SUSCEPTIBILITY OF DIAPREPES ABBREVIATUS (COLEOPTERA: CURCULIONIDAE) TO A COMMERCIAL PREPARATION OF BACILLUS THURINGIENSIS SUBSP.

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1 330 Florid Entomologist 85(2) June 2002 SUSCEPTIBILITY OF DIAPREPES ABBREVIATUS (COLEOPTERA: CURCULIONIDAE) TO A COMMERCIAL PREPARATION OF BACILLUS THURINGIENSIS SUBSP. TENEBRIONIS A. A. WEATHERSBEE III, 1 Y. Q. TANG, 2,3 H. DOOSTDAR 3 AND R. T. MAYER 1 1 USDA, ARS, U.S. Horticulturl Reserch Lortory, 2001 South Rock Rod, Fort Pierce, FL Visiting scientist, Biologicl Control Reserch Institute, Fujin Agriculturl University, Fuzhou, Fugin Chin 3 Visiting scientist, USDA, ARS, U.S. Horticulturl Reserch Lortory, 2001 South Rock Rd., Fort Pierce, FL ABSTRACT A commercil preprtion of the microil entomopthogen, Bcillus thuringiensis susp. tenerionis (Btt) ws evluted for iologicl ctivity ginst the Diprepes root weevil, Diprepes revitus (L.). A reduction in survivl ws oserved for neontl lrve exposed to insect diet incorported with Btt nd in potted citrus treted with Btt soil ppliction. A tretment-induced, weight gin reduction for neontes ws indicted only in the diet ssy. Lrve exposed t 5 weeks old to diet treted with Btt demonstrted dose-dependent mortlity response. The men ges for lrvl deth rnged from 111 to 128 dys mong tretments. The LC 50 for lrve in this ge group ws 6.2 ppm [AI] nd the slope of the proit line ws The mortlity response of lrve exposed t 12 weeks old lso ws dose dependent nd the men ges for lrvl deth rnged from 130 to 141 dys mong tretments. The LC 50 for lrve in this ge group ws 25.4 ppm [AI] nd the slope of the proit line ws The delyed ptterns of mortlity tht we oserved mong lrve treted t 5 nd 12 weeks old indictes tht disese is slow to develop in older lrve ut tht deth occurs efore mturtion is completed. Key Words: Diprepes revitus, Bcillus thuringiensis, entomopthogen, citrus, Diprepes root weevil RESUMEN Se evluó un formulción comercil del entomoptógeno microil, Bcillus thuringiensis susp. tenerionis (Btt) por su ctividd iológic en contr del gorgójo Diprepes revitus (L.). Se oservó un reducción en l sorevivenci de lrvs neonts expuests un diet rtificil que conteni Btt, y de lrvs en potes con roles trtdos con Btt. Un reducción del peso gndo por ls lrvs neonts fue inducid por el trtmiento solmente en el cso del ensyo con diet rtificil. Lrvs expuests diet trtd ls 5 semns de edd mnifestron un mortlidd proporcionl l dosis. El rngo del promedio del número de dis hst l muerte de ls lrvs fue de dis entre trtmientos. L DL 50 pr ls lrvs de este grupo fue de 6.2 ppm (IA) y el pendiente de l line proit fue de L mortlidd de lrvs expuests ls 12 semns de edd fue dependiente de l dosis y l edd promedi de l muerte vrió entre 130 y 141 dis entre trtmientos. L DL 50 pr ls lrvs de este grupo fue de 25.4 ppm (IA) y el pendiente de l line proit fue de El ptron de mortlidd que oservmos entre lrvs trtds ls 5 y ls 12 semns de edd indic que l enfermedd se desrrolló lentmente en ls lrvs myores pero l muerte ocurrió ntes de l mdurción. The most economiclly importnt insect pest of citrus in Florid is the Diprepes root weevil, Diprepes revitus (L.). Losses to citrus growers re estimted in excess of $75 million yerly (Anonymous 1997). This root weevil lso ttcks sugr cne, vegetle crops, nd ornmentl plntings in res tht re infested. The suter- Mention of trdemrk or proprietry product does not constitute gurntee or wrrnty of the product y the U.S. Deprtment of Agriculture nd does not imply its pprovl