enhancement of nucleopolyhedrovirus infectivity against mamestra brassicae (lepidoptera: noctuidae)...

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Enhancement of Nucleopolyhedrovirus Infectivity AgainstMamestra brassicae (Lepidoptera Noctuidae) by ProteinsDerived from Granulovirus and a Fluorescent BrightenerAuthor(s) Shigeyuki Mukawa and Chie GotoSource Journal of Economic Entomology 100(4)1075-1083 2007Published By Entomological Society of AmericaDOI httpdxdoiorg1016030022-0493(2007)100[1075EONIAM]20CO2URL httpwwwbiooneorgdoifull1016030022-0493282007291005B10753AEONIAM5D20CO3B2

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BIOLOGICAL AND MICROBIAL CONTROL

Enhancement of Nucleopolyhedrovirus Infectivity Against Mamestrabrassicae (Lepidoptera Noctuidae) by Proteins Derived from

Granulovirus and a Fluorescent Brightener

SHIGEYUKI MUKAWA AND CHIE GOTO1

Insect Pest Management Research Team National Agricultural Research CenterKannondai Tsukuba Ibaraki 305-8666 Japan

J Econ Entomol 100(4) 1075ETH1083 (2007)

ABSTRACT The synergistic enhancement of nucleopolyhedrovirus (NPV) infection by granulo-viruses(GVs) iswell documented andaGVgranuleproteinnamedviral enhancinhasbeen identiTHORNedas an active contributor to this effect We detected the presence of two proteins with molecular massof 93 and 108 kDa in granules of a GV isolated from Xestia c-nigrum (L) (XecnGV) as candidates forenhancin and we conTHORNrmed that at least the 108-kDa protein enhances the infectivity of Mamestrabrassicae nucleopolyhedrovirus (MabrNPV) We tested the effect of virion-free proteins obtainedfrom XecnGV granules (GVPs) on MabrNPV infection and we made a comparison with an enhancingchemical the stilbene-derived szliguorescent brightener Tinopal Bioassay was performed employing thediet contamination method by using second instars of Mamestra brassicae (L) (Lepidoptera Noc-tuidae) The enhancing effects of GVPs (01 mgg diet) and Tinopal (1 mgg diet) were estimatedto be 707ETH815-fold and 269ETH337-fold respectively as calculated from the LC50 values of MabrNPVwith or without the additives The additives reduced the lethal time of MabrNPV-infected larvae andthey caused death at a younger instar These results suggest that GVPs can enhance MabrNPVinfection as effectively as Tinopal

KEYWORDS nucleopolyhedrovirus granulovirus szliguorescent brightenerMamestra brassicae viralenhancement

Baculoviruses including nucleopolyhedrovirus (NPV)and granulovirus (GV) have been recognized as pos-sible microbial insecticides that are highly host spe-ciTHORNc and thus safe to nontarget organisms Howeverhigh production costs and narrow spectrum have lim-ited their use as biological control agents Moreoverthe larvae of most target pests have a short period ofhigh susceptibility Efforts have been made to en-hance baculovirus efTHORNcacy by increasing host suscep-tibility with chemicals such as phosphatidylcholine(Yamamoto and Tanada 1978) boric acid (Cisneros etal 2002) and neem extract (Shapiro et al 1994) Sev-eral stilbene-derived szliguorescent brighteners greatlyenhance the virulence of Lymantria dispar NPVagainst its homologous host (Shapiro and Robertson1992) The effects of brighteners in other NPVETHhostsystems are well documented (eg Hamm and Sha-piro 1992 Vail et al 1996 Zou and Young 1996 Mo-rales et al 2001) Fluorescent brighteners facilitate thearrival of NPV at midgut of the host epithelial cells bydisrupting the peritrophic matrix which serves as abarrier topathogenicmicroorganisms(WangandGra-nados 2000 Mukawa et al 2003 Okuno et al 2003) Inaddition szliguorescent brighteners weaken host resis-

tanceby inhibiting theapoptosisofNPV-infectedmid-gut cells (Dougherty et al 2006) and by blocking thesloughing of infected primary target cells in the mid-gut (Washburn et al 1998)

