Louisin Stte University LSU Digitl Commons LSU Mster's Theses Grdute School 2010 Toxicity interction of fipronil nd imidcloprid ginst Coptotermes formsnus Pn Luo Louisin Stte University nd Agriculturl nd Mechnicl College, compsscu@gmil.com Follow this nd dditionl works t: http://digitlcommons.lsu.edu/grdschool_theses Prt of the Entomology Commons Recommended Cittion Luo, Pn, "Toxicity interction of fipronil nd imidcloprid ginst Coptotermes formsnus" (2010). LSU Mster's Theses. 1246. http://digitlcommons.lsu.edu/grdschool_theses/1246 This Thesis is brought to you for free nd open ccess by the Grdute School t LSU Digitl Commons. It hs been ccepted for inclusion in LSU Mster's Theses by n uthorized grdute school editor of LSU Digitl Commons. For more informtion, plese contct gcoste1@lsu.edu.
TOXICITY INTERACTION OF FIPRONIL AND IMIDACLOPRID AGAINST COPTOTERMES FORMOSANUS A Thesis Submitted to the Grdute Fculty of the Louisin Stte University nd Agriculturl nd Mechnicl College in prtil fulfillment of the requirements for the degree of Mster of Science in The Deprtment of Entomology By Pn Luo B.S., Chin Agriculturl University, 2008 August 2010
ACKNOWLEDGMENTS I would like to express pprecition to my mjor professor, Dr. Gregg Henderson. I thnk him for his support for the cdemic reserch nd invluble dvice in the experimentl design nd dt nlysis. Gret pprecitions re expressed for his ptience nd expertise in editing the thesis. In ddition, his intellectul honesty in science nd detil-oriented working style gretly impressed me nd will be my guidnce in my future creer. Thnks to my committee members, Drs. Michel J. Stout nd Wyne Krmer for their suggestions nd support for my reserch nd thesis. Very specil thnks re due to Bl K. Gutm nd Dr. Lixin Mo for the encourgement nd dvice in both cdemic nd non-cdemic res during my time t LSU. Thnks lso to Nikhil Ngendr, Ynxi Liu, Dr. Zhorigetu Chen nd Xioyu Feng for their ssistnce nd dvice for the reserch. I lso thnk fellow students nd friends Yunlong Yng, Mthew Gimmel, Grnt Aucoin, Json Hmm, Xun Chen, Jennifer Gordon, Ktherine Prys, Poornim Jysimh nd Dr. Xioyi Wu for the generl help during my grdute study. Specil thnks to my friends in other deprtments such s Chenfei Go, Fei Wng nd Feifei Hn for their support nd friendship during my sty t LSU. Finlly, I sincerely pprecite the support nd encourgement from my fmily nd reltives, especilly my wife, Meijio Zhou nd prents, Guihe Luo nd Ronghui Li. Without their help, I would not hve finished grdute studies brod. ii
TABLE OF CONTENT ACKNOWLEDGMENTS... ii LIST OF TABLES... iv LIST OF FIGURES... v ABSTRACT... vi CHAPTER 1. INTRODUCTION... 1 References... 6 CHAPTER 2. CHANGES IN SURVIVORSHIP OF COPTOTERMES FORMOSANUS DUE TO DIFFERENT COMBINATIONS OF FIPRONIL AND IMIDACLOPRID... 10 Introduction... 11 Mterils nd Methods... 12 Results... 15 Discussion... 18 References... 23 CHAPTER 3. CHANGES IN SURVIVORSHIP OF COPTOTERMES FORMOSANUS DUE TO GRADIENT COMBINATIONS AND LOW-CONCENTRATION COMBINATIONS OF FIPRONIL AND IMIDACLOPRID... 26 Introduction... 27 Mterils nd Methods... 28 Results... 34 Discussion... 42 References... 46 CHAPTER 4. CHANGES IN SURVIVORSHIP AND BEHAVIOR IN COPTOTERMES FORMOSANUS DUE TO GRADIENT CONCENTRATIONS OF IMIDACLOPRID COMBINED WITH A FIXED AMOUNT OF FIPRONIL... 49 Introduction... 50 Mterils nd Methods... 51 Results... 54 Discussion... 57 References... 64 CHAPTER 5 SUMMARY AND CONCLUSIONS... 67 VITA... 70 iii
LIST OF TABLES Tble 2.1 List of tretments nd their concentrtions in solutions nd substrtes... 13 Tble 2.2 Men percentge of mortlity (±SEM) of termites in untreted rens... 19 Tble 3.1 List of tretments nd their concentrtions in solutions nd substrtes in grdient combintion biossys... 31 Tble 3.2 List of tretments nd their concentrtions in solutions nd substrtes in lowconcentrtion biossys... 31 Tble 3.3 Percentge of mortlity of termites in untreted Petri dishes... 42 Tble 4.1 List of tretments nd their concentrtions in solutions nd substrtes... 53 iv
LIST OF FIGURES Fig 2.1 A seled Petri dish (left) nd the storge drwer (right)... 14 Fig 2.2 Survivl numbers in different tretments t different times... 16 Fig 2.3 Survivl numbers in ech tretment t different times... 17 Fig 3.1 A Petri dish with excvtion holes in the low-concentrtion biossy... 33 Fig.3.2 Survivl numbers in different tretments t different times... 35 Fig 3.3 Survivl numbers in ech tretment t different times... 36 Fig 3.4. Survivl numbers in different tretments t ech time... 38 Fig 3.5. Survivl numbers in ech tretment t different times... 39 Fig 3.6. Excvtion holes in different tretments... 41 Fig 4.1 Survivl numbers in different tretments t different times... 55 Fig 4.2 Survivl numbers in ech tretment t different times... 56 Fig 4.3 Excvtion holes in different tretments... 58 Fig 4.4 The reltionship of chnges in numbers of excvtion holes nd chnges in percentge mortlity of termites... 59 Fig 4.5 The mechnism of imidcloprid leding to lower mortlity in the combintions thn in fipronil lone... 60 Fig 4.6 The threshold concentrtion of imidcloprid tht hd negtive effects on fipronil... 61 v
ABSTRACT The Formosn subterrnen termite, Coptotermes formosnus Shirki (Isopter: Rhinotermitide), is considered one of the most destructive structurl pests in the world, especilly in wrm nd humid res. Insecticide ppliction is n effective strtegy in termite control. In recent yers, non-repellent insecticides hve become populr for their high efficcy due to delyed toxicity nd horizontl trnsfer. Fipronil (registered nme Termidor ) nd imidcloprid (registered nme Premise ) hve been pplied to the perimeter of millions of houses in the United Sttes. Fipronil nd imidcloprid hve different modes of ction which my produce synergistic effect when combined. There hve been no studies on the toxicity interction of fipronil nd imidcloprid ginst termites including the Formosn subterrnen termite. The originl objective of the reserch ws to determine whether combintions of the termiticides led to enhnced toxicity ginst Formosn subterrnen termites. Combintions of the non-repellent insecticides were treted on filter pper nd snd for evlution. After timed exposures, ny living termites were trnsferred to untreted Petri dishes. Mortlity of termites ws recorded before nd fter the trnsfer. Lower mortlity ws observed when imidcloprid ws mixed with fipronil compred to fipronil lone. Mortlity ws incresed by the mixture over imidcloprid lone. To vlidte these results, more combintions were introduced in the second vi