to the exclusion of other products tht my lso e suitle. rnen lrve extensively dmge host plnt root systems on which they feed. Lrvl feeding in citrus predisposes root tissues to infections y pthogens such s Phytophthor spp. (Rogers et l. 1996), induces decline in tree helth nd fruit production, nd my eventully kill the tree (Schroeder & Sutton 1977). In severe cses of uncontrolled weevil popultions, entire citrus groves re destroyed (pers. os.). Entomopthogens hve received considerle ttention s potentil iocontrol gents of root weevil lrve. Bevers et l. (1983) nd Román

2 Wethersee et l.: Susceptiility of D. revitus to B. thuringiensis 331 nd Bevers (1983) initilly demonstrted the pthogenicity nd sesonl prevlence of severl nturlly occurring fungi nd nemtodes ttcking D. revitus. Since then, mny others hve demonstrted the potentil for iologicl control of root weevil lrve y entomopthogens (Duncn et l. 1996; Figuero & Román 1990; Quintell & Mc- Coy 1997, 1998; Schroeder 1987, 1994). Nemtode pplictions were recommended for control s recently s 1999, ut the insecticidl compound, ifenthrin, is currently the only mteril listed in the Florid Citrus Pest Mngement Guide for control of lrvl stges (Futch et l. 2001). Bcteril entomopthogens hve not een previously investigted s potentil iocontrol gents for Diprepes root weevil. The microil pthogen, Bcillus thuringiensis susp. tenerionis (Btt), ws shown to hve ctivity ginst representtives of the order Coleopter including the fmilies Chrysomelide, Bostrichide, nd Curculionide (Herrnstdt et l. 1986; Krieg et l. 1983, 1987; Beegle 1996; Sde et l. 1996). The cterium produces crystlline δ-endotoxin during the process of sporultion (Hofte & Whiteley 1989). Upon ingestion y susceptile insect host, the endotoxin is proteolyticlly ctivted in the midgut nd cuses feeding inhiition, septicemi, nd eventul deth (Knowles 1994). We investigted commercilly ville Btt product, Novodor (Novo Nordisk, North Chicgo, IL), to determine if it hd iologicl ctivity ginst D. revitus, envisioning tht demonstrtion of ctivity my provide the impetus to develop this nd possily other B. thuringiensis susp. for field ppliction. Insect Rering MATERIALS AND METHODS Neontl, 5-week-old, nd 12-week-old lrve of D. revitus were otined from lortory colony mintined y the U.S. Horticulturl Reserch Lortory, Fort Pierce, FL. Lrve were rered on commercilly-prepred insect diet (Product No. F1675, Bio-Serv, Frenchtown, NJ) plced within seled diet cups (PC100, 30 ml cups nd lids, Jet Plstic, Hrrisurg, PA). Preprtion of insect diet nd rering of lrve were similr to the methods descried y Bevers (1982) with temperture nd moisture content of diet optimized for lrvl development (Lpointe 2000, Lpointe & Shpiro 1999). Weevil lrve used in the iossys were mintined in diet cups stored in plstic trys in growth chmer t 26 C nd 24-h drk cycle. Diet Incorportion of Btt Prepred insect diet ws heted to 90 C for 15 min., covered with foil, nd llowed to cool to 56 C in heted wter th efore incorporting tretments. A commercil preprtion of B. thuringiensis susp. tenerionis (Novodor 3% [AI], [30 mg spores nd δ-endotoxin crystls per ml product]) ws incorported into the diet t rtes of 0, 0.3, 3.0, 30, nd 300 ppm (µg AI/ml diet). s were incorported into the gr-lden diet with the id of heted, stirrer plte. The resulting mixtures were pipetted into diet cups (15 ml diet per cup), llowed to solidify nd cool to room temperture, nd then were closed with lid. All steps fter heting of the diet were performed