The GVs from Pseudaletia unipuncta (Haworth)Trichoplusia ni (Hubner) Xestia c-nigrum (L) Heli-coverpa armigera (Hubner) and Spodoptera frugi-perda (JE Smith) enhance the infectivity of NPVs(Tanada 1959 Derksen and Granados 1988 Goto 1990Shapiro 2000a) This viral enhancement is due to aprotein named a synergistic factor viral enhancingfactor or enhancin which is a component of thegranules of the GVs (Tanada et al 1973 Hashimoto etal 1991 Corsaro et al 1993) PuriTHORNed enhancin proteinacts as a metalloprotease and degrades the peritrophicmatrix proteins resulting in the disruption of the peri-trophic matrix (Derksen and Granados 1988 Leporeet al 1996 Wang and Granados 1997) There is alsoevidence that this protein achieves its enhancing ef-fect by increasing the fusion of the viral envelope withthe midgut cell plasma membrane (Tanada et al 19751980 Uchima et al 1989 Kozuma and Hukuhara 1994)Whole-genome analysis ofX c-nigrumGV (XecnGV)has revealed that the genome contains four homologsof enhancinOne of them is highly homologous to theenhancins ofH armigeraGVT niGV andP unipuncta1 Corresponding author e-mail cgotoaffrcgojp

0022-0493071075ETH1083$04000 2007 Entomological Society of America

GV (Hayakawa et al 1999) although no informationis available concerning the products of these genes Inthis study we show the presence of a protein withenhancing effects on NPV infection in the XecnGVgranulesMamestra brassicae (L) (Lepidoptera Noctuidae)

which is closely related to the bertha armywormMa-mestra configurata Walker of North America is animportant insect pest of vegetables and ornamentalplants in Europe and Asia Several NPVs have beenisolated fromM brassicae in Europe and Japan (Arugaet al 1960 Vlak and Groner 1980 Brown et al 1981)There are hopes that these viruses named MabrNPVscan be used agents for biological control of M bras-sicae (Akutsu 1972 Brown et al 1981 Evans and Al-laway 1983) and several other lepidopteran pests be-cause it has a fairly wide host range (Doyle et al 1990)We have genetically identiTHORNed a Japanese isolate(MabrNPV T) as a variant of M configurata NPV(MacoNPV B) (Mukawa and Goto 2006) As a THORNrststep toward practical application of MabrNPV T wetested the infectivity of this NPV against M brassicaelarvae and compared the enhancing effect of virion-free proteins obtained from granules (GVPs) and thatof a szliguorescent brightener Tinopal UNPA-GX

Materials and Methods

Insects Nucleopolyhedrovirus and Additives Mbrassicaewas collected in Tsukuba Ibaraki Japan andmaintained on artiTHORNcial diet (Insecta LFS NihonNosan Kogyo Co Ltd Yokohama Japan) for 10 yrMythimna separata (Walker) also was collected inTsukuba and maintained on another artiTHORNcial diet forNoctuidae (Okada 1990)

TheMbrassicaeNPV (MabrNPV) T was isolated byAkutsu (1972) at the Tokyo Metropolitan AgriculturalExperiment Station The virus was propagated in lar-vae ofM brassicaeMabrNPV-infected cadavers mac-erated in distilled water were THORNltered through twolayers of gauze and centrifuged at 150 g for 1 min toremove the debris The supernatant was centrifugedthree times at 3000 g for 15 min in distilled waterto pellet the polyhedral inclusion bodies (PIBs) Thepellet was resuspended in 05 M NaCl in 10 mM Tris-HCl pH 75 incubated for 1 h at room temperatureand centrifuged at 3000 g for 15 min The pellet wasthen resuspended and washed three times as de-scribed above with 10 mM Tris-HCl pH 75 A sus-pension of this pellet was layered on the top of ninevolumes of Percoll (GE Healthcare Little ChalfontBuckinghamshire United Kingdom) solution adjustedto pH 75 with 1 M HCl and then centrifuged at44500 g for 30 min The band containing PIBs ofMabrNPV was collected and washed three times bycentrifugation at 3000 g in 10 mM Tris-HCl pH 75The concentration of PIBs in the stock suspension wasdetermined using a Thoma hemocytometer under aphase-contrast microscope The suspension wasstored at 4C until use

We used a X c-nigrum GV (XecnGV) -4 cloneoriginating from an X c-nigrum larva collected at Me-

muro Hokkaido Japan (Goto et al 1985 Hayakawa etal 1999) The virus was propagated in X c-nigrumlarvae and the homogenized cadavers were THORNlteredthrough six layers of gauze and a layer of absorbentcotton The THORNltrate was centrifuged at 1000 g for 5min The granules in the supernatant were puriTHORNedseveral times by differential centrifugations at 3000 g for 5 min and at 10000 g for 30 min to remove darkcontaminants A suspension of the pellet was mixedwith the same volume of glycerol and centrifuged at13000 g for 30 min The pellet was washed twice bycentrifugation at 10000 g for 30 min with distilledwater Granules were obtained by a discontinuoussucrose density gradient centrifugation consisting of50ETH80 (wtvol) solutions (Yamamoto and Tanada1978) and then washed twice with distilled water anddesiccated at 4C The granules were suspended indistilled water and NaOH was added (THORNnal conc 20mM) to dissolve the occlusion matrix The dissolvedviral suspension was neutralized with 1 M HCl andbuffered with 1100 volumes of 1 M Tris-HCl pH 80The XecnGV virions and undissolved substrates werepelleted by centrifugation at 70000 g for 20 min Thesupernatant containing GV proteins (GVPs) was col-lected and stored at 4C until use