nd third set of experiments. Besides the recording of mortlity, the number of excvtion holes mde by termites in snd ws lso counted to determine whether excvtion ctivity ws relted to mortlity effects. A second objective ws to seek threshold level whereby the efficcy of fipronil becomes negtively impcted by imidcloprid presence. A threshold of between 15 nd 25 ppm imidcloprid dded to 100 ppm fipronil reduced the efficcy of fipronil. An increse in the number of excvtion holes ws significntly ssocited with rising mortlity, indicting imidcloprid ffected the uptke of fipronil by reducing termite excvtion behvior of treted soil. In prcticl terms nd of potentil concern for homeowners, the studies suggest tht the efficcy of Termidor pplied round the perimeters of houses my be negtively ffected by the presence of Premise. vii
CHAPTER 1 INTRODUCTION 1
Termites belong to the order Isopter nd re some of the most importnt structurl pests. According to the clssifiction proposed by Snyder (1949) nd Emerson (1955), there re six fmilies of Isopter in the world: Mstotermitide, Klotermitide, Hodotermitide, Rhinotermitide, Serritermitide nd Termitide. Four fmilies hve been described in the United Sttes: Klotermitide, Hodotermitide, Rhinotermitide nd Termitide (Snyder 1949, Emerson 1955). There re forty to fifty species of termites distributed in the United Sttes; however, only two drywood termites, Cryptotermes brevis (Wlker) nd Incisitermes minor (Hgen), one tree termite, Nsutitermes costlis (Holmgren) nd five subterrnen termites, Coptotermes formosnus Shirki, C. gestroi (Wsmnn), Reticulitermes hesperus Bnks, R. flvipes (Kollr) nd R. virginicus (Bnks), re generlly considered importnt economic pests (Su nd Scheffrhn 1990, Culliney nd Grce 2000, Scheffrhn et l. 2002, Cbrer et l. 2005). The economic loss due to termites hs been estimted t 11 billion dollrs per yer nd hs incresed s the stndrd of living hs improved (Su 2002, Oi et l. 2003). Among these termite species, C. formosnus is known s the most destructive species in the United Sttes becuse it is the most ggressive nd hs lrger colony popultions; however, due to its wide distribution in the U.S., R. flvipes is regrded the single most economiclly importnt species (Su nd Scheffrhn 1990, Culliney nd Grce 2000). 2
Coptotermes formosnus is sometimes clled the Orientl subterrnen termites or Asin subterrnen termites indicting its origin (Su nd Tmshiro 1987). The first record of C. formosnus in the continentl United Sttes ws from shipyrd in Houston, Texs in 1965 (Bel 1967). It ws reported tht Formosn subterrnen termites cused billions of dollrs in dmge nd control costs nnully ntionwide, with 300 million dollrs in property dmge, preventive mesures nd structurl repir in New Orlens lone (USDA-ARS 2007). Insecticide ppliction is n effective strtegy for termite control (Gold et l. 1996, Henderson 2003). Fipronil ws first introduced to the United Sttes in 1996 by Rhone Poulenc Ag. Compny (U.S. EPA Office of Prevention, Pesticide nd Toxic Substnces 1996). It is used s termite control product, for fire nts, cockroches, turf insects nd fles (U.S. EPA Office of Prevention, Pesticide nd Toxic Substnces 1996b, Cox 2005). In ddition, it is pplied to crops such s corn nd cotton for plnt protection (Overmyer et l. 2005). The first fipronil product in termite control in the United Sttes ws Termidor which ws pproved by the EPA in 1999 (PANNA 2009). Compred to fipronil, imidcloprid hs longer history- its first synthesis nd mode of ction were reported in 1984 (Schroder nd Flttum 1984). Premise is the first trdemrk of the imidcloprid product for termite control in the United Sttes mrketed by Byer Corportion in the mid-1990s. Besides its uses in termite, nt nd fle control, it is lso pplied 3
to griculturl products to control sucking nd chewing insect species. Its ppliction formultions include folir sprys, soil tretments nd seed dressings (Elbert et l. 1998). Fipronil belongs to the phenylpyrzole clss of insecticide (IRAC 2008). It is known to inhibit the neurotrnsmitter γ-minobutyric cid (GABA) in both insects nd vertebrtes (Hosie et l. 1995, Tingle et l. 2003). The exct binding sites re still unknown (Le Corronc et l. 2002). Recently glutmte-gted chloride chnnels (GluCl), chloride chnnels unique to insects, were lso found to be inhibited by fipronil (Iked et l. 2003). The mode of ction my ply criticl role in the high selective toxicity of fipronil in insects but not mmmls (Zho et l. 2004). Imidcloprid directly binds to postsynptic nicotinic cetylcholine receptors (nachr) which cuses its toxic effects in insects s well s vertebrtes (Elbert et l. 1998). It is the first insecticide found to block nachr completely nd irreversibly in insects; the binding is 1,000 times stronger in insects thn in vertebrtes which endows its high insect selectivity (Methfessel 1992). Fipronil nd imidcloprid, s non-repellent insecticides, hve ttrcted more interest thn trditionl repellent insecticides (Krd 2001, Henderson 2003, Ibrhim et l. 2003, Shelton nd Grce 2003). Non-repellent insecticides often mintin the property of non-repellency even t high concentrtions (up to 500 prt per million in Reticulitermes hesperus) (Srn nd Rust 2007). The trnsfer of fipronil nd imidcloprid mong termite workers s well s between 4
workers nd soldiers hs been studied (Thorne nd Breisch 2001, Ibrhim et l. 2003, Srn nd Rust 2007). There is liner reltionship between dose uptke nd insecticide contct time in subterrnen termites (Srn nd Rust 2007). Body contct (including grooming) plys the min role in lethl dose horizontl trnsfer compred to the trnsmission by trophllxis (Srn nd Rust 2007). The trnsfer from soldiers to workers is significntly higher thn workers to soldiers (Ibrhim et l. 2003). A study on distnce of horizontl trnsfer in the field showed tht the lethl effects in Formosn subterrnen termites my be limited however (Su 2005). Since Termidor nd Premise represent two of the most populr termiticides in the termite control mrket it is common for pest control compnies treting the perimeter of houses to use either of them. If the owners of house chnges hnds, or if pest control contrcts re shifted from one compny to nother, house might be multi-treted over time with Termidor nd Premise. This would suggest tht mixture of Premise nd Termidor would likely occur in the field. However, there hve been no studies on the toxicity interction of fipronil nd imidcloprid ginst Formosn subterrnen termites. I hypothesized tht there ws toxicity interction effects between fipronil nd imidcloprid nd tht termite behvior my ply n importnt role in their combined toxicity effects. In this thesis studies on termite survivorship to different rtios of combintions of fipronil nd imidcloprid were conducted. In ddition, termite behviorl response to the 5