in lminr flow, clen ench to void contmintion. Evlution of Btt Activity The iologicl ctivity of Novodor ws evluted initilly ginst neontl lrve exposed to treted insect diet. Neontes were riefly surfce sterilized in 5% lech solution nd rinsed with sterile, deionized wter prior to eing plced in diet cups. The tretments included the 5 rtes of tretment-incorported diet descried ove nd there were 3 replictions of ech tretment. A tretment comprised 18 diet cups, ech infested with 5 neontl lrv. The numers nd weights of surviving lrve in ech diet cup were ssessed fter 6 weeks of exposure to treted diet. The ctivity of Novodor lso ws evluted ginst 5-week old weevil lrve. Lrve used in the experiment weighed ~110 mg efore exposure to tretments. s were prepred s ove nd replicted 6 times. Ech tretment comprised 18 diet cups, ech contining 1 lrv. Mortlity, ge t deth, nd weights of eclosed dults were recorded twice weekly. The experiment ws terminted fter 5 months when most lrve hd either died or mtured to dults. Novodor ctivity lso ws evluted ginst 12-week old lrve tht weighed ~510 mg efore exposure to tretments. The tretments were incorported in diet t the rtes descried ove. There were 3 replictions of tretments, ech comprising 30 lrve confined individully to diet cups. Mortlity, ge t deth, nd weights of eclosed dults were recorded twice weekly. The experiment ws terminted fter 3 months when most lrve hd either died or mtured to dults. Another experiment ws conducted to determine the effect of Novodor suspensions pplied s soil tretments ginst neontl lrve feeding on potted citrus roots. The citrus plnts used in the study were 1 yer old Cleoptr Mndrin (Citrus reshni Hort., ex Tn.) rootstock potted in 473 cm 3 continers of potting soil (Metromix 500, Scotts, Mrysville, OH). The tretments included 4 rtes of Novodore (0, 3.0, 30, nd 300 ppm [(g AI/ ml DI wter]) pplied s 50 ml suspensions to the soil of ech continer. Two drench pplictions were mde 14 dys prt. There were 6 replictions of ech tretment. A tretment comprised 1

3 332 Florid Entomologist 85(2) June 2002 potted citrus plnt infested with 20 neontes immeditely fter the first drench ppliction. All tretments were mintined in growth chmer t 26 C with photoperiod of 14:10 (L:D) h. The numers nd weights of surviving lrve were ssessed fter 6 weeks. Dt Anlyses nd Sttistics Percentge dt were djusted for control mortlity using the Aott (1925) formul nd trnsformed (rcsine) efore nlyses. Dt were nlyzed y the Generl Liner Models Procedure, nd differences mong tretment mens were determined y Tukey s studentized rnge test (SAS Institute 1990). Differences mong mens were considered significnt t proility level of 5 percent (P 0.05). Proit nlyses were conducted using the Proit Procedure (SAS Institute 1990) to generte LC 50 vlues nd slopes of proit lines for lrvl mortlity due to tretments. Untrnsformed mens were presented in the dt tles. Neontl Lrve RESULTS The survivl of root weevil lrve exposed s neontes to diet treted with Novodor ws significntly reduced (F = 9.36; df = 4, 262; P < ) fter 6 weeks. While 2 of 5 lrve survived fter 6 weeks in the control group, only 1 lrve survived on verge in the 300 ppm tretment (Tle 1). The low survivl rte oserved for control lrve is ddressed in the discussion section. The fresh weights of surviving lrve were significntly reduced (F = 8.46; df = 4, 262; P < ) y tretments indicting tht lrvl feeding or nutrient ssimiltion my hve een inhiited. Surviving lrve in the 300 ppm tretment weighed 53% less thn those in the control group (Tle 1). 