The szliguorescent brightener used in this experimentwas Tinopal (Fluorescent brightener 28 TinopalUNPA-GX Sigma-Aldrich St Louis MO)Isolation and Purification of Enhancing Proteins

The proteins with NPV enhancing activity were pu-riTHORNed from XecnGV granules following the method ofHara et al (1976) with some modiTHORNcations The GVgranules (10ETH20 mgml) were dissolved in 002 MNaOH at 4C and the solution was adjusted to a THORNnalconcentration of 50 mM Tris-HCl pH 76ETH80 thencentrifuged at 100000 g for 15 min to remove virusparticles and undissolved substrates Five to 10 ml ofsupernatant was loaded onto a 25- by 60-cm SephaclylS-300 SuperTHORNne gel THORNltration column (PharmaciaUppsala Sweden) equilibrated with 50 mM Tris-HClpH 80 and eluted with the same buffer at a szligow rateof 09 mlmin Samples of 36 ml of efszliguent werecollected and their absorbance at 280 nm was mea-sured To analyze the enhancing activity newlymolted THORNfth instars ofM separata were fed on a smallpiece of diet contaminated with a known amount ofMabrNPV PIBs and 3ETH5 l of each sample Larvae thathad completely consumed the diet within 24 h weretransferred to glass tubes (18 mm in diameter 120 mmin height) with paper plugs for individual rearing onvirus-free diet Twenty to 24 larvae were used forchecking the activity of each fraction and they wereobserved daily until death or pupation at 23C undera photoperiod of 168 (LD) h The samples with en-hancing activity were pooled and concentrated indialyzing tubing under reduced pressure and thenthey were THORNltered again through the same columnThe second efszliguent was checked and the fractionwith enhancing activity was pooled and concentratedin the same manner and then it was dialyzed against1 mM Tris-HCl pH 80 The obtained solution wasabsorbed on a DEAE-cellulose column (16 by 14 cm)

1076 JOURNAL OF ECONOMIC ENTOMOLOGY Vol 100 no 4

and eluted with a linear sodium chloride gradient(0ETH05 M) in 1 mM Tris-HCl at a szligow rate of 05mlmin Protein concentration was estimated by mea-suring absorbance at 280 nm (optical density[OD]280) and 260 nm (OD280) according to the fol-lowing formula

concn (mgml) 15 OD280 075 OD260

SDS-Polyacrylamide Gel Electrophoresis (PAGE)SDS-PAGE was performed as described by Laemmli(1970) Proteins were visualized using Coomassie Bril-liant Blue R staining The molecular mass of the pro-teins was determined by comparing them to standards(Bio-Rad Japan Tokyo Japan)Bioassay for Evaluation of Viral Enhancement ofGVPs and Tinopal For the comparison of enhancingeffect of GVPs and Tinopal we added 500 l of the PIBsuspension of MabrNPV in 10 mM Tris-HCl pH 75with or without 1 mgml GVPs or 1 (wtvol) Tinopalto 45 g of insect diet and we mixed it well at roomtemperature The treated diet was spread onto a 6-cm-

diameter petri dish and the open dish was set into a9-cm petri dish containing a moisturized THORNlter paperForty-one to 43 newly molted second instars of Mbrassicae were introduced to the petri dish and theywere allowed to feed for 48 h at 25C under a photo-period of 168 (LD) h Next 36 of the larvae weretransferred to 21-ml (37-mm-diameter 23-mm-height) plastic cups for individual rearing on virus-free diet The concentrations of MabrNPV used in theexperiments were 104 1045 105 1055 106 and 1065

(10000 31600 100000 316000 1000000 and3160000) PIBs per g of diet without any additives1015 102 1025 103 1035 104 and 1045 (316 100 3161000 3160 10000 and 31600) PIBs per g of diet withGVPs and 102 1025 103 1035 104 1045 and 105 (100316 1000 3160 10000 31600 and 100000) PIBs perg of diet with Tinopal The THORNnal concentrations ofGVPs and Tinopal in the treated diet were 01 and 1mgg respectively Control larvae were fed on diettreated with distilled water or one of the two additivesExperiments were replicated three times Larvae were