toxicnts ws observed nd possible explntions of chnges in survivorship due to different combintions re discussed. References Bel, R. H. 1967. Formosn invder. Pest Control 35: 13-17. Cbrer BJ, Su N.-Y., Scheffrhn R. 2005. UF/IFAS reserchers find nother termite in south Florid s destructive s Formosn "super termite". UF/IFAS News. http://news.ifs.ufl.edu/2005/04/08/double-trouble-ufifs-reserchers-find-nothertermite-in-south-florid-s-destructive-s-formosn-super-termite. Accessed on June 9, 2010. Cox, C. 2005. Fipronil. Journl of Pesticide Reform 25: 10. Culliney, T. W. nd J. K. Grce. 2000. Prospects for the biologicl control of subterrnen termites (Isopter: Rhinotermitide), with specil reference to Coptotermes formosnus Bulletin of Entomologicl Reserch 90: 9-21 Elbert, A., R. Nuen, nd W. Leichet. 1998. Insecticides with novel modes of ction, pp. 50-73. In I. Ishy nd D. Degheele [eds.]. Springer, New York. Emerson, A. E. 1955. Geogrphicl origins nd dispersions of termite gener. Fieldin Zoology 37: 465-520. Gold, R. E., H. N. Howell, B. M. Pwson, nd M. S. Wright. 1996. Persistence nd biovilbility of termiticides to subterrnen termites (Isopter: Rhinotermitide) from five soil types nd loctions in Texs. Sociobiology 28: 337-363. Henderson, G. 2003. Liquid lerning. Pest Control 31: 48-50. Hosie, A. M., H. A. Bylis, S. D. Buckinghm, nd D. B. Sttelle. 1995. Actions of the insecticide fipronil on dieldrin-sensitive nd -resistnt GABA receptors of Drosophil melnogster. British Journl of Phrmcology 116: 909-912. 6
Ibrhim, S. A., G. Henderson, nd H. Fei. 2003. Toxicity, repellency, nd horizontl trnsmission of fipronil in the Formosn subterrnen termite (Isopter: Rhinotermitide). Journl of Economic Entomology 96: 461-467. Iked, T., X. Zho, Y. Kono, J. Z. Yeh, nd T. Nrhshi. 2003. Fipronil modultion of glutmte-induced chloride currents in cockroch thorcic gnglion neurons. Neuro Toxicology 24: 807-815. IRAC. 2008. IRAC mode of ction clssifiction, http://www.irconline.org/documents/moa%20clssifiction_aug08_v6.1.pdf. Accessed on Jnury 20, 2010. Krd, B. 2001. Gulfport studies sty the course. Pest Control 69: 30-33, 73. Le Corronc, H., P. Alix, nd B. Hue. 2002. Differentil sensitivity of two insect GABA-gted chloride chnnels to dieldrin, fipronil nd picrotoxinin. Journl of Insect Physiology 48: 419-431. Methfessel, C. 1992. Action of imidcloprid on the nicotinic cetylcholine receptors in rt muscle. Pflnzenschutz-Nchrichten Byer 45: 369-380. Myles, T. G., nd W. L. Nutting. 1988. Termite eusocil evolution: re-exmintion of Brtz's hypothesis nd ssumptions. The Qurterly Review of Biology 63: 1-23. Oi, F. M., J. L. Cstner, nd P. G. Koehler. 2003. The Estern subterrnen termite, http://edis.ifs.ufl.edu/in031. Accessed on Mrch 20, 2010. Overmyer, J. P., B. N. Mson, nd K. L. Armbrust. 2005. Acute toxicity of imidcloprid nd fipronil to nontrget qutic insect, Simulium vitttum Zetterstedt cytospecies IS-7. Bulletin of Environmentl Contmintion nd Toxicology 74: 872-879. PANNA. 2009. PAN pesticides dtbse - pesticide products. Pesticide ction network, North Americ. http://www.pesticideinfo.org/detil_product.jsp? REG_NR=00796900210&DIST_NR=007969. Accessed on Mrch 20, 2010. 7
Srn, R. K., nd M. K. Rust. 2007. Toxicity, uptke, nd trnsfer efficiency of fipronil in Western subterrnen termite (Isopter: Rhinotermitide). Journl of Economic Entomology 100: 495-508. Scheffrhn, R. H., B. J. Cbrer, W. H. Kern, nd N.-Y. Su. 2002. Nsutitermes costlis (Isopter: Termitide) in Florid: first record of non-endemic estblishment by higher termite. Florid Entomologist 85: 273-275. Schroder, M. E., nd R. F. Flttum. 1984. The mode of ction nd neurotoxic properties of the nitromethylene heterocycle insecticides. Pesticide Biochemistry nd Physiology 22: 148-160. Shelton, T. G., nd J. K. Grce. 2003. Effects of exposure durtion on trnsfer of nonrepellent termiticides mong workers of Coptotermes formosnus Shirki (Isopter: Rhinotermitide). Journl of Economic Entomology 92: 456-460. Snyder, T. E. 1949. Ctlog of the termites (Isopter) of the world. Smithsonin Miscellneous Collections 112: 1-490. Su, N.-Y. 2002. Novel technologies for subterrnen termite control. Sociobiology 40: 95-101. Su, N.-Y., nd R. H. Scheffrhn. 1990. Economiclly importnt termites in the United Sttes nd their control. Sociobiology 17: 77-94. Su, N.-Y., nd M. Tmshiro. 1987. An overview of the Formosn subterrnen termite in the world, pp. 3-15. In: Tmshiro M., Su N.-Y. [eds.], Biology nd control of the Formosn subterrnen termite. College of Tropicl Agriculturl nd Humn Resources, University of Hwii, Honolulu, HI. Su, N.-Y. 2005. Response of the Formosn subterrnen termites (Isopter: Rhinotermitide) to bits or nonrepellent termiticides in extended forging rens. Journl of Economic Entomology 98: 2143-2152. Thorne, B. L., nd N. L. Breisch. 2001. Effects of sublethl exposure to imidcloprid on subsequent behvior of subterrnen termite Reticulitermes virginicus (Isopter: Rhinotermitide). Journl of Economic Entomology 94: 492-498. 8
Tingle, C. C. D., J. A. Rother, C. F. Dewhurst, S. Luer, nd W. J. King. 2003. Fipronil: environmentl fte, ecotoxicology, nd humn helth concerns. Reviews of Environmentl Contmintion nd Toxicology: 1-66. U.S. EPA Office of Prevention, Pesticide nd Toxic Substnces. 1996. New pesticide fct sheet. Office of Pesticide Progrms, pp. 1-10. U.S. EPA Office of Prevention, Pesticide nd Toxic Substnces. 1996b. Fipronil pet products - review of domestic niml sfety studies with spot-on formultion nd use informtion with spry formultion. Office of Pesticide Progrms, pp. 1-7. USDA-ARS. 2007. Fungl fom seeks nd destroys termites. http://www.rs.usd.gov/is/ar/rchive/sep07/termites0907.htm. Accessed on Mrch 20, 2010. Zho, X., J. Z. Yeh, V. L. Slgdo, nd T. Nrhshi. 2004. Fipronil is potent open chnnel blocker of glutmte-ctivted chloride chnnels in cockroch neurons. The Journl of Phrmcology nd Experimentl Therpeutics 310: 192-201. 9
