, ,441 6,457 1,374 Total female 5,526 44,703 3, % Female (38.6%) (49.4%) (46.7%) (57.2%) Total male 8,799 45,738 3, Mean total ( SE) , F , 62 P Male vs female Male vs female Male vs female Male vs female Male vs female Mean female ( SE) , , Mean male ( SE) , , F F F F , 29 1, 45 1, 35 1, 13 P P P P in activity, stabilization 1.5 mo posttreatment, and signiþcant increases to full recovery by 4 mo (Fig. 4; F ; df 8, 89; P 0.001). SRRC Service Building Yard Alates. No signiþcant difference occurred in the mean total number of alates captured outdoors during 2009, 2010, 2011, and indoors during 2011 (Table 6). There was a numerical increase in the number of alates captured in 2010 followed by a numerical reduction of alates in No signiþcant difference occurred in the number of males and females collected outdoors in 2009, 2010, 2011, and indoors in 2011 (Table 6). University of New Orleans Liberal Arts Building Atrium (LAB). There were nonsigniþcant numerical posttreatment termite reductions compared with 2010 and pretreatment 2011 (Table 2; Fig. 3; F 2.510; df 3, 259; P 0.059). SigniÞcant differences occurred between the mean number of termites collected from individual LAB monitors posttreatment in 2011 (Table 7). Termites abandoned three LAB monitors posttreatment, namely, U322 (treated), U316, and U317, which were signiþcant reductions from their respective pretreatment individual monitors (Table 7). The remaining seven active monitors, with abandoned monitors excluded, were not signiþcantly different from each other (Table 7). Located furthest from the treatment, monitors U318 and U319 (73.5 and 78.5 m from treatment, respectively) Þrst posttreatment collections (7 d) produced 7,053 and 13,243 termites, respectively, which included 67 intoxicated workers. Healthy LBNs were also present posttreatment (Table 4). A cluster of several hundred obviously smaller workers were present as part of collections at 52 and 99 d posttreatment (Table 4). A signiþcant correlation existed between individual posttreatment LAB monitor distance from treatment and number of termites it produced (r 0.801; P ; N 10). Mean worker weights from different LAB monitors collected at the same time were signiþcantly different both pre- and posttreatment (Table 8). Mean worker weights collected from the same monitors on different dates were also signiþcantly different for 8 of 10 monitors (Table 8). At 1 mo posttreatment (June 2011), worker weights were signiþcantly reduced in four of six monitors compared with pretreatment weights from the same monitor, most of which increased back to pretreatment weights at 5 mo (Table 8). Mean ( SE) posttreatment soldier proportions ( %) increased signiþcantly from pretreatment, % (F ; df 1, 37; P 0.001). Fig. 3. Number of Formosan subterranean termite by Þpronil spot-treated colony (mean SE). Scale bars without the same letter are signiþcantly different by protected LSD (P 0.05). PT above the 2010 SRRC bar indicates Posttreatment for this scale bar only.

7 April 2014 OSBRINK ET AL.: EFFECTS OF FIPRONIL ON C. formosanus COLONIES 733 Fig. 4. Acoustical activity of a Þpronil spot-treated Formosan subterranean termite colony. Means without the same letter are signiþcantly different by protected LSD (P 0.05). City Park s Diagonal Drive (DIAG). There was a signiþcant posttreatment reduction in termite numbers (Table 2; Fig. 3; F ; df 1, 109; P 0.001). A posttreatment signiþcant difference also occurred between mean termite numbers from individual DIAG monitors in 2011 (Table 9), with four of Þve monitors with mean termite production of 1,000. Posttreatment, all Þve individual monitors produced numerically less termites than pretreatment, signiþcant only with C330 (Table 9). Monitor C353 produced signiþcantly more termites than the other DIAG monitors, and was located furthest (55.0 m) from the treatment (Table 9). Overall, termite production of posttreatment DIAG remained reduced with no signiþcant increase for 172 d; however, monitor C353 consistently produced over twice the DIAG monitor average (Table 10). Posttreatment termites numbers from individual DIAG monitors were signiþcantly correlated with monitor distance from the treatment (r 0.943; P ; N 5). Pretreatment DIAG worker weight had signiþcant differences between monitors with C15 and C17 with smaller workers (Table 11). Posttreatment collections in June and July 2011 had no signiþcant worker weight differences between monitors collected at the same times. However, later collections had signiþcant intermonitor worker weight differences with C353 containing the largest workers (Table 11). DIAG individual monitors C15, C17, and C353 worker weights increased signiþcantly over time (Table 11). DIAG soldier mean ( SE) proportions posttreatment ( %) were signiþcantly greater than pretreatment ( %), with similar increases from untreated Quad Oak during the same time periods, with Ð % (F ; df 3, 57; P 0.001). Magnolia Stand (MAG). No signiþcant difference occurred between annual termite collection from Table 7. Total number of Formosan subterranean termite collected by monitor from LAB colony receiving a fipronil spot treatment (mean SE) Monitor 2010Ð2011 Pretreatment 2011 Posttreatment 2011 Posttreatment (adjusted) LAB U50 3, Aa 3, ,293.9ABa F df 1, 15 P , ,293.9A U315 5, ,102.8Aa Ba F df 1, 15 P , A U316 5, ,515.1Aa Cb U b T 10 P Removed U317 2, Aa Cb F df 1, 15 P Removed U318 4, ,246.4Aa 5, ,229.9ABa F df 1, 15 P , ,229.9A U319 3, Aa 6, ,243.5Aa F df 1, 15 P , ,243.5A U320 4, ,018.2Aa 4, ,105.4ABa F df 1, 15 P , ,105.4A U322 a 5, ,330.3Aa Cb F df 1, 15 P Removed U323 2, Aa 2, ,258.6ABa F df 1, 15 P A U324 3, Aa 2, ,025.1Ba F df 1, 15 P A F F F df 9, 119 df 9, 39 df 6, 27 P P P Means in a column followed by the same capital letter are not signiþcantly different, LSD Test (P 0.05). Means in a row followed by the same small letter are not signiþcantly different, LSD Test (P 0.05). a Treated monitor. b MannÐWhitney rank sum test, normality test failed (P 0.05).

8 734 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 2 Table 8. Formosan subterranean termite worker weight (mg) from fipronil spot treated LAB colony monitors (mean SE) Monitor Pretreatment Posttreatment 3/2011 4/2011 6/ /2011 LAB U BDa BCDEa DEb CGHIa F df 3, 15 P U DGa BDGIa ND BFHa F df 2, 11 P U BCGa EFHIJa ND ND F df 1, 7 P U CDa HKb ND ND F df 1, 7 P U DEc Aa BCDd Ab F df 3, 15 P U Aa ADa Ab DIb F df 3, 15 P U Fb ABa Bb AFa F df 3, 15 P U322 a Eb BCDFa ND ND F df 1, 7 P U323 ND ND BCEb BFGa F df 1, 7 P U Ba GJKb Fc Eb F df 3, 15 P F F F F df 8, 35 df 8, 35 df 5, 23 df 6, 27 P P P P Means in a column followed by the same capital letter are not signiþcantly different, LSD Test (P 0.05). Means in a row followed by the same small letter are not signiþcantly different, LSD Test (P 0.05). ND, no data. a Treated monitor. MAG in 2008Ð2011 pretreatment (2010 and pretreatment 2011 combined) and in 2011 posttreatment (Table 2). Individual MAG monitor termite productions were not signiþcantly different from each other preor posttreatment (Table 9). No individual MAG monitors had signiþcant differences between a monitorõs pre- and posttreatment termite production (Table 9). MAG did produce signiþcantly less termites at 1 d posttreatment, which increased with the next two collections (Table 10). No signiþcant correlation was found between total number of termites collected from speciþc MAG monitors and its distance from the Þpronil spot treatment (r ; P 0.888; N 5). MAG worker weights from pretreatment and most posttreatment collections were signiþcantly different between some monitors collected at the same time (Table 12). Numerous pre- and posttreatment collections contained larger workers ( 4 mg). Worker weights from three of Þve individual MAG monitors were signiþcantly different between collection dates from the same monitor, with posttreatment workers tending to be smaller than pretreatment collections (Table 12). Monitor C302 was the only monitor with no mean worker collections 4 mg (Table 12). MAGÕs mean ( SE) soldier proportions were not signiþcantly different between pre- ( %) and posttreatment ( %) collections (F ; df 1, 69; P 0.899). Quadruplex Oak Tree Untreated (QO). There was no signiþcant change in mean ( SE) number of termites collected from untreated QO whether grouped for comparison with SRRC (2009 Ð2010 pretreatment, 2010 posttreatment, and 2011 post- Table 9. Total number of Formosan subterranean termite collected by monitor from colonies receiving fipronil spot treatments (mean SE) Monitor 2010Ð2011 Pretreatment 2011 Posttreatment DIAG C15 a 3, Aa Ba F df 1, 21 P C17 2, Aa Ba F df 1, 21 P C330 2, Aa Bb F df 1, 21 P C331 2, Aa Ba F df 1, 21 P C353 4, ,090.1Aa 2, Aa F df 1, 21 P F F df 4, 79 df 4, 29 P P MAG C56 5, ,024.1Aa 6, ,587.3Aa F df 1, 13 P C302 5, ,396.8Aa 2, Aa F df 1, 13 P C304 a 2, Aa 4, ,894.7Aa F df 1, 13 P C305 4, Aa 4, ,280.3Aa F df 1, 13 P C360 3, ,337.2Aa 3, ,145.9Aa F df 1, 13 P F F df 4, 44 df 4, 24 P P Means in a column followed by the same capital letter are not signiþcantly different, LSD Test (P 0.05). Means in a row followed by the same small letter are not signiþcantly different, LSD Test (P 0.05). a Treated monitor.