Fig 1 PuriTHORNcation of enhancing proteins of XecnGV granules (A) Elution proTHORNles of Sephacryl S-300 THORNltration ofdissolved XecnGV granules and enhancing activity of fractions on NPV infection Samples (3 l per larva) were combinedwith a suspension of MabrNPV to give a THORNnal dose of 07 105 PIBs per larva (solid columns and as zero) or 55 104 PIBsper larva (open columns) and inoculated to THORNfth instars ofM separata Letters with a double arrow correspond to the fractionsshown in Fig 1B (B) SDS-PAGE analysis of Sephacryl S-300 THORNltration Lane 1 molecular marker proteins lane 2 initialsupernatant of dissolved granules lanes 3ETH9 the fractions of AETHG respectively The arrowheads a and b indicate 108- and93-kDa proteins (C) SDS-PAGE analysis of the DEAE-cellulose puriTHORNed 93- and 108-kDa proteins Lanes 1 initial supernatantof dissolved granules lane 2 93-kDa protein lane 3 108-kDa protein and lane 4 granulin

August 2007 MUKAWA AND GOTO XECNGV AND A BRIGHTENER AS Mamestra NPV ENHANCERS 1077

observed daily for mortality until day 13 after inocu-lation Larvae with typical symptoms of NPV infec-tion such as a waxy appearance and liquefaction of thecadaver were recorded as being infected Tissuesmears were prepared from larvae that had died in the13 d after inoculation without symptoms of NPV in-fection to test for the presence of PIBs under a phase-contrast microscope Larvae that died for reasonsother than NPV infection were excluded from thefollowing analysisData AnalysisMortality data were analyzed using a

probit analysis (Finney 1978) against a common log-arithm of all virus concentrations with or withoutone of the additives by using the POLO-PC program(LeOra Software 1987) The activity ratio with 95conTHORNdence limits (CLs) was calculated as the ratio ofmedian lethal concentration (LC50) values accordingto the method of Robertson and Preisler (1992) Datafor the lethal time THORNtted to a log normal distributionwere analyzed by means of a parametric survival anal-ysis using JMP software version 501 (SAS Institute2002) Larvae surviving for 13 d after inoculation wereincluded in the analysis as censored cases Regressionanalysis was performed with data on the lethal time oflarvae that had died from NPV infection (Farrar andRidgway 1998)

Results

Isolation of Enhancing Protein in the Granules ofXecnGV The supernatant of the granule solution wasTHORNltered through a Sephaclyl S-300 column The efszligu-ent containing two proteins with molecular masses of108 and 93 kDa (fraction C hereafter) had NPV en-hancing ability (Fig 1A and B) Fraction C was con-centrated under pressure and then it was THORNlteredthrough a Sephaclyl S-300 column again for furtherpuriTHORNcation of these two proteins Bioassay conTHORNrmedthe enhancing activity to be present in the fractionconsisting of mainly these two proteins (Table 1) Thisfraction of the second gel THORNltration was concentratedunder pressure and absorbed on a DEAE-cellulosecolumn The column was then eluted with a linearsodiumchloridegradient(0ETH05M) in1mMTris-HClThe 93-kDa protein was eluted in the THORNrst large peakat 025ETH03 M NaCl and the 108-kDa protein waseluted in the third large peak at 038ETH045 M NaCl The

SDS-PAGE analysis results suggested that the frac-tions obtained in the THORNnal puriTHORNcation step were ho-mogeneous (Fig 1C) Each of the fractions was dia-lyzed against 10 mM Tris-HCl pH 80 to check theenhancing activity by bioassay The 108-kDa proteinfraction showed high NPV-enhancing activity (Table1) whereas the activity of the 93-kDa protein was notobvious (data not shown)Effect of GVPs on Susceptibility of Second Instars

Among the larvae in the control group only one larvadied on day 13 in the third trial with GVPs We per-formed a probit analysis without taking natural re-sponse into account because no PIB was detected ina tissue smear of the larva The addition of GVPs (01mgg diet) to the inoculum reduced the LC50 valuesof MabrNPV from 60000 to 740 PIBs per g of diet from52000 to 730 PIBs per g of diet and from 170000 to2300 PIBs per g of diet in the THORNrst second and thirdtrial respectively (Table 2) The addition of GVPs didnot cause any signiTHORNcant change in the slope of theprobit mortality line in the THORNrst and third trials (like-lihood ratio parallelism test 2 0066 P 0797 and2 3174P 0075 respectively) In the second trialthe addition of GVPs resulted in a signiTHORNcantly steeperslope (likelihood ratio parallelism test 2 5921 P0015)

For the analysis of the instar of death and lethaltime we excluded data for treatments where the high-est mortality did not reach 50 (Fig 2) All the larvaeinfected with MabrNPV died during the third orfourth stadium In the treatment of MabrNPV alonemost larvae died during the fourth stadium at thelower concentrations and the ratio of larvae that diedduring the third stadium increased gradually as theNPV concentration increased from 105 to 1065 PIBsper g of diet (Fig 2A) In the treatment of MabrNPVin combination with GVPs 85 of infected larvaedied during the third stadium regardless of NPV con-centration and the mean larval mortality of each treat-ment increased gradually as NPV concentration in-creased from 103 to 1045 PIBs per g of diet (Fig 2A)