CHAPTER 2 CHANGES IN SURVIVORSHIP OF COPTOTERMES FORMOSANUS DUE TO DIFFERENT COMBINATIONS OF FIPRONIL AND IMIDACLOPRID 10
Introduction Both fipronil nd imidcloprid hve become populr for their non-repellent nd efficcious nture (Su 2005, Rust nd Srn 2008). The fetures of non-repellency, delyed toxicity nd horizontl trnsfer, led to higher control efficcy for termite popultions thn do trditionl repellent pyrethroids or cutely toxic orgnophosphorous termiticides (Krd 2001, Wgner 2003, Hu 2005, Tsunod 2006). Fipronil inhibits the neurotrnsmitter γ-minobutyric cid (GABA) nd glutmte-gted chloride chnnels (GluCl) while imidcloprid binds to the postsynptic nicotinic cetylcholine receptors (nachr) (Abbink 1991, Hosie et l. 1995, Iked et l. 2003, Tingle et l. 2003). Different modes of ction indicte synergistic effects my exist if both compounds re dded together. As these compounds represent two of most common termiticides in the termite control mrket tody, numerous studies hve been conducted on the toxicity of both fipronil nd imidcloprid in the lbortory nd field (Ibrhim et l. 2003, Osbrink et l. 2005, Su 2005). However, it is surprising tht no reserch hs been conducted on their toxicity interction. Lbortory experiments were conducted to test mortlity in the Formosn subterrnen termite Coptotermes formosnus Shirki to fipronil, imidcloprid nd their combintions. Mortlity ws lso evluted when termites were trnsferred to untreted Petri dishes following their survivl in termiticide tretments. The objective of the experiment ws to determine whether there ws 11
synergism or not between fipronil nd imidcloprid ginst field-collected Formosn subterrnen termites in lbortory rens. Mterils nd Methods Termites. Formosn subterrnen termites, C. formosnus were collected using crtetrpping technique (Smith et l. 2004, Gutm nd Henderson 2010) from Brechtel Prk in New Orlens, Louisin in October, 2008. The crte trp ws kept in 140-L trsh cn with lid nd ws stored in the urbn entomology lbortory under room conditions (26-28 C, 70-80% RH). Prior to the strt of the trils, helthy nd ctive workers nd soldiers were trnsferred from crte trp (one colony) to plstic continer with moist brown pper towels (Tork Universl hnd towel, SCA Tissue North Americ, Neenh, WI). One colony ws used in the biossy. Termiticides. The two termiticides tested in the trils were fipronil (Fipronil Tech., BAS 350I, BASF Corportion, Reserch Tringle Prk, NC) nd imidcloprid (Premise 75, Byer Corportion, Knss City, MO). Biossys. Fifty grms of utoclved dry snd (construction snd, Louisin Cement Products, LLC., Bton Rouge, LA) were loded in ech Petri dish (100 mm 15 mm, Medegn Medicl Products, Gllwy, TN) nd filter pper (55mm in dimeter, Grde 1 nd Grde 2, Whtmn) ws plced on top of the snd. Termiticide solutions were prepred by dissolving preweighed termiticides with wter in 100 ml volumetric flsks. In order to ensure fipronil ws 12
mixed in wter completely (no visible prticles), the volumetric flsks were plced in n ultrsonic bth (Brnsonic 1510R-MT ultrsonic clener, Dnbury, CT) working t 70 W nd 42 khz for 20-30 minutes. The four termiticide solutions prepred for the biossy nd the concentrtions of ech termiticide in the substrte re shown in Tble 2.1. A 10 ml solution ws dded to the filter pper in the Petri dishes nd 10 ml wter only ws dded s the control. Tble 2.1. List of tretments nd their concentrtions in solutions nd substrtes. Tretments Concentrtions in solutions (ppm) Concentrtions in substrtes (ppm) 100 ppm F* 100 F 16.67 F 100 ppm F + 100 ppm I* 100 F + 100 I 16.67 F + 16.67 I 50 ppm F + 50 ppm I 50 F + 50 I 8.33 F + 8.33 I 100 ppm I 100 I 16.67 I Control 0 0 *F: fipronil; I: imidcloprid. Fifty termites, (45 workers nd 5 soldiers) were introduced from the plstic continer forementioned to ech treted Petri dish by soft fine brush. Then every Petri dish ws seled with Prfilm (Prfilm M, Pechiney Plstic Pckging, Chicgo, IL) nd stored in n isolted chmber t room temperture (21 23 C) (Fig. 2.1). Ech tretment ws tested t four exposure times, 24, 41, 51 nd 65 hours nd two replictes were performed. Two Petri dishes of ech tretment were rndomly tken out of the chmber t ech time period. The number of surviving termites, defined s ny prt of the termite moving with or without the stimulus from brush ws recorded. 13
Fig. 2.1. A seled Petri dish (left) nd the storge drwer (right). After counting surviving termites, they were trnsferred using clen forceps to untreted Petri dishes (100 mm 15 mm, Medegn Medicl Products, Gllwy, TN) which were pre- loded with 50 g utoclved dry snd, filter pper (55mm in dimeter, Grde 1 nd Grde 2, Whtmn) nd 10 ml wter. All toxicnt-free Petri dishes were lbeled with the termiticide termites were previously exposed to nd the exposure time (24, 41, 51 or 65 hours). They were then stored bck in the drk drwer t room temperture (21 23 C). Surviving termites in Petri dishes were exmined gin for mortlity between 24 hours nd 52 hours fter plcement into untreted dishes. Dt nlysis. A two-wy ANOVA ws used to nlyze the vrince of survivl numbers mong different concentrtion combintions nd different times by using generlized liner model (PROC GLM) in SAS softwre (SAS 9.1, SAS Institute, NC). The mens of survivl numbers were seprted using Lest Significnt Difference test (LSD) (α=0.05). 14
In the rens where termites were trnsferred to untreted Petri dishes, two-wy ANOVA ws used to nlyze the vrince of mortlity percentges (= Ded termite numbers / Totl termite numbers trnsferred) in different tretments for ech time. The mens of mortlity percentges were seprted by LSD (α=0.05). Results There ws significnt interction between fipronil nd imidcloprid. In ddition, significnt interction between tretment nd time ws found (F = 9.83; df = 12, 39; P < 0.0001). Tretment effect. There ws significnt difference in survivl numbers mong the tretments (F = 214.04; df = 4, 39; P < 0.0001) (Figure 2.2). Among the four different termiticides tretments, fipronil lone cused the lowest survivl while imidcloprid lone resulted in the highest survivl numbers t 24, 41, 51 nd 65 hours. The survivl numbers in the tretment of 100 ppm fipronil plus 100 ppm imidcloprid were significntly lower thn in the tretment of 100 ppm imidcloprid but significntly higher thn in the tretment of 100 ppm fipronil t 41, 51 nd 65 hours. There ws no significnt difference in survivl numbers between tretments of 100 ppm imidcloprid nd control t 24, 41 nd 51 hours (Figure 2.2). Time effect. There ws significnt difference in survivl numbers ws found mong observtion times (F = 54.63; df = 3, 39; P < 0.0001) (Fig. 2.3). The survivl numbers were significntly reduced in the tretment of 100 ppm fipronil s erly s 24 hours, while there ws 15