9 April 2014 OSBRINK ET AL.: EFFECTS OF FIPRONIL ON C. formosanus COLONIES 735 Table 10. (mean SE) Detailed post-treatment total no. of Formosan subterranean termite from colonies receiving fipronil spot treatments Posttreatment (d) DIAG C353 no. termites MAG MAG notes 1 Ð Ð c Ð a 1213 Ð Ð a 914 3, ,208.9b 16 LBN 69 Ð Ð 6, a 57 LBN 88 1, a , ,087.6a 510 LBN 116 1, a 4335 Ð Ð , ,062.5a 107 LBN a 1629 Ð F F df 5, 29 df 4, 24 P P Means in a column followed by the same letter are not signiþcantly different, LSD Test (P 0.05). LBN, large brachypterous nymphs; Ð no data. treatment; F 0.996; df 2, 167; P 0.372) or grouped for comparisons with LAB, DIAG, and MAG (2010Ð2011 pretreatment and 2011 posttreatment; F 0.147; df 1, 99; P 0.702; Table 2; Fig. 3). Mean weights of QO workers collected at the same time had signiþcant differences between monitors on three of four collection dates (Table 13). Mean worker weights from the same monitor at different collection dates had signiþcant differences with the smallest consistently coming from the Þrst collection (June 2010). QO soldier proportions were not signiþcantly different between collections grouped for comparisons with SRRC (2009Ð2010, 2010, and 2011), but were signiþcantly different when grouped as 2010Ð2011 ( %) and 2011 ( %) for comparisons with LAB, DIAG, and MAG (F ; df 1, 27; P 0.001). Discussion Colony effects of a Þpronil spot treatment appeared greatest in DIAG and least in MAG. Abandonment of Þpronil-treated monitors limit toxicant exposure and colony effect. In contrast, sustained consumption of effective termite baits delivers enough toxicant to crash a population. In the absence of avoidance, effective chitin synthesis inhibitors have relatively doseindependent times to intoxication in contrast to ÞpronilÕs dose-dependent times to intoxication (Su 2005, Remmen and Su 2005b). Fipronil is considered nonrepellent because termites initiate tunneling behavior in treated substrates, but tunneling and substrate penetration are limited by dose-dependent rapid intoxication occurring within hours (Osbrink and Lax 2002b, Hu 2005, Remmen and Su 2005a). Abandoned treated monitors adjacent to active termite populations imply functional repellency. Su (2005) suggests a nonrepellent toxicant may cause secondary repellency from the accumulation of decomposing poisoned termites in galleries near the treatment. Abandonment of Þpronil treated monitors and the resulting colony survival indicates that Þpronil does not Þt a liquid-bait model (Osbrink et al. 2005, Su 2005). Colony survival reported in the current study were consistent with observations (W.L.A.O., unpublished data) obtained by monitoring a pest management professionalõs treatment (Þpronil 0.125%) of a storage shed infested with the SRRC C. formosanus colony (7 April 2009). Independent SRRC C. formosanus colony monitors located within 10 m of the treated shed indicated no negative colony effects. Secondary repellency may explain the absence of colony effect, as dead termites accumulate during construction of replacement galleries through substrates disrupted by trenching (Su et al. 1982, Su 2005). SRRC colonyõs infestation of an adjacent untreated storage shed was also uninterrupted after shed and monitor treatments (W.L.A.O., unpublished data). Table 11. Formosan subterranean termite worker weight (mg) from fipronil spot-treated colonies (means SE) Monitor Pretreatment Posttreatment 3/2011 4/2011 6/2011 7/ / /2011 DIAG C15 a ND Bc ND Ab ND Ba F df 2, 11 P C Bc Bc Ab Ab ND Ba F df 4, 19 P C Aa Ab ND ND Bb ND F df 2, 11 P C Ab Ab Aa Ab Bb ND F df 4, 19 P C Ab Ab Ab Ab Aa Aa F df 5, 23 P F F F F F F df 3, 15 df 4, 19 df 2, 11 df 3, 15 df 2, 11 df 2, 11 P P P P P P Means in a column followed by the same capital letter are not signiþcantly different, LSD Test (P 0.05). Means in a row followed by the same small letter are not signiþcantly different, LSD Test (P 0.05). a Treated monitor; ND no data.