Parametric survival analysis was performed with thedata from viral concentration 105 PIBs per g of diet(MabrNPV alone) and 103 PIBs per g of diet(MabrNPV plus GVPs) The lethal time was nega-tively correlated with the common logarithm of viralconcentration (likelihood ratio test 2 25578 df

Table 1 Enhancement of MabrNPV infection to fifth instars of M separata by addition of the samples obtained in each step of thepurification procedure

PuriTHORNcation procedure of additivesTotal protein of additives

(ng per larva)MabrNPV

(PIBs per larva)n Infection ()

Sephaclyl S-300 THORNltrationControl 0 55 104 23 43Initial supernatant of dissolved granules 4000 55 104 24 750First gel THORNltration 3200 55 104 23 739Second gel THORNltration 730 55 104 24 708

DEAE-cellulose column chromatographyControl 0 55 104 24 42108-kDa protein 69 55 104 24 583Control 0 14 105 18 56108-kDa protein 138 14 105 18 833


1 P 005) The lethal time differed signiTHORNcantlybetween treatments with or without GVPs (likelihoodratio test 2 25665 df 1 P 005) and an

interaction between viral concentration and GVPstreatment was detected (likelihood ratio test 2 864 df 1 P 005) The lethal time did not differsigniTHORNcantly among the trials (likelihood ratio test2 214 df 2 P 034) However the interactionsbetween viral concentration and trial and betweenGVPs treatment and trial were signiTHORNcant (likelihoodratio test 2 3964 df 2 P 005 and 2 2386df 2 P 005 respectively) The interaction amongviral concentration GVPs treatment and trial was notsigniTHORNcant (likelihood ratio test 2 210 df 2 P035) Most larvae inoculated with MabrNPV plusGVPs died earlier than those inoculated withMabrNPV alone regardless of the much higher con-centration of NPV in the latter (Fig 2B)Effect of Tinopal on Susceptibility of Second In-stars No control larvae treated with or without Tino-pal died in the 13 d after inoculation The addition ofTinopal (1 mgg diet) to the inoculum reduced theLC50 values of MabrNPV from 140000 to 5300 PIBsper g of diet from 210000 to 6100 PIBs per g of dietand from 100000 to 3500 PIBs per g of diet in the THORNrstsecond and third trials respectively (Table 3) A like-lihood ratio parallelism test of the probit analysis re-vealed that the slope of the probit mortality lines forMabrNPV alone and MabrNPV plus Tinopal did notdiffer signiTHORNcantly in any of the three trials (2 3604P 0058 2 2181 P 0140 and 2 0429 P 0513 respectively)

For the analysis of the instar of death and the lethaltime we excluded data from treatments in which thehighest mortality did not reach 50 (Fig 3) Mostlarvae infected with MabrNPV died during the third orfourth stadium but only one larva inoculated at eachof 105 and 106 PIBs per g of diet in the third trial ofMabrNPV-alone treatment died during the THORNfth sta-dium In the MabrNPV-alone treatment the ratio oflarvae that died during the third stadium graduallyincreased as the NPV concentration increased from105 to 1065 PIBs per g of diet (Fig 3A) In the treat-ment of MabrNPV in combination with Tinopal 70of infected larvae died during the third stadium re-gardless of NPV concentration and the mean larvalmortality increased gradually as NPV concentrationincreased from 1035 to 105 PIBs per g of diet (Fig 3A)

Table 2 Logndashdosendashprobit parameters for MabrNPV with or without GVPs against M brassicae second instars

Treatment n Slope SE Intercept SELC50 (95 CL)

(PIBs per g diet)2 (df)

Activity ratioa

(95 CL)

Trial 1MabrNPV alone 212 139 017 164 082 60 104 (24 104ETH12 105) 768 (4)MabrNPV GVPs 248 145 015 085 044 74 102 (53 102ETH10 103) 331 (5) 815 (442ETH1469)



a Activity ratios were calculated after THORNtting regression lines with a common slope of 142 011 for the THORNrst trial and 149 012 for thethird trial The regression lines in the second trial could not be THORNtted in parallel so the activity ratio was calculated as described by Robertsonand Preisler (1992)