Survivl numbers 50 40 30 20 b 24 hrs b 41 hrs 51 hrs 65 hrs d d d c c c cd d 10 b b b 0 Control 100 F* 100 F + 100 I* 50 F + 50 I 100 I Control 100 F 100 F + 100 I 50 F + 50 I 100 I Control 100 F 100 F + 100 I Groups with different retments 50 F + 50 I 100 I Control 100 F 100 F + 100 I 50 F + 50 I 100 I Fig. 2.2. Survivl numbers in different tretments t different times. Mens with the sme letter re not significntly different. * F: fipronil; I: imidcloprid. 16
Control 100 F* 100 F + 100 I* 50 F + 50 I 100 I Survivl numbers 50 40 30 20 b b b b b c b 10 b b c 0 24 41 51 65 24 41 51 65 24 41 51 65 24 41 51 65 24 41 51 65 Times (hrs) Fig. 2.3. Survivl numbers in ech tretment t different times. Mens with the sme letter re not significntly different. * F: fipronil; I: imidcloprid. 17
no significnt difference in the tretments of 100 ppm imidcloprid up to 51 hours (Fig. 2.3). The chnges in survivl numbers in the tretment of 100 ppm fipronil plus 100 ppm imidcloprid mong different times were not significntly different (F = 3.65; df = 3, 7; P = 0.1216). There ws no significnt difference in survivl numbers in the control (F = 0.46; df = 3, 7; P = 0.7243) (Fig. 2.3). In ddition, some termites were hevily infected with fungi, especilly when they were exposed to termiticides for long time (i.e. 65 hours). In the trnsfer study, termites previously treted with combintions of 100 ppm fipronil plus 100 ppm imidcloprid nd 50 ppm fipronil plus 50 ppm imidcloprid for 24, 41 nd 51 hours showed higher percentge mortlity thn those in the tretment of 100 ppm imidcloprid lone (Tble 2.2). The mortlity in tretments of 100 ppm fipronil plus 100 ppm imidcloprid with 41 hours or 51 hours exposure time nd fter round 50 hours in untreted Petri dishes ws much higher thn in tretments with 24 hours exposure time. A similr trend ws lso found in the tretment of 100 ppm imidcloprid lone (Tble 2.2). Discussion Compred to the high mortlity led by fipronil, the toxicity of imidcloprid ws both reduced nd delyed. One of the most interesting results found in the study ws tht mortlities in the combintions of fipronil nd imidcloprid were lower thn in fipronil lone but higher thn in imidcloprid lone. These results cused me to reject the hypothesis tht synergistic effects 18
Tble 2.2. Men percentge of mortlity (±SEM) of termites in untreted rens. Exposure time in the previous Time in the non-toxic Previous tretments Percentge of mortlity (men ± SEM) (%) tretment (hrs) Petri dishes 24 30 Control 7.184 ± 3.0165 * 100 ppm F 89.775 ± 1.895 b 100 ppm F+ 100 ppm I 14.447 ± 3.733 50 ppm F+ 50 ppm I 60.914 ± 4.392 c 100 ppm I 11.818 ± 9.480 F = 143.89; df = 4,9; P < 0.0001 41 52 Control 6.066 ± 0.184 100 ppm F 81.250 ± 6.250 b 100 ppm F+ 100 ppm I 95.000 ± 5.000 b 50 ppm F+ 50 ppm I 77.737 ± 9.444 b 100 ppm I 71.485 ± 23.755 b F = 8.42; df = 4,9; P = 0.0191 51 52 Control 5.100 ± 5.102 100 ppm F 91.665 ± 8.335 b 100 ppm F+ 100 ppm I 86.150 ± 4.330 bc 50 ppm F+ 50 ppm I 93.165 ± 3.975 b 100 ppm I 56.054 ± 17.760 c F = 15.74; df = 4,9; P = 0.0049 * Within ech time period, mens in the sme column followed by different letters indicted significnt differences between tretments. Mens were seprted by Lest Significnt Difference test (LSD) (α=0.05) existed between fipronil nd imidcloprid. The result tht imidcloprid lone led to the lowest mortlities mong the four termiticide tretments nd tht no significnt differences between tretments of imidcloprid nd controls were observed indicted low toxicity of imidcloprid in generl (Fig. 2.1). This phrmcodynmic pttern of imidcloprid on termites ws lso noted in 19
other studies. For exmple, Thorne nd Breisch (2001) observed tht the effect of imidcloprid ws so negligible tht the symptoms would dispper if termites were not exposed to imidcloprid for sufficient mount of time. Due to the reltively low toxicity of imidcloprid it ws not surprising tht the mortlity in the combintions would be incresed when fipronil ws dded. Thorne nd Breisch (2001) lso noticed delyed toxicity of imidcloprid nd reported tht deth cused by imidcloprid took severl dys. In ddition, Hgsm (2003) reported tht complete mortlity of Reticulitermes hesperus Bnks required 14 dys when they were exposed to 500 ppm imidcloprid for 2 hours. Therefore in the present study, it ws not surprising tht both the increse of time exposure to the termiticides nd the time in untreted Petri dishes cused mortlities in chemicl combintions nd imidcloprid lone to increse (Tble 2.2). Although imidcloprid showed reduced nd delyed toxicity, behviorl chnges cused by imidcloprid were pprent nd much fster thn in other tretments. Immobility ws noticed in termites either treted with imidcloprid lone or combintions of fipronil nd imidcloprid s erly s two hours posttretment (unreported results from preliminry experiment). Henderson (2003) reported tht significntly decresed digging nd wlking behviors were found when Formosn subterrnen termites were exposed to imidcloprid t 9 hours, while the effect of fipronil on digging nd wlking behviors ws not significntly different from control groups. 20