5-Week Old Lrve Insect diet treted with Novodor cused significnt increse (F = 76.57; df = 4, 20; P < ) in mortlity of lrve exposed t the ge of 5 weeks. Mortlity of lrve exposed to the 3 ppm tretment (44%) ws significntly (P 0.05) greter thn tht of the controls (15%). Lrvl mortlity in the 30 ppm tretment exceeded 50% (Tle 2). The clculted LC 50 for lrve in this ge group ws 6.2 (95% FL = ) ppm [AI]. The slope of the proit line ws 2.29 (SE = 0.31) (χ 2 = 43.07; df = 1; P < ). s lso cused significnt reduction (F = 6.25; df = 4, 20; P = ) in the men deth ges of lrve. The deth ges for lrve mintined on treted diets rnged from 111 to 128 d while tht of control lrve ws 154 d. Lrve in the 30 nd 300 ppm tretments died significntly (P 0.05) erlier thn the control lrve (Tle 2). The mortlity response we oserved occurred lter thn is typicl for insects susceptile to B. thuringiensis; nevertheless, the response ws consistent, dosedependent, nd occurred erlier thn tht oserved for the controls. We oserved tht disese nd deth occurred in treted insects during the lter stges of lrvl development nd erly phses of puption (Fig. 1). The presence of Btt ws confirmed y isolting the cteri from internl tissues of ded lrve. Bcteril isoltes were cultured in 40 ml LB roth in 125 ml ffled flsk mintined in n incutor shker t 28 C nd 200 rpm until sporultion phse. Cultures were exmined using Hoffmn contrst microscopy t 1000 mgnifiction to identify Btt endospores nd δ-endotoxin crystls. The men fresh weights of dults tht eclosed on treted diets lso were significntly reduced (F = 3.39; df = 4, 20; P = ). Adult weevils tht survived the 300 ppm tretment weighed 15% less thn control dults, indicting tht inhiition of feeding or disruption of nutrient ssimiltion my hve occurred mong survivors of tretments (Tle 2). 12-Week Old Lrve A significnt increse (F = 8.82; df = 4, 8; P < ) in mortlity ws oserved for 12-week old lrve fed Novodor treted diet. Mortlity in the 30 ppm tretment (53%) ws significntly (P TABLE 1. SURVIVAL AND AVERAGE FRESH WEIGHT OF D. ABBREVIATUS LARVAE EXPOSED AS NEONATES (5 PER DIET CUP) FOR 6 WEEKS TO INSECT DIET INCORPORATED WITH NOVODOR. Numer of surviving lrve ± SE (n = 3) Weight (mg) of surviving lrve ± SE (n = 3) ± ± ± ± ± ± ± ± ± ± 20.9 AI refers to the concentrtion (µg/ml) of ctive ingredient, comprising spores nd δ-endotoxin of B. thuringiensis vr. tenerionis, in prepred diet.

4 Wethersee et l.: Susceptiility of D. revitus to B. thuringiensis 333 TABLE 2. MORTALITY AND AGE AT DEATH OF LARVAE, AND FRESH WEIGHT OF ECLOSED ADULTS, FOR D. ABBREVIATUS EXPOSED AS 5-WEEK-OLD LARVAE TO INSECT DIET INCORPORATED WITH NOVODOR. % Mortlity ± SE Deth ge (d) ± SE Adult wt. (mg) ± SE (n = 6),c (n = 6),c (n = 6),c ± ± ± ± ± ± ± ± ± ± 4.9 c ± ± ± 4.4 d ± ± 19.5 AI refers to the concentrtion ((g/ml) of ctive ingredient, comprising spores nd δ-endotoxin of B. thuringiensis vr. tenerionis, in prepred diet. Mortlity, ge t deth, nd fresh weights of eclosed dults were ssessed twice weekly during the experiment. c 0.05) greter thn tht for the control group (6%) (Tle 3). The clculted LC 50 for lrve in this ge group ws 25.4 (95% FL = ) ppm [AI]. The slope of the proit line ws 2.75 (SE = 0.44) (χ 2 = 47.37; df = 1; P < ). The men ge t which mortlity occurred lso ws significntly reduced y tretments (F = 4.54; df = 4, 8; P = ). The men deth ge for lrve in the 300 ppm tretment (130 d) ws significntly (P 0.05) less thn tht for control lrve (153 d) (Tle 3). s did not significntly