Fig 2 Effects of GVPs on MabrNPV infection of secondinstars ofM brassicae The THORNnal concentration of GVPs was 01mgg diet Only the treatments with high mortality are shown inthis Figure (A) Proportion of mortality by instar The threecolumnsrespectivelyshowthedatafortheTHORNrst secondandthirdtrials at each concentration (B) Mean SE of lethal timeRegression lines are shown if the regression models were signif-icant(least-squaresTHORNttingP005)Theregressionequation forthe MabrNPV-alone treatment at each trial was lethal time 1286 083 (log10PIBs) 1175 071 (log10PIBs) 985 042 (log10PIBs) respectively and that for MabrNPV plusGVPs treatment for the THORNrst and third trials was lethal time 844037(log10PIBs)787044(log10PIBs)respectively


Parametric survival analysis was performed basedon the data from viral concentration of 105 PIBs perg of diet (MabrNPV alone) and 1035 PIBs per g of

diet (MabrNPV plus Tinopal) According to the like-lihood ratio test the lethal time was negatively cor-related with the common logarithm of viral concen-tration (2 22299 df 1 P 005) The lethal timediffered signiTHORNcantly between treatments with orwithout Tinopal (2 23406 df 1 P 005) andthe interaction between viral concentration and Ti-nopal treatment was detected (2 515 df 1 P005) However the lethal time differed signiTHORNcantlyamong trials (2 1159 df 2 P 005) Theinteraction between viral concentration and trial wassigniTHORNcant (2 770 df 2 P 005) whereas thatbetween Tinopal treatment and trial was not signiTHORN-cant (2 538 df 1 P 007) The interactionamong viral concentration Tinopal treatment andtrial was not signiTHORNcant (2 289 df 2 P 024)Larvae inoculated with MabrNPV plus Tinopal diedearlier than those inoculated with MabrNPV alone ina comparison of treatments with equivalent mortality(Fig 3B)

Discussion

Enhancement of viral infection is an important con-sideration in the practical use of baculoviruses forinsect pest control Several chemicals have been re-ported as enhancers of NPV infection and the pow-erful effects of stilbene-derived szliguorescent brighten-ers such as Tinopal are well documented (eg Hammand Shapiro 1992 Vail et al 1996 Zou and Young 1996Morales et al 2001) The synergistic enhancement ofNPV infection by GVs has been known for 50 yr(Tanada 1985) and an active component of this effectwas identiTHORNed from granules of P unipunctaGV and Tni GV (Hashimoto et al 1991 Roelvink et al 1995Gijzen et al 1995) Another GV XecnGV also has theeffect of enhancing NPV infection (Goto 1990) Itsgenome contains at least four homologs of vef orenhancin genes (Hayakawa et al 1999) although noinformation is available on the protein components ofXecnGV granules Of these four genes XecnGV en-hancin-3 (orf154) shows the highest homology to en-hancins of other GVs especially to that ofH armigeraGV (HaGV) (Hayakawa et al 1999) Enhancins can begrouped into three classes with a molecular mass of104 kDa 108ETH110 kDa and 120 kDa (Roelvink et al

Table 3 Logndashdosendashprobit parameters for MabrNPV with or without Tinopal against M brassicae second instars



Activity ratioa

(95 CL)

Trial 1MabrNPV alone 201 169 019 374 102 14 105 (48 104ETH37 105) 1315 (4)MabrNPV Tinopal 236 126 013 030 049 53 103 (24 103ETH12 104) 1131 (5) 269 (99ETH701)



a Activity ratios in each trial were calculated after THORNtting regression lines with a common slope of 142 011 162 012 and 177 014respectively

Fig 3 Effects of Tinopal on MabrNPV infection of secondinstars of M brassicae The THORNnal concentration of Tinopal was01 (1 mgg diet) Only treatments with high mortality areshown in this Figure (A) Proportion of mortality by instar Thethree columns respectively show the data for the THORNrst secondand third trials at each concentration (B) Mean SE of lethaltime Regression lines are shown if the regression models weresigniTHORNcant(least-squaresTHORNttingP005)Theregressionequa-tion for the MabrNPV-alone treatment for each trial was lethaltime 1054 035 (log10PIBs) 1346 092 (log10PIBs)1519 [minus 123 (log10PIBs) respectively and that forMabrNPV plus Tinopal treatment for the second trial was lethaltime 835 032 (log10PIBs)


1995) HaGV enhancin is a representative of the sec-ond class (Roelvink et al 1995) We conTHORNrmed bySDS-PAGE analysis in this study the presence of twoproteins of molecular mass close to those of enhancinsthat are components of XecnGV granules and weshowed that a 108-kDa protein had marked enhancingactivity of MabrNPV infection It is highly possiblethat this 108-kDa protein is closely related to that ofHaGV Further study is needed to elucidate the effectsof each enhancin homolog protein of XecnGV andpossible interactions among them