Experiments on Reticulitermes virginicus (Bnks) lso showed immobility symptoms cused by imidcloprid s erly s 4 hours posttretment (Thorne nd Breisch 2001). It ws reported tht both fipronil nd imidcloprid were minly tken up nd trnsferred by body contct (Hgsm 2003, Srn nd Rust 2007). Since imidcloprid gretly reduces termite wlking nd digging behvior, the uptke nd trnsfer of the toxicnt my be inhibited (Henderson 2003). I propose tht the presence of imidcloprid reduces the chnce of termites coming in contct with nd tking up the more toxic fipronil, leding to lower mortlities when imidcloprid nd fipronil re combined. More fungus ws found in Petri dishes treted with imidcloprid nd high mortlities were ccompnied with this fungl ssocition. It ws reported tht termites reduced removl of fungl spores when in the presence of imidcloprid, resulting in susceptibility to entomopthogenic fungi (Rmkrishnn et l. 1999). This feture of imidcloprid could be complimentry chrcter for the low toxicity. In both directly treted nd untreted rens fter exposure, the tretment with fipronil lone yielded higher mortlity thn the other tretments (Fig. 2.2, Tble 2.2). The treted rens tested the direct effects of fipronil on termites, while untreted rens were used to check the effects of fipronil-residue on termites. Since fipronil is non-repellent termiticide, termites my enter treted res indvertently. After exposure to termiticides hving delyed toxicity they 21
could continue to forge for n extended period of time. The results from untreted rens indicted tht termites tht initilly escped deth in treted rens eventully died. When the toxicity of fipronil in this study ws compred with other publictions, lower level of mortlity ws noted. For exmple, Bgneres et l. (2009) reported tht 77% nd 90% mortlity in Americn nd French Reticulitermes flvipes (Kollr) ws obtined when they were exposed to 1 ppm fipronil in snd fter 24 hours, while this study showed 16 ppm (the concentrtion in the snd nd filter pper when 100 ppm fipronil solution ws dded) yielded only 30% mortlity of C. formosnus t 24 hours (Fig. 2.2). The difference in the mortlity of R. flvipes nd C. formosnus could be linked to the vrition in susceptibility in the two species R. flvipes is considered more susceptible thn C. formosnus to fipronil (Remmen nd Su 2005). Although the results showed imidcloprid reduced the efficcy of fipronil nd fipronil enhnced the toxicity of imidcloprid, the experimentl design could be chllenged by the limited concentrtions in the biossy - only two concentrtions (100 ppm nd 50 ppm) of fipronil nd imidcloprid were tested. More combintions of fipronil nd imidcloprid t different concentrtions re needed to vlidte the results. Also in the trnsfer study I vried the time in my evlution of toxicity between tretments. Observtion time in the untreted Petri dishes should hve used constnt time. 22
References Abbink, J. 1991. The biochemistry of imidcloprid. Pflnzenschutz-Nchrichten Byer 44: 183-194. Bgnères, A. G., A. Pichon, J. Hope, R. W. Dvis, nd J. L. Clément. 2009. Contct versus feeding intoxiction by fipronil in Reticulitermes termites (Isopter: Rhinotermitide): lbortory evlution of toxicity, uptke, clernce, nd trnsfer mong individuls. Journl of Economic Entomology 102: 347-356. Gutm, B. K., nd G. Henderson. 2010. Impct of snd moisture level on food consumption nd distrbution of Formosn subterrnen termites (Isopter: Rhinotermitide) with different soldier percentge. Journl of Entomologicl Science. Accepted for publiction 10 June 2010. Hgsm, K. 2003. Utiliztion nd movement of toxicnts nd nutrients nd their effects on the Western subterrnen termite, Reticulitermes hesperus Bnks (Isopter: Rhinotermitide). pp. 1-143, Ph.D. disserttion. Deprtment of Entomology, University of Cliforni, Riverside. Henderson, G. 2003. Liquid lerning. Liquid lerning 31: 48-50. Hosie, A. M., H. A. Bylis, S. D. Buckinghm, nd D. B. Sttelle. 1995. Actions of the insecticide fipronil on dieldrin-sensitive nd- resistnt GABA receptors of Drosophil melnogster. British Journl of Phrmcology 116: 909-912. Hu, X. P. 2005. Evlution of efficcy nd nonrepellency of indoxcrb nd fipronil-treted soil t vrious concentrtions nd thicknesses ginst two subterrnen termites (Isopter: Rhinotermitide). Journl of Economic Entomology 98: 509-517. Ibrhim, S. A., G. Henderson, nd H. Fei. 2003. Toxicity, repellency, nd horizontl trnsmission of fipronil in the Formosn subterrnen termite (Isopter: Rhinotermitide). Journl of Economic Entomology 96: 461-467. 23
Iked, T., X. Zho, Y. Kono, J. Z. Yeh, nd T. Nrhshi. 2003. Fipronil modultion of glutmte-induced chloride currents in cockroch thorcic gnglion neurons. NeuroToxicology 24: 807-815. Krd, B. 2001. Gulfport studies sty the course. Pest Control 69: 30-33, 73. Osbrink, W. L. A., M. L. Cornelius, nd A. R. Lx. 2005. Effect of imidcloprid soil tretments on occurrence of Formosn subterrnen termites (Isopter: Rhinotermitide) in independent monitors. Journl of Economic Entomology 98: 2160-2168. Rmkrishnn, R., D. R. Suiter, C. H. Nktsu, R. A. Humber, nd G. W. Bennett. 1999. Imidcloprid-enhnced Reticulitermes flvipes (Kollr) (Isopter: Rhinotermitide) susceptibility to the entomopthogen Metrhizium nisoplie (Metsch.) Sorokin. Journl of Economic Entomology 92: 1125-1132. Remmen, L. N., nd N.-Y. Su. 2005. Time trends in mortlity for thimethoxm nd fipronil ginst Formosn subterrnen termites nd Estern subterrnen termites (Isopter: Rhinotermitide). Journl of Economic Entomology 98: 911-915. Rust, M. K., nd R. K. Srn. 2008. Toxicity, repellency, nd effects of cetmiprid on Western subterrnen termite (Isopter: Rhinotermitide). Journl of Economic Entomology 101: 1360-1366. Srn, R. K., nd M. K. Rust. 2007. Toxicity, uptke, nd trnsfer efficiency of fipronil in Western subterrnen termite (Isopter: Rhinotermitide). Journl of Economic Entomology 100: 495-508. Smith, W. R., T. L. Amburgey, G. Henderson, nd D. R. Ring. 2004. Fcility for conducting field tests on Coptotermes formosnus t Louisin Stte University Agriculturl Center. Forest Products Journl 54: 26-28. Su, N. -Y. 2005. Response of the Formosn subterrnen termites (Isopter: Rhinotermitide) to bits or nonrepellent termiticides in extended forging rens. Journl of Economic Entomology 98: 2143-2152. 24
Thorne, B. L., nd N. L. Breisch. 2001. Effects of sublethl exposure to imidcloprid on subsequent behvior of subterrnen termite Reticulitermes virginicus (Isopter: Rhinotermitide). Journl of Economic Entomology 94: 492-498. Tingle, C. C. D., J. A. Rother, C. F. Dewhurst, S. Luer, nd W. J. King. 2003. Fipronil: environmentl fte, ecotoxicology, nd humn helth concerns. Reviews of Environmentl Contmintion nd Toxicology: 1-66. Tsunod, K. 2006. Trnsfer of fipronil, nonrepellent termiticide, from exposed workers of Coptotermes formosnus (Isopter: Rhinotermitide) to unexposed workers. Sociobiology 47: 563-575. Wgner, T. L. 2003. U.S. Forest service termiticide tests. Sociobiology 41: 131-141. 25