10 736 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 2 Table 12. Formosan subterranean termite worker weight (mg) from fipronil spot-treated colonies (mean SE) Monitor Pretreatment Posttreatment 3/2011 6/2011 8/2011 9/ /2011 MAG C Ba Ba Aa Ca Ca F df 4, 19 P C Ba Bd Ad Dc Ce F df 4, 19 P C304 a Ba ND Aa Aa Aa F df 3, 15 P C Aa Bc Ab Bb Ab F df 4, 19 P C Ba Aab Ab Bab Bc F df 4, 19 P F F F F F df 4, 19 df 3, 15 df 4, 19 df 4, 19 df 4, 19 P P P P P Means in a column followed by the same capital letter are not signiþcantly different, LSD Test (P 0.05). Means in a row followed by the same small letter are not signiþcantly different, LSD Test (P 0.05). a Treated monitor; ND no data. Examination of individual colony monitors provided further insight into colony effects of Þpronil. Generally, ÞpronilÕs population effects decreased as distance from treatment increased. Termites abandoned all directly treated monitors and the two monitors closest (6.7 and 12.6 m) to LABÕs treatment. Termite numbers were also reduced in the monitors closest to SRRC (3.2 m) and DIAG treatments (0.3, 0.7, and 1.0 m). Treatment effects were less at greater treatment distances at SRRC (5.6 m), LAB (10.4Ð78.0 m), MAG (23.7Ð109.3 m), and DIAG (55.0 m). Consistent with distance as a limiting factor of ÞpronilÕs effects, the least affected MAG colonyõs treatment was over triple the distance from its closest independent monitor compared the other treated colonies ( 23 m). DIAGÕs spot treatment had the greatest colony effect and was closest to the primary nest. Four of DIAGÕs Þve monitors (including treatment) were located around the base of the same southern live oak and 1 m from each other. Such a cluster of productive monitors, two of which produced signiþcantly smaller workers pretreatment, suggest DIAGÕs primary nest was located in this tree (King and Spink 1969). Hardwood trees appear to be the preferred location of the primary nest of C. formosanus (Ehrhorn 1934, Osbrink et al. 1999, Osbrink and Lax 2002b). Thus, termites were directly treated close to the queen, early instars, and large numbers of nest mates, optimizing conditions for horizontal transfer (Potter and Hillery 2002, Ibrahim et al. 2003, Shelton and Grace 2003, Hu 2005). Fipronil intoxicated termites collected 70 m from LAB treatment differs from other studies where C. formosanus or Reticulitermes spp. traveled 5 m because of loss of mobility (Potter and Hillery 2002, Su 2005, Ripa et al. 2007, Saran and Rust 2007). In contrast, Vargo and Parman (2012) reported elimination of a R. flavipes colony whose colony range extended to a monitor 13.5 m from the Þpronil soil treatment. However, colony collapse could explain monitor abandonment without the toxicant being distributed to all monitors. Similarly in this current study, abandoned LAB monitor (12.6 m) might be explained by the secondary repellency of dead termites from the treated monitor blocking common galleries leading to the abandoned monitor (Su et al. 1982, Su 2005). This current study differs from others in that spot treatments were applied to active monitors and included direct spray (involuntary treatment) of live termites. Osbrink and Lax (2003) reported collections of imidacloprid-intoxicated termites some distance ( 10 m) from imidacloprid tree treatments. Direct toxicant application to termites during foam treatments of tree voids resulted in large population effects, whereas no population effects were detected after soil treatments, which initially provided no toxicant contact (Osbrink and Lax 2003, Osbrink et al. 2005). Extended movement of intoxicated termites may be explained when a reduced Þpronil dose increases its dose-dependent time to intoxication (Remmen and Su 2005b). Wet treated termites may brießy facilitate horizontal transfer in a manner analogous Table 13. Formosan subterranean termite worker weight (mg) from untreated colony (mean SE) Monitor Untreated 6/ /2010 3/2011 8/2011 QO C Ab Bab Aa Ba F df 3, 15 P C Ac ABb Aa Bb F df 3, 15 P C Bc Aa Ab ND F df 2, 11 P C Ac ABb Ab Aa F df 3, 15 P F F F F df 3, 15 df 3, 15 df 3, 15 df 2, 11 P P P P Means in a column followed by the same capital letter are not signiþcantly different, LSD Test (P 0.05). Means in a row followed by the same small letter are not signiþcantly different, LSD Test (P 0.05); ND no data.