XecnGV exerts an interfering effect on NPV infec-tion after inoculation with intact GV granules andNPV polyhedra against mid instars of X c-nigrum(Goto 1990) This interfering effect of XecnGV alsowas observed in inoculation of MabrNPV polyhedracombined with granules of XecnGV in larvae of Mbrassicae and H armigera even though they are non-permissive hosts of XecnGV (CG unpublished data)Hackett et al (2000) reported the possibility of asimilar effect of HaGV on Helicoverpa zeaNPV infec-tion to H zea (Boddie) larvae To avoid this kind ofinterfering effect of GVs on NPV infection we pre-pared GVPs without virions from XecnGV granulesWe evaluated the ability of GVPs to enhanceMabrNPV infection of young M brassicae larvae bycomparison with Tinopal a well-known enhancingchemical The activity ratios of enhancement with 01mgg diet of GVPs and 1 mgg diet of Tinopal were707ETH815-fold and 269ETH337-fold respectively Theoverlap of 95 conTHORNdence intervals among all activityratios indicates that the viral enhancement resultingfrom addition of GVPs is equivalent to that of TinopalDetermination of the optimum concentrations ofthese additives will be necessary to establish cost-effective pest control formulation of NPVs

In our bioassay of both GVPs and Tinopal the in-teractions between the viral concentration and thepresence of additives were detected by means of asurvival analysis The results revealed that the slopesin the regression of lethal time on viral concentrationwere signiTHORNcantly different between the treatmentwith MabrNPV alone and the treatment withMabrNPV plus additives The regression lines ob-tained in all the six trials of the sole treatment ofMabrNPV exhibited negative slopes This is consistentwith a previous report showing that increased virusdosage causes a reduction in lethal time (van Beek etal 1988) However the regression slopes were lesssteep or insigniTHORNcant in the treatments with MabrNPVplus either additive however the slopes were signif-icantly different among trials even for identical treat-ments This heterogeneity might be caused by the longobservation intervals

The lethal time of NPV-infected larvae of somenoctuids is reduced by adding szliguorescent brightenerto the inoculum (Shapiro and Hamm 1999 Shapiro2000b Shapiro and Argauer 2001) Boughton et al(2001) have suggested that this results from massiveinitial infection with virions in the midgut in the pres-ence of brightener In contrast the addition of szliguo-rescent brightener has no obvious inszliguence on the

lethal time of larvae if the concentration of NPVs isadjusted to give the same mortality as treatment withNPVs alone (Boughton et al 2001 Martotildenez et al2003) These reports use the droplet feeding methodfor the bioassay in which the larvae are exposed toinoculum over a short period Our bioassay which hasa long inoculation period of 48 h shows that larvaeinoculated with MabrNPV and an additive died sig-niTHORNcantly earlier than those inoculated with MabrNPValone even in treatments resulting in equivalent mor-tality Both GV enhancin and szliguorescent brightenersaffect the integrity of the midgut peritrophic matrix(Derksen and Granados 1988 Wang and Granados1997 2000 Mukawa et al 2003 Okuno et al 2003)Several reports indicate some additional effects ofTinopal on the larval midgut such as inhibition ofapoptosis (Dougherty et al 2006) prevention of thesloughing of infected cells (Washburn et al 1998) andreduction of midgut luminal pH (Sheppard et al 1994Sheppard and Shapiro 1994) These effects may facil-itate the earlier deaths of the infected larvae seen inour bioassay It also is reported that enhancins facil-itate the uptake of NPV virions by the midgut cells bymediating the fusion of the viral envelope and the cellplasma membrane (Tanada et al 1975 1980 Uchimaet al 1989 Kozuma and Hukuhara 1994) Reduction oflethal time and death in younger instars caused by theaddition of GVPs suggest that GVPs have an additionaleffect on NPV infection of the midgut cells Details ofthe enhancement of NPV infection by GVPs need tobe investigated especially in the midgut cells aftervirus entry

We have identiTHORNed the active components of viralenhancement in the XecnGV granules and demon-strated that the GVPs increase the susceptibility ofMbrassicae to orally inoculated MabrNPV in the sameway as does Tinopal Moreover the GVPs and Tinopallead to earlier death of MabrNPV-infected larvae inour bioassay using the diet contamination methodOur results suggest that these additives improve theefTHORNciency of MabrNPV opening the potential for useof NPVs at lower concentrations

Acknowledgments

We are grateful to Takayuki Mitsunaga for valuable sug-gestions on the statistical analysis and Yoshito Suzuki forcritical reading of the manuscript We thank Shinya Tsuda forhelping the protein puriTHORNcation This research was supportedby a grant from the Agriculture Forestry and Fisheries Re-search Council of Japan

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Received 4 January 2007 accepted 7 May 2007


BIOLOGICAL AND MICROBIAL CONTROL

Enhancement of Nucleopolyhedrovirus Infectivity Against Mamestrabrassicae (Lepidoptera Noctuidae) by Proteins Derived from