CHAPTER 3 CHANGES IN SURVIVORSHIP OF COPTOTERMES FORMOSANUS DUE TO GRADIENT COMBINATIONS AND LOW-CONCENTRATION COMBINATIONS OF FIPRONIL AND IMIDACLOPRID 26
Introduction Coptotermes formosnus Shirki is one of the most destructive structurl pests in the United Sttes (Su nd Scheffrhn 1990). Louisin is hevily infested stte. For exmple, the economic loss cused by Formosn subterrnen termites is estimted t 300 million dollrs in New Orlens lone (USDA-ARS 2007). Two of most populr insecticides in the termite control mrket, fipronil nd imidcloprid hve been exhustively studied for uptke by individuls s well s horizontl trnsfer within colony (Bskrn et l. 1999, Rmkrishnn et l. 2000, Ibrhim et l. 2003, Osbrink et l. 2005, Su 2005, Tsunod 2006, Tomlski et l. 2010). Studies using 14 C reveled tht contct ws the min pthwy for the uptke of fipronil nd imidcloprid nd cuticulr trnsport ws necessry for toxicnt trnsfer in colony (Hgsm 2003, Srn nd Rust 2007, Bgnères et l. 2009). In lbortory biossys, fipronil nd imidcloprid showed non-repellency nd delyed toxicity (Hgsm 2003, Ibrhim et l. 2003, Remmen nd Su 2005, Srn nd Rust 2007). Studies on behvior chnges to termiticides showed tht imidcloprid significntly inhibited termite digging, tunneling nd wlking behviors (Thorne nd Breisch 2001, Hgsm 2003, Henderson 2003). A soil penetrtion study showed concentrtion nd thickness of treted substrtes ffected termite mortlity (Hu 2005). Efficcy nd degrdtion of fipronil nd imidcloprid in the field were lso reported (Bskrn et l. 1999, Osbrink et l. 2005, Su 2005). 27
Though fipronil nd imidcloprid shre delyed toxicity nd non-repellency which mke them successful in tody s termite control mrket, they hve different modes of ction. Fipronil binds to GABA receptors nd glutmte-gted chloride chnnels while imidcloprid trgets cetylcholine receptors (Abbink 1991, Hosie et l. 1995). This difference led me to initilly hypothesize tht there ws synergistic effect between them. The experiment in chpter Ⅱ showed tht fipronil incresed the toxicity of imidcloprid while imidcloprid reduced the toxicity of fipronil. The objective of this chpter ws to vlidte nd extend the results of chpter Ⅱ. Biossys evluting the mortlity of Formosn subterrnen termites exposed to grdient concentrtion combintions of fipronil nd imidcloprid nd low-concentrtion combintions of fipronil nd imidcloprid were conducted. In ddition, in order to explore the reltionship between behvior nd mortlity, excvtion holes, defined s discrete cvities in the snd long the inner mrgin of the Petri dish, were recorded in the low-concentrtion combintion biossy. Mterils nd Methods Termites. Formosn subterrnen termites, C. formosnus were collected using crtetrpping technique (Smith et l. 2004, Gutm nd Henderson 2010) in New Orlens, Louisin in 2008. The crte trp ws kept in 140-L trsh cn with lid nd ws stored in the urbn entomology lbortory under constnt conditions (26-28 C, 70-80% RH). Prior to the strt of the trils, helthy nd ctive workers nd soldiers were trnsferred from crte trp (one colony) 28
to plstic continer with moist brown pper towels (Tork Universl, SCA Tissue North Americ, Neenh, WI). Termiticides. The two termiticides tested in the trils were fipronil (Fipronil Tech., BAS 350I, BASF Corportion, Reserch Tringle Prk, NC) nd imidcloprid (Premise 75, Byer Corportion, Knss City, MO). Grdient combintions biossy. Fifty grms of utoclved dry snd (construction snd, Louisin Cement Products, LLC., Bton Rouge, LA) were loded in ech Petri dish (100 mm 15 mm, Medegn Medicl Products, Gllwy, TN) nd filter pper (55mm in dimeter, Grde 1 nd Grde 2, Whtmn) ws plced on top of the snd. Termiticide solutions were prepred by dissolving pre-weighed termiticides with wter in 100 ml volumetric flsks. The termiticide weight, concentrtions in the solution nd concentrtions in the snd nd Petri dishes in ech tretment re shown in Tble 3.1. In order to ensure fipronil ws mixed with wter completely (no visible prticles), the volumetric flsks were plced in n ultrsonic bth (Brnsonic 1510R- MT ultrsonic clener, Dnbury, CT) working t 70 W nd 42 khz for 20-30 minutes. A 10 ml solution from ech termiticide formultion ws dded to the filter pper in the Petri dishes nd 10 ml wter only ws dded s the control. Fifty termites (45 workers nd 5 soldiers) were introduced from the plstic continer forementioned to ech treted Petri dish by soft fine brush. One colony ws used in the 29
biossy. Then every Petri dish ws seled with Prfilm (Prfilm M, Pechiney Plstic Pckging, Chicgo, IL) nd stored in n isolted chmber t room temperture (21 23 C). Ech tretment ws tested t three exposure times, 24, 48 nd 66 hours nd three replictes were performed. Three Petri dishes in ech tretment were rndomly tken out of the chmber t ech time period. The number of surviving termites, defined s ny prt of the termite moving with or without the stimulus from brush ws recorded. Low-concentrtion combintion biossys. Fifty grms of utoclved dry snd ws loded in every Petri dish (100 mm 15 mm, Medegn Medicl Products, Gllwy, TN) nd filter pper (55mm in dimeter, Grde 1 nd Grde 2, Whtmn) were plced on top of the snd. Termiticide solutions were prepred by dissolving pre-weighed termiticides with wter in 100 ml volumetric flsks. The termiticide weight, concentrtions in the solution nd concentrtions in the snd nd Petri dishes in ech tretment re shown in Tble 3.2. A 10 ml solution from ech termiticide formultion ws dded to the filter pper in the Petri dishes nd 10 ml wter only ws dded s the control. Fifty termite workers were introduced from the plstic continer forementioned ( different colony from the colony in grdient combintion biossy) to ech treted Petri dish by soft fine brush. Every Petri dish ws then seled with Prfilm (Prfilm M, Pechiney Plstic Pckging, Chicgo, IL) nd stored in n isolted chmber t room temperture (21 23 C). 30