11 April 2014 OSBRINK ET AL.: EFFECTS OF FIPRONIL ON C. formosanus COLONIES 737 to wet paint. Rapid cascades of multiple liquid transfers may progressively deliver decreased doses extending ÞpronilÕs intoxication period to over a week (Remmen and Su 2005b). Intoxicated LAB termites collected 7 d posttreatment does not contradict this model. Termites that were reported to travel 5 m received higher doses of Þpronil, resulting in relatively rapid ( 1 d) mobility loss (Potter and Hillery 2002, Su 2005, Ripa et al. 2007, Saran and Rust 2007, Neoh et al. 2013). Overall, intracolony mean worker weight differences were surprisingly variable between simultaneous monitor collections as well as from the same monitor from different collection dates. Su and Scheffrahn (1988) found mean body weights of C. formosanus foragers within a colony are fairly homogeneous in south Florida. Because C. formosanus was introduced to south Florida relatively recently ( 1980), all colonies may have originated from a single introduction, and thus possess similar genetic composition (Su and Scheffrahn 1988). Waller and La Fage (1987) suggest an annual cycle of worker weight, as decreasing winter egg production increases population age (size). No such pattern suggested itself in this current study. Intracolony groupings of similar sized foragers may suggest feeding site Þdelity of cohorts of similar age or possible similar degrees of inbreeding (Kaib et al. 1996, Evans et al. 1999, Husseneder et al. 2008). Husseneder et al. (2008) found foraging worker body weight within C. formosanus colonies located in New Orleans, LA, are negatively correlated with the degree of inbreeding in individuals relative to others in their colony (Husseneder et al. 2008). Intracolony inbreeding differences are a colony life-history trait where primary reproductives are eventually replaced by their multiple offspring whose progeny possess greater and varied nest mate relatedness (Thorne 1997, Vargo and Husseneder 2009). In addition, colonies in this current study all had histories of the brachypterous nymph production required for replacement reproductives to be present (Raina et al. 2004; W.L.A.O., unpublished data). DIAG colonyõs rapid (months) increase of worker weights contrasted with the other treated colonies and may reßect termination of reproduction and disproportionately high mortality of early instars after the Þpronil treatment of live termites adjacent to the primary nest as discussed above. Decreased queen egg production can result from reduced seasonal temperatures, intoxication, death, or resource deprivation (Waller and La Fage 1987). Possible explanations facilitating early instar mortality include their mass exposure while congregated in the primary nest and greater toxicant sensitivity (King and Spink 1969, Koehler et al. 1993). Increased C. formosanus worker size were also reported after chitin synthesis inhibitor exposure and was attributed to increased mortality of smaller, earlier instars, caused, however, by increased frequency of molting failure when compared with later, larger instars (King and Spink 1974, Osbrink et al. 2011). Grace et al. (1995) found slow (years) worker mass increase, as C. formosanus colonies declined with no clear understanding of mechanisms involved. C. formosanus colony elimination and reinvasion studies have reported that worker weights may differ between the eliminated colony and the colony that reoccupies the vacated area (Messenger et al. 2005, Mullins et al. 2011). First indications of postelimination reinvasion may be slow (months) or rapid (days) with a progressive increase in activity, depending on local termite pressure (Grace et al. 1996, Messenger et al. 2005). In the current study, most monitors remained active and provided no opportunity for occupation by another colony. Thus, in the current study, worker weight changes are not explained by colony replacement. Posttreatment collections with clusters of obviously smaller workers or soldiers accompanying average termites may have been produced by a new supplemental or replacement reproductive. Posttreatment, workers may begin to forage from the nest earlier to replace those lost. Small workers may indicate close proximity to the nursery and eggs production site (King