ffect the fresh weights of eclosed dults in this ge group (F = 1.34; df = 4, 8; P = ), likely ecuse lrve used in the experiment hd ttined ner mximum weights efore exposure to tretments. Soil s Survivl of neontes on potted citrus ws significntly reduced (F = 4.87; df = 3, 15; P < ) y soil tretments with Novodor. Lrvl survivl ws significntly (P 0.05) less in ll tretments s compred to the controls fter 6 weeks exposure to treted soils nd citrus roots (Tle 4). The low rte of survivl for control lrve is ddressed in the discussion section. In contrst to the results of the diet iossy ginst neontes, the fresh weights of surviving lrve were not significntly reduced (F = 1.02; df = 3, 15; P = ) in the potted citrus iossy (Tle 4). Some lrve my hve een le to void exposure to Btt where soil drenches resulted in unequl dispersion of tretments. DISCUSSION Fig. 1. Helthy (left) nd disesed (right) lrve nd pupe of D. revitus due to feeding on diet incorported with Novodor. The results of our experiments indicte tht neontl, 5-week-old, nd 12-week-old lrve of D. revitus were susceptile to the iologicl effects of Novodor. Activity for B. thuringiensis ginst older lrvl stges of insects is not normlly expected; nevertheless, the dose-dependent mortlities we oserved for lrve exposed t 5 nd 12 weeks old were reproducile. The low survivl rtes we oserved for neontes in our controls re typicl in experiments such s this, where multiple lrve re collectively grouped to chllenge plnts or generte dt for iossys of diet-incorported mterils. Low survivl rtes for neontl D. revitus hve een reported y others (Schroeder & Sieurth 1997, Quintell & McCoy 1997), nd re due to nturl mortlity fctors including ggressive interctions mong lrve confined together (Lpointe & Shpiro 1999). The response we oserved for older lrve to Novodor my e ttriuted to the finding tht midgut trypsin ctivity increses to mximum in D. revitus lrve t the ge of pproximtely 7 weeks (Yn et l. 1999). Yn et l. lso inferred tht the midgut of D. revitus lrve

5 334 Florid Entomologist 85(2) June 2002 TABLE 3. MORTALITY AND AGE AT DEATH OF LARVAE, AND FRESH WEIGHT OF ECLOSED ADULTS, FOR D. ABBREVIATUS EXPOSED AS 12-WEEK-OLD LARVAE TO INSECT DIET INCORPORATED WITH NOVODOR. % Mortlity ± SE Deth Age (d) ± SE Adult Wt. (mg) ± SE (n = 3),c (n = 3),c (n = 3),c ± ± ± ± ± ± ± 21.5 c ± ± ± 15.4 c ± ± ± 12.5 c ± ± 13.5 AI refers to the concentrtion (µg/ml) of ctive ingredient, comprising spores nd δ-endotoxin of B. thuringiensis vr. tenerionis, in prepred diet. Mortlity, ge t deth, nd fresh weights of eclosed dults were ssessed twice weekly during the experiment. c is proly lkline, s shown for mny Lepidopter nd Dipter, since the enzyme ctivity oserved ws most ctive t ph of Alkline soluiliztion nd proteolytic clevge y serine proteses, such s trypsin, re required for ctivtion of mny B. thuringiensis δ-endotoxins (Pietrntonio et l. 1993) including those produced y Btt. The Cry3 toxin of Btt my undergo sustntil clevge t the N-terminus without loss of iologicl ctivity (Crroll et l. 1989). It hs een suggested tht these dditionl protese clevge sites my fcilitte insertion of portion of the protein into the trget memrne of the midgut epithelium (Knowles 1994) nd susequent pore formtion. The mortlity responses we oserved for lrve treted t 5 nd 12 weeks old my hve een product of pproprite levels of ph nd serine proteses in midgut tissues during those stges of development. The delyed period to lrvl deth my hve een function of the ility of D. revitus to