Tble 3.1. List of tretments nd their concentrtions in solutions nd substrtes in grdient combintion biossys. Tretments Insecticide weight (mg) Concentrtions in solutions (ppm) Concentrtions in snd nd filter pper (ppm) 100 ppm F* 10.48 (F) 100 (F) 16.68 (F) 80 ppm F + 20 ppm I* 8.43 (F) + 2.67 (I) 80 (F) + 20 (I) 13.42 (F) + 3.34 (I) 60 ppm F + 40 ppm I 6.16 (F) + 5.28 (I) 60 (F) + 40 (I) 9.80 (F) + 6.60 (I) 40 ppm F + 60 ppm I 4.30 (F) + 7.93 (I) 40 (F) + 60 (I) 6.84 (F) + 9.91 (I) 20 ppm F + 80 ppm I 2.17 (F) + 10.65 (I) 20 (F) + 80 (I) 3.45 (F) + 13.31 (I) 100 ppm I 13.41 (I) 100 (I) 16.76 (I) Control 0 0 0 *F: fipronil; I: imidcloprid Tble 3.2. List of tretments nd their concentrtions in solutions nd substrtes in the low-concentrtion biossys. Tretments Insecticide weight (mg) Concentrtions in solutions (ppm) Concentrtions in snd nd filter pper (ppm) 10 ppm F* 1.05 (F) 10 (F) 1.67 (F) 10 ppm F + 10 ppm I* 1.05 (F) + 1.33 (I) 10 (F) + 10 (I) 1.67 (F) + 1.66 (I) 5 ppm F + 5 ppm I 0.52 (F) + 0.67 (I) 5 (F) + 5 (I) 0.83 (F) + 0.83 (I) 10 ppm I 1.33 (I) 10 (I) 1.66 (I) Control 0 0 0 *F: fipronil; I: imidcloprid 31
Ech tretment ws tested t three exposure times, 72, 96 nd 120 hours nd three replictes were performed. Three Petri dishes in ech tretment were rndomly tken out of the chmber t ech time period. The number of surviving termites, defined s ny prt of the termite moving with or without the stimulus from brush ws recorded. In ddition, the number of excvtion holes, defined s discrete cvities in the snd long the inner mrgin of the Petri dish ws recorded (Fig. 3.1). After recording survivorship in ech Petri dish, surviving termites were trnsferred using clen forceps to untreted Petri dishes (100 mm 15 mm, Medegn Medicl Products, Gllwy, TN) which were pre-loded with 50 g utoclved dry snd, filter pper (55mm in dimeter, Grde 1 nd Grde 2, Whtmn) nd 10 ml wter. All untreted Petri dishes were lbeled with the previous Petri dish tretments nd exposure time (72, 96 nd 120 hrs) nd were stored in the drk drwer t room temperture (21 23 C). Surviving termites were counted gin in Petri dishes fter 44 hours. Dt nlysis. A two-wy ANOVA ws used to nlyze the vrince of surviving numbers between different tretments t ech time nd ech tretment mong different times by using generlized liner model (PROC GLM) in SAS softwre (SAS 9.1, SAS Institute, NC). The mens of survivl numbers were seprted using Lest Significnt Difference test (LSD) (α=0.05). 32
Fig. 3.1. A Petri dish with excvtion holes in the low-concentrtion biossy. The number of excvtion holes between different times nd between different tretments ws nlyzed by two-wy ANOVA. The mens of hole numbers were seprted by Lest Significnt Difference test (LSD) (α=0.05). A Simple Liner Regression (SLR) ws used to nlyze the reltionship between the mortlity nd the number of excvtion holes for different termiticide tretments t ech time period. In the trnsfer study of low-concentrtion biossy, the percentge of mortlity = ded termite numbers/ totl termite numbers trnsferred. 33
Results Grdient combintion biossy. There ws significnt interction between tretment nd time (F = 6.05; df = 12, 62; P < 0.0001). A significnt difference in survivl numbers ws found mong the tretments tested t ech time period (F = 21.00; df = 6, 62; P < 0.0001) (Figure 3.2). The survivl numbers in the tretment of 80 ppm fipronil plus 20 ppm imidcloprid were significntly higher thn in the tretment of 100 ppm fipronil lone t 48 nd 66 hours but not t 24 hours (Figure 3.2). Among the combintion tretments, the survivl numbers in 40 ppm fipronil plus 60 ppm imidcloprid were lowest but still higher thn in fipronil lone t 48 nd 66 hours (Figure 3.2). When the dt were nlyzed within ech tretment mong different times, significnt difference in survivl numbers ws found (F = 39.59; df = 2, 62; P < 0.0001) (Fig. 3.3). The survivl numbers were significntly reduced in the tretment of 100 ppm fipronil nd tretment of 20 ppm fipronil plus 80 ppm imidcloprid t ll time intervls (Fig. 3.3). The chnges of survivl numbers in the tretment of 100 ppm imidcloprid nd tretment of 80 ppm fipronil plus 20 ppm imidcloprid mong different times were not significntly different (100 ppm imidcloprid: F =1.02; df = 2, 8; P = 0.4163; 80 ppm fipronil plus 20 ppm imidcloprid: F = 0.38; df = 2, 8; P =0.6994) (Fig. 3.3). There ws no significnt difference in survivl numbers in the controls (F = 0.86; df = 2, 8; P = 0.4705). 34
50 40 b bc 24 hrs 48 hrs 66 hrs bc bc c c bc bc bc bc bc bd b bc bd d d Survivl numbers 30 20 c 10 0 100 F* 80 F 60 F +20 I* +40 I 40 F +60 I 20 F +80 I 100 I Con* 100 F 80 F +20 I 60 F +40 I 40 F +60 I 20 F +80 I 100 I Con 100 F 80 F +20 I 60 F +40 I 40 F +60 I 20 F +80 I 100 I Con Different tretment t different time Fig. 3.2. Survivl numbers in different tretments t different times. Mens with the sme letter re not significntly different. *F: fipronil; I: imidcloprid; Con: control. 35
50 45 40 100 F* 80 F + 20 I* 60 F + 40 I 40 F + 60 I 20 F + 80 I 100 I Control b b b c Survivl numbers 35 30 25 20 15 b b 10 5 0 c 24 48 66 24 48 66 24 48 66 24 48 66 24 48 66 24 48 66 24 48 66 Times (hrs) Fig. 3.3. Survivl numbers in ech tretment t different times. Mens with the sme letter re not significntly different. * F: fipronil; I: imidcloprid. 36
Low-concentrtion biossy. There ws significnt interction between tretment nd time (F = 3.22; df =8, 44; P = 0.009). A significnt difference in survivl numbers ws found mong the tretments t ech time period (F = 23.41; df = 4, 44; P < 0.0001) (Figure 3.4). The survivl numbers in the tretment of 5 ppm fipronil plus 5 ppm imidcloprid were significntly higher thn in the tretment of 10 ppm fipronil plus 10 ppm imidcloprid but lower thn 10 ppm imidcloprid t 96 hours (Figure 3.4). Among ll termiticide tretments t 120 hours, the survivl numbers in the tretment of 5 ppm fipronil plus 5 ppm imidcloprid were significntly higher thn other termiticide tretments. There ws no significnt difference between tretments of 10 ppm fipronil nd 10 ppm fipronil plus 10 ppm imidcloprid t ll time intervls. No significnt difference ws found between termiticide tretments t 72 hours. One of the Petri dishes treted with imidcloprid lone ws hevily infested by fungi (Figure 3.4). When the dt were nlyzed within ech tretment mong different times, significnt difference in survivl numbers ws found (F = 9.61; df = 2, 44; P = 0.0006) (Fig. 3.5). The survivl numbers in the tretment of 10 ppm fipronil dropped to less thn 10% t 72 hours nd 100% mortlity ws pproched t 96 nd 120 hours. In contrst, the survivl number in tretment of 10 ppm imidcloprid ws much higher t 72 nd 96 hours nd were not significntly decresed until 120 hours. There ws no significnt difference in the combintion of 5 ppm fipronil plus 5 ppm imidcloprid mong ll time intervls (F = 1.86; df = 2, 8; P = 0.2351); while 37