Granulovirus and a Fluorescent Brightener

SHIGEYUKI MUKAWA AND CHIE GOTO1

Insect Pest Management Research Team National Agricultural Research CenterKannondai Tsukuba Ibaraki 305-8666 Japan

J Econ Entomol 100(4) 1075ETH1083 (2007)

ABSTRACT The synergistic enhancement of nucleopolyhedrovirus (NPV) infection by granulo-viruses(GVs) iswell documented andaGVgranuleproteinnamedviral enhancinhasbeen identiTHORNedas an active contributor to this effect We detected the presence of two proteins with molecular massof 93 and 108 kDa in granules of a GV isolated from Xestia c-nigrum (L) (XecnGV) as candidates forenhancin and we conTHORNrmed that at least the 108-kDa protein enhances the infectivity of Mamestrabrassicae nucleopolyhedrovirus (MabrNPV) We tested the effect of virion-free proteins obtainedfrom XecnGV granules (GVPs) on MabrNPV infection and we made a comparison with an enhancingchemical the stilbene-derived szliguorescent brightener Tinopal Bioassay was performed employing thediet contamination method by using second instars of Mamestra brassicae (L) (Lepidoptera Noc-tuidae) The enhancing effects of GVPs (01 mgg diet) and Tinopal (1 mgg diet) were estimatedto be 707ETH815-fold and 269ETH337-fold respectively as calculated from the LC50 values of MabrNPVwith or without the additives The additives reduced the lethal time of MabrNPV-infected larvae andthey caused death at a younger instar These results suggest that GVPs can enhance MabrNPVinfection as effectively as Tinopal

KEYWORDS nucleopolyhedrovirus granulovirus szliguorescent brightenerMamestra brassicae viralenhancement

Baculoviruses including nucleopolyhedrovirus (NPV)and granulovirus (GV) have been recognized as pos-sible microbial insecticides that are highly host spe-ciTHORNc and thus safe to nontarget organisms Howeverhigh production costs and narrow spectrum have lim-ited their use as biological control agents Moreoverthe larvae of most target pests have a short period ofhigh susceptibility Efforts have been made to en-hance baculovirus efTHORNcacy by increasing host suscep-tibility with chemicals such as phosphatidylcholine(Yamamoto and Tanada 1978) boric acid (Cisneros etal 2002) and neem extract (Shapiro et al 1994) Sev-eral stilbene-derived szliguorescent brighteners greatlyenhance the virulence of Lymantria dispar NPVagainst its homologous host (Shapiro and Robertson1992) The effects of brighteners in other NPVETHhostsystems are well documented (eg Hamm and Sha-piro 1992 Vail et al 1996 Zou and Young 1996 Mo-rales et al 2001) Fluorescent brighteners facilitate thearrival of NPV at midgut of the host epithelial cells bydisrupting the peritrophic matrix which serves as abarrier topathogenicmicroorganisms(WangandGra-nados 2000 Mukawa et al 2003 Okuno et al 2003) Inaddition szliguorescent brighteners weaken host resis-

tanceby inhibiting theapoptosisofNPV-infectedmid-gut cells (Dougherty et al 2006) and by blocking thesloughing of infected primary target cells in the mid-gut (Washburn et al 1998)

The GVs from Pseudaletia unipuncta (Haworth)Trichoplusia ni (Hubner) Xestia c-nigrum (L) Heli-coverpa armigera (Hubner) and Spodoptera frugi-perda (JE Smith) enhance the infectivity of NPVs(Tanada 1959 Derksen and Granados 1988 Goto 1990Shapiro 2000a) This viral enhancement is due to aprotein named a synergistic factor viral enhancingfactor or enhancin which is a component of thegranules of the GVs (Tanada et al 1973 Hashimoto etal 1991 Corsaro et al 1993) PuriTHORNed enhancin proteinacts as a metalloprotease and degrades the peritrophicmatrix proteins resulting in the disruption of the peri-trophic matrix (Derksen and Granados 1988 Leporeet al 1996 Wang and Granados 1997) There is alsoevidence that this protein achieves its enhancing ef-fect by increasing the fusion of the viral envelope withthe midgut cell plasma membrane (Tanada et al 19751980 Uchima et al 1989 Kozuma and Hukuhara 1994)Whole-genome analysis ofX c-nigrumGV (XecnGV)has revealed that the genome contains four homologsof enhancinOne of them is highly homologous to theenhancins ofH armigeraGVT niGV andP unipuncta1 Corresponding author e-mail cgotoaffrcgojp

0022-0493071075ETH1083$04000 2007 Entomological Society of America




















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enhancement of nucleopolyhedrovirus infectivity against mamestra brassicae (lepidoptera: noctuidae)...

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