survive for long periods without food or wter (personl oservtion) nd the slow development of disese. Any future evlution of Btt on lrvl D. revitus, nd possily other insects, should consider the importnce of suitle midgut environments for endotoxin ctivtion nd the generl hrdiness of the insect. We oserved dose-dependent responses for mortlity, ge t deth, nd fresh weights of dult survivors from lrve treted t the ge of 5 weeks. Although the mortlity response developed slowly, the men ges t deth were consistently erlier mong treted s compred to control lrve. Also, the fresh weights of dults tht survived tretments were reduced compred to those of the control, indicting tht midgut dmge or feeding inhiition my hve occurred mong lrve tht survived tretments. A dose-dependent mortlity response lso ws oserved for lrve treted t the ge of 12 weeks. Lrve treted t this stge of development often died s pupe, further supporting the contention tht disese ws slow to develop. The fresh weights of the dults tht developed from lrve treted t 12 weeks old were not ffected y tretments. Lpointe (2000) reported tht lrvl weights of D. revitus were mximum t 12 weeks old, nd then declined during further development, thus explining the lck of weight response for lrve in this ge group. There is much yet to e lerned out the ctivtion nd inding ptterns of B. thuringiensis endotoxins, nd the susequent onset of disese in susceptile insects. We oserved tht disese ws slow to develop in D. revitus. The results of our experiment indicte tht there my e gerelted fctors tht influence lrvl D. revitus response to Btt endotoxins. There my lso e other endotoxins witing discovery tht re etter dpted to ctivtion nd inding y lrvl D. revitus. This report provides the impetus to further explore these processes in the Di- TABLE 4. SURVIVAL AND MEAN FRESH WEIGHT OF D. ABBREVIATUS LARVAE EXPOSED AS NEONATES (20 PER PLANT) FOR 6 WEEKS TO POTTED CITRUS TREATED WITH NOVODOR. Numer of surviving lrve ± SE (n = 6) Weight (mg) of surviving lrve ± SE (n = 6) ± ± ± ± ± ± ± ± 12.1 AI refers to the concentrtion (µg/ml) of ctive ingredient, comprising spores nd δ-endotoxin of B. thuringiensis vr. tenerionis, in prepred diet.

6 Wethersee et l.: Susceptiility of D. revitus to B. thuringiensis 335 prepes root weevil nd other insects. Our demonstrtion of Novodore ctivity ginst different lrvl stges of D. revitus is encourging enough to wrrnt dditionl testing. ACKNOWLEDGMENTS The uthors thnk Ginny Schmit nd Krin Crosy (USDA, ARS, U.S. Horticulturl Reserch Lortory, Fort Pierce, FL) for technicl support nd ssistnce with insect rering nd preprtion of insect diets. REFERENCES CITED ABBOTT, W. S A method for computing the effectiveness of n insecticide. J. Econ. Entomol. 18: ANONYMOUS Diprepes Tsk Force Minutes, July 17, University of Florid. Lke Alfred, FL. BEAVERS, J. B Biology of Diprepes revitus (Coleopter: Curculionide) rered on rtificil diet. Florid Entomol. 65: BEAVERS, J. B., C. W. MCCOY, AND D. T. KAPLAN Nturl enemies of suterrnen Diprepes revitus (Coleopter: Curculionide) lrve in Florid. Environ. Entomol. 12: BEEGLE, C. C Efficcy of Bcillus thuringiensis ginst Lesser Grin Borer, Rhyzoperth dominic (Coleopter: Bostrichide). Biocontrol. Sci. Tech. 6: CARROLL, J., J. LI, AND D. J. ELLAR Proteolytic processing of coleoptern-specific δ-endotoxin produced y Bcillus thuringiensis vr. tenerionis. Biochem. J. 261: DUNCAN, L. W., C. W. MCCOY, AND A. C. TERRANOVA Estimting smple size nd persistence of entomogenous nemtodes in sndy soils nd their efficcy ginst the lrve of Diprepes revitus in Florid. J. Nemtol. 