and Spink 1974). In this current study, numbers of antennal segments were not quantiþed for instar determination. Soldier proportion differences had no clear association with treatments even though C. formosanus soldiers were found to be more Þpronil tolerant than workers (Osbrink et al. 2001). Coptotermes spp. soldiers occupy (defend) foraging sites earlier and evacuate later than most workers, thus small collection numbers contain higher soldier proportions (Arab et al. 2012; W.L.A.O., unpublished data). Waller and La Fage (1988) reported seasonal ßuctuation of soldier proportions peaking during spring dispersal swarms (Waller and La Fage 1988); however, no such pattern was obvious in this current study. Nest oak AED readings indicated that termite activity plummeted and remained low at 1 and 2 d posttreatment but did not disappear. Osbrink and Cornelius (2013a) hypothesized that tarsal claw substrate interactions of moving termites create the primary signal detected by AED. Therefore, a reduction in termite numbers or their movement will lower AED counts. Possible explanations for the large posttreatment signal reduction would include the accumulation of dead and moribund termites in the tree and movement of survivors away from the dead. Thus, signal-producing live termites left the trunk lumen, a good signal conductor, and entered or passed through the carton nest in the soil, a poor signal conductor, just below the trunk (Osbrink et al. 1999, 2008; Mankin et al. 2002; Osbrink and Lax 2002a). This scenario would enhance colony survival by reducing the horizontal transfer of a toxicant (Ibrahim et al. 2003, Shelton and Grace 2003, Tsunoda 2006, Mulrooney et al. 2007, Saran and Rust 2007, Spomer et al. 2008, Bagneres et al. 2009). Posttreatment low AED readings are consistent with the immobilization effect of Þpronil reported by Saran and Rust (2007) and Su (2005). SRRC colony survived, re-

12 738 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 2 covered, and produced strong AED signals within 2 mo. Tree voids occupied by C. formosanus are often large enough to facilitate compartmentalization and isolation of toxicant and dead termites (Osbrink et al. 1999, Osbrink and Lax 2002a). Posttreatment production of large nymphs and alates in three of four treated colonies indicated a continued capability to produce dispersal ßights and establish new colonies (Raina et al. 2004). Absence of posttreatment changes in alate sex ratio trapped outdoors and indoors indicated possible queen survival because replacement reproductives from orphaned C. formosanus, Coptotermes lacteus (Froggatt), and Coptotermes acinaciformis (Froggatt) colonies produced predominantly male alates as a mechanism to reduce inbreeding (Su and Scheffrahn 1987, Lenz and Runko 1993, Crosland et al. 1994, Roisin and Lenz 2002, Husseneder et al. 2006). In conclusion, distance appears to be a factor limiting ÞpronilÕs colony effects. This study indicates that C. formosanus may not be a good candidate for exterior perimeter and localized interior treatment because of the large range and size of their colonies. Applied evaluations of speciesðpesticideðsubstrate dynamics provide critical insights where performance characterization facilitates successful implementation of the best control strategies. Acknowledgments We greatly appreciate suggested manuscript improvements provided by N. Y. Su, S. Skoda, J. Bland, D. Thomas, and M. Tarver. References Cited Arab, A., Y. Blanco, and A. M. Costa-Leonardo Dynamics of foraging and recruitment behavior in the Asian subterranean termite Coptotermes gestroi (Rhinotermitidae). Psyche 2012: ID Bagneres, A. G., A. Pichon, J. Hope, R. W. Davis, and J.-L. 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Owens Reinvasion and colony expansion of Coptotermes formosanus (Isoptera: Rhinotermitidae) after areawide elimination. J. Econ. Entomol. 104: 1687Ð1697. Mulrooney, J. E., R. D. Hasse, T. L. Wagner, and P. D. Gerard Activity of Reticulitermes flavipes (Isoptera: Rhinotermitidae) exposed to nestmates treated with slowacting nonrepellent termiticides. Sociobiology 50: 71Ð86. Neoh, K.-B., C.-C. Lee, and C.-Y. Lee Effects of termiticide exposure on mutual interactions between the treated and untreated workers of the Asian sub-