28: FIGUEROA, W., AND J. ROMÁN Biocontrol of the sugrcne rootstlk orer, Diprepes revitus (L.) (Coleopter: Curculionide), with entomophilic nemtodes. J. Agric. Univ. P.R. 74: FUTCH, S. H., J. L. KNAPP, AND C. W. MCCOY Fct Sheet ENY-611, 2001 Florid Citrus Pest Mngement Guide, FCES, IFAS, University of Florid. Revised: Octoer HERRNSTADT, C., G. G. SOARES, E. R. WILCOX, AND D. L. EDWARDS A new strin of Bcillus thuringiensis with ctivity ginst coleoptern insects. Biotechnology. 4: HÖFTE, H., AND H. R. WHITELEY Insecticidl crystl proteins of Bcillus thuringiensis. Microiol. Rev. 53: KNOWLES, B. H Mechnism of ction of Bcillus thuringiensis insecticidl δ-endotoxins. Adv. Insect. Physiol. 24: KRIEG, V. A., A. M. HUGER, AND W. SCHNETTER Bcillus thuringiensis vr. Sn Diego strin M-7 is identicl to the formerly in Germny isolted B. thuringiensis vr. tenerionis strin BI J. Appl. Entomol. 104: KRIEG, V. A., A. M. HUGER, G. A. LANGENBRUNCH, AND W. SCHNETTER Bcillus thuringiensis vr. tenerionis, new pthotype effective ginst lrve of Coleopter. Z. Angew. Entomol. 96: LAPOINTE, S. L Therml requirements for the development of Diprepes revitus (Coleopter: Curculionide). Environ. Entomol. 29: LAPOINTE, S. L., AND J. P. SHAPIRO Effect of soil moisture on development of Diprepes revitus (Coleopter: Curculionide). Florid Entomol. 82: PIETRANTONIO, P. V., B. A. FEDERICI, AND S. S. GILL Interction of Bcillus thuringiensis endotoxins with the insect midgut epithelium, pp In N. E. Beckge, S. N. Thompson, nd B. A. Federici (eds.), Prsites nd Pthogens of Insects, Vol 2. Acdemic Press, New York. QUINTELLA, E. D., AND C. W. MCCOY Pthogenicity enhncement of Metrhizium nisoplie nd Beuveri ssin to first instrs of Diprepes revitus (Coleopter: Curculionide) with sulethl doses of imdcloprid. Environ. Entomol. 26: QUINTELLA, E. D., AND C. W. MCCOY Synergistic effect of imdcloprid nd two entomopthogenic fungi on the ehvior nd survivl of lrve of Diprepes revitus (Coleopter: Curculionide) in soil. J. Econ. Entomol. 91: ROGERS, S., J. H. GRAHAM, AND C. W. MCCOY Insect-plnt pthogen interctions: Preliminry studies of Diprepes root weevil injuries nd Phytophthor infections. Proc. Fl. Stte Hort. Soc. 109: ROMÁN, J., AND J. B. BEAVERS A survey of Puerto Ricn soils for entomogenous nemtodes which ttck Diprepes revitus (L.) (Coleopter: Curculionide). J. Agric. Univ. P.R. 67: SAADE, F. E., G. B. DUNPHY, AND R. L. BERNIER Response of the Crrot weevil, Listronotus oregonensis (Coleopter: Curculionide), to strins of Bcillus thuringiensis. Biologicl Control. 7: SAS INSTITUTE SAS/STAT user s guide for personl computers, vol. 6. SAS Institute, Cry, NC. SCHROEDER, W. J Lortory iossys nd field trils of entomogenous nemtodes for control of Diprepes revitus (Coleopter: Curculionide) in citrus. Environ. Entomol. 16: SCHROEDER, W. J Comprison of two steinernemtid species for control of the root weevil Diprepes revitus. J. Nemtol. 26: SCHROEDER, W. J., AND P. J. SIEBURTH Impct of surfctnts on control of the root weevil Diprepes revitus lrve with Steinernem riorvis. J. Nemtol. 29: SCHROEDER, W. J., AND R. A. SUTTON Citrus root dmge nd the sptil distriution of eggs of Diprepes revitus. Florid Entomol. 60: 114. YAN, X. H., H. L. DE BONDT, C. C. POWELL, R. C. BUL- LOCK, AND D. BOROVSKY Sequencing nd chrcteriztion of the citrus weevil, Diprepes revitus, trypsin cdna: Effect of Aedes trypsin modulting oosttic fctor on trypsin iosynthesis. Eur. J. Biochem. 262: