Detection of genes encoding antimicrobial peptides in Mexican strains of Trichoplusia ni (Hübner) exposed to Bacillus thuringiensis

Detection of genes encoding antimicrobial peptides in Mexican strains of Trichoplusia ni (Hübner) exposed to Bacillus thuringiensis

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  Detection of genes encoding antimicrobial peptides in Mexicanstrains of   Trichoplusia ni   (Hu¨bner) exposed to  Bacillus thuringiensis P. Tamez-Guerra a,* , J.A. Valadez-Lira a , J.M. Alcocer-Gonza´lez a , B. Oppert b ,R. Gomez-Flores a , R. Tamez-Guerra a , C. Rodrı´guez-Padilla a a Departmento de Microbiologı´ a e Inmunologı´ a, Facultad de Ciencias Biolo´  gicas, Universidad Auto´ noma de Nuevo Leo´ n (FCB-UANL),AP 46-F, 66450 San Nicola´ s de los Garza, N.L., Mexico b USDA-ARS Grain Marketing and Production Research Center, 1515 College Avenue, Manhattan, KS 66502, USA Received 15 October 2007; accepted 13 February 2008Available online 20 February 2008 Abstract The systemic immune response of   Trichoplusia ni   after  Bacillus thuringiensis  (Bt) exposure was evaluated by comparing the expressionof genes encoding antimicrobial peptides (AMPs) in Bt-susceptible and -resistant  T. ni   strains that were either exposed or not to Xen-Tari  (Bt-XT). AMP genes were detected by RT-PCR using primers for attacin, gloverin, lebocin, lysozyme, and peptidoglycan recog-nition peptide (PGRP). In general, AMP genes were detected more frequently in Mexican field strains previously exposed to Bt (SALXand GTOX) than in a Mexican laboratory strain (NL), but expression was similar to the AMP expression in USA laboratory strains (USand USX). Among the AMPs, transcripts for lebocin were the least detected (11.7%) and those for lysozyme were the most detected(84.8%) in all samples. Lebocin was detected only in 2nd instar and pupa. All untreated controls expressed attacin. Attacin and gloverinwere not detected in any midgut sample, and their highest detection was in pupa. Lysozyme was rarely detected in 2nd instar larvae fromany strain or treatment but was detected in almost all midgut and hemolymph samples. Overall, AMPs were found more in  T. ni   strainspreviously exposed to Bt-XT, especially lebocin and globerin (1.8-fold increase) and PGRP (3.8-fold increase). The data suggest that theexpression of AMPs in  T. ni   correlates to previous Bt exposure.   2008 Elsevier Inc. All rights reserved. Keywords: Trichoplusia ni   immune response;  Bacillus thuringiensis ; Antimicrobial peptides; Bt-susceptibility 1. Introduction Insects represent three-fourths of all animal species andhave confronted many potentially pathogenic microorgan-isms, including those used in pest control. However, insectshave developed protective mechanisms to evade the patho-genic effects of microbes (Vilmos and Kurucz, 1998; Gilles-pie et al., 1997). Insect defense mechanisms are diverse andinvolve cellular and systemic type reactions (Jarosz, 1996;Hultmark et al., 1983; Marchini et al., 1993). Cellular-med-iated reactions mainly involve phagocyte cells and the for-mation of a capsule produced by hemolymph cells (Jarosz,1996). The systemic responses usually involve a rapid syn-thesis of small cationic peptides, such as defensins, cecro-pins, and attacins (Marchini et al., 1993; Natori, 1995).Following a bacterial infection, antimicrobial peptides areproduced in the insect fat body (analogous to the liver inmammals) and in hemolymph cells, and accumulate inthe hemolymph of the infected insect (Gillespie et al.,1997; Hoffmann, 1997). Bacillus thuringiensis  (Bt) has been the most commer-cially used bioinsecticide among entomopathogenic micro-organisms. The role of insect immune protectivemechanisms to evade Bt infection is unknown, but the pro-duction of inhibitory factors from Bt strains can interferewith the insect immune response (Edlund et al., 1976).Insect immunity was reported to play an important rolein the overall pathogenicity of another bacterium,  Serratiamarcescens  (Flyg et al., 1980). However,  S. marcensces 0022-2011/$ - see front matter    2008 Elsevier Inc. All rights reserved.doi:10.1016/j.jip.2008.02.008 * Corresponding author. Fax: +5281 8352 4212. E-mail address: (P. Tamez-Guerra).  Available online at Journal of Invertebrate Pathology 98 (2008) 218–227 Journal of  I NVERTEBRATE P  ATHOLOGY   spreads through the hemolymph, whereas Bt entersthrough the digestive system.Insects with different Bt-susceptibility have been demon-strated to have variations in (1) toxin receptors in the mid-gut (DeMaagd et al., 2001), or (2) the production of digestive proteases resulting in differences in enzymaticactivity levels (Oppert et al., 1997). However, other protec-tive mechanisms may be responsible for varying suscepti-bility to bacteria harboring Bt toxins in insects(Tabashnik et al., 1997). Therefore, we compared theexpression of genes encoding antibacterial peptides(AMPs) in strains of the cabbage looper  Trichoplusia ni  (Hu¨bner) with varying susceptibility to Bt protoxins andtoxins. 2. Materials and methods  2.1. InsectsTrichoplusia ni   strains were selected based on differencesin susceptibility to Bt toxins and protoxins (Tamez-Guerraet al., 2006) (Table 1). Strains included NL (a Mexican strain collected in Nuevo Leon and reared in our labora-tory for 3 years), US (kindly provided by Dr. Behle fromthe National Centre for Agriculture Utilization Research,United States Department of Agriculture, Peoria, IL),and two wild strains from east central Me´xico, GuanajuatoState (SAL and GTO). Selected strains were exposed in thelaboratory to XenTari   (Bt-XT) (Valent Biosciences Cor-poration, Libertyville, Illinois, E.U.A.; imported and dis-tributed in Mexico by DuPont Me´xico, S.A. de C.V.)over several generations (Tamez-Guerra et al., 2006) andwere coded with an ‘‘X ”  (USX, SALX, and GTOX). Allinsect populations were reared on artificial diet (McGuireet al., 1997) at 25  C ± 2  C, 55 ± 10% relative humidityand 16 h light/8 h dark photoperiod.  2.2. Bioassays We determined the presence of genes encoding AMPs ineach  T. ni   strain under different Bt toxin exposures, usingtwo bioassays: an overlayer bioassay and a stained drop-let-feeding bioassay (Tamez-Guerra et al., 2006; Behleet al., 2000). Bt-XT treatments consisted of five doses of XenTari  , based on international units (IU), as follows:0, 500, 500*(exposed to Bt-XT until larvae reached a spe-cific instar and analyzed 20 h after exposure), 2500, or3500 IU/ml. Other treatments consisted of a fresh cultureor a mixture of spore–crystals from Bt var  thuringiensis BtUANL01001 (Btt) and  Escherichia coli   strain DH5- a (EC), either alone or with 500 IU/ml Bt-XT. The only   strain that was susceptible to both Cry protoxin andtoxin was US (Tamez-Guerra et al., 2006), and thereforethe response of US to  E. coli   exposure was consistentlytested for microbial peptide production. We tested freshculture and spore–crystals of Btt to compare the infectivestage (fresh culture) versus the insecticidal toxins presentin crystals.  Escherichia coli   was tested because it is aGram-negative bacterium commonly used as a positivecontrol for AMP detection by PCR (Sugiyama et al.,1995; Axen et al., 1997; Lundstrom et al., 2002; Haraand Yamakawa, 1995; Spies et al., 1986). Btt and  E. coli  were kindly provided by the Laboratory of Immunology,Biological Sciences College, Autonomous University of Nuevo Leon, MX. Btt was srcinally isolated from soil inGuanajuato, MX, whereas  E. coli   was srcinally obtainedfrom ATCC (Manassas, VA).  Escherichia coli   and Btt cul-tures (100  l l) were at an optical density 600 nm  of 0.6 U whenharvested (Gene Quant Pro, Amersham Bioscience, Brazil).To obtain a spore–crystal mixture from a slant sample, Bttwas inoculated in a Petri dish on nutrient agar (Spectrum)and incubated at 28  C. After 5 days of culture, if sporesand crystals were observed, culture was collected into30 ml of sterile saline solution (0.1 M sodium chlorideand 0.01% Triton-X 100 in distilled water) and washedthree times, mixing it in distilled water and centrifugingat 20,000  g   for 30 min (Beckman, Avanti, J-25I). Spore– crystals were lyophilized (Labconco 77500-20, KansasCity, MO). The dried spore–crystal mixture was tested ata dose of 100 mg/ml.Treatments of bacterial challenge consisted of Btt, Btt-EC, Bt-XT, and EC in the overlayer bioassay in selectedstrains and developmental stages. Treatments were mixedin PBS, and 50  l l of each treatment were applied to2 cm 2 artificial diet mixed with 0.1% bovine serum albumin(BSA) in 12-well trays (Costar). PBS only was applied as acontrol (Tamez-Guerra et al., 2006). Doses were air-driedfor 2 h and infested with one  T. ni   larva per well. For theBt-XT treatments, larvae were fed artificial diet until theyreached a specific instar, when they were transferred totreated diet and were collected and analyzed after 20 h Table 1 Trichoplusia ni   strains used in this study, and comparison of relativesusceptibilities to Cry 1Aa, Cry1Ab, and Cry1Ac toxins and protoxins a Strain code b Source Resistance ratio c NL Laboratory UANL-FCB, Mexico 91, pAa vs. USUS USDA-ARS, Peoria, IL, USA NoneUSX 4.5, pAa vs. US21, pAc vs. NLSALX Field, Salvatierra, Gto. Mexico 6.2, Ab vs. US16, Ab vs. USGTOX Field, San Luis de la Paz, Gto., Mexico 16, pAa vs. US40, Ab vs. US50, pAb vs. NL87, pAc vs. NL22, pAc vs. US a Data from Tamez-Guerra et al., 2006. b Strains that were selected in the laboratory with XenTari were denotedby the designation of the strain followed by an ‘‘X ” . c Resistance ratio was based on confidence limits of the LC 50  value. Aa,Ab, or Ac, and pAa, pAb, and pAc = resistance to Cry1Aa, Cry1Ab, orCry1Ac toxins or protoxins, respectively, compared with laboratorystrains (NL or US), as indicated. P. Tamez-Guerra et al./Journal of Invertebrate Pathology 98 (2008) 218–227   219  exposure, based on previous reports of AMP gene expres-sion time analysis (Chowdhury et al., 1995; Liu et al.,2000). For pupae analysis, larvae were fed on treated dietthroughout larval development and were collected as pupa.Each bioassay was performed on different days with tripli-cate determinations.Neonates were exposed to Bt-XT in a droplet-feedingbioassay. Neonates were fed on a colored solution with dis-tilled water or a solution containing specific doses of eachtreatment. Positively treated larvae were selected by colorfrom each treatment and were incubated in 14:10 light:darkphotoperiod at 28  C to the specific instar or pupae stage.  2.3. Antimicrobial peptide (AMP) gene detection RT-PCR was used to detect genes encoding AMP in   exposed to different bacterial treatments. Samplesincluded the whole body of 2nd, 3rd, and 4th instar larvaeand pupae (Kang et al., 1996a), and hemolymph or midgutof 4 th instar larvae, exposed or not to the treatmentsdescribed above (Wang et al., 2004; Hara and Yamakawa,1995). The fat body was not analyzed separately, but wasconsidered as the contributing factor if hemolymph andmidgut were negative. In general, whole body, hemolymphor midgut samples were obtained after fasting larvae for 2– 4 h, as recommended for midgut enzyme detection (Lamet al., 2000). For whole larvae or pupae, three individualsfrom each selected instar were homogenized in 1.5 ml of pre-chilled, 50 mM Tris–HCl buffer pH 8.0, containing0.01 M CaCl 2  (PRO-250, PRO Scientific, Monroe, CT).From these samples, 50–100 mg were homogenized with1 ml of TRI reagent (Molecular Research Center, Inc.,Connecticut, OH). Midgut mRNA was obtained by a pre-vious procedure (Chomczynski and Sacchi, 1987). In brief,three midguts with food bolus were dissected from 4thinstar larvae and homogenized and added to 1 ml of TRIreagent. Hemolymph was obtained by a syringe from sam-ples after the midgut was removed and added to pre-chilledTRI reagent (1:5 v:v). Samples were incubated 5 min atroom temperature. In each sample, 0.2 ml of chloroformwas added and vigorously mixed for 15 s and incubatedat room temperature for 2–3 min. Samples were then cen-trifuged at 12,000  g   for 8 min at 2–8  C. The upper layer(transparent phase) was isolated and transferred into anew tube, 500  l l of isopropanol was added, and the samplewas mixed in a vortex, incubated at room temperature for5–10 min, and centrifuged at 12,000  g   for 8 min. The super-natant was discarded, and the remaining pellet with RNAwas washed with 1 ml of ethanol 75% in DEPC water (milliQ water mixed vigorously with 0.1% diethylpyrocarbonatefor 2 h and autoclaved). The sample was centrifuged for5 min at 7500  g  . The supernatant was discarded and the pel-let was air-dried for 5–10 min. The pellet was dissolved bypipetting in 50–200  l l of DEPC water and was incubated at55–60  C for 10 min. AMP gene detection was performedin triplicate from randomly selected larvae from the samegeneration.RT-PCR was used to synthesize complementary DNA(cDNA) from RNA. In each tube, 10  l l of 5  reaction buf-fer (250 mM Tris–HCL, pH 8.3, 375 mM KCl, and 1.5 mMMgCl 2 ), 1  l l of 50 mM dithiothreitol, 1  l l of 1 U of RNA-ase inhibitor, 2  l l of 800  l M of dNTPs, 2  l l of 2.5  l M of primer dT12-18, and 1  l l of 200 U of Moloney murine leu-kaemia virus (MMLV) reverse transcriptase (PROMEGA)were added to 5  l g of RNA samples. This mixture wasadjusted to 50  l l with DEPC water and was incubated at37  C for 2 h. The enzyme was inactivated by increasingthe temperature to 60  C for 10 min.To identify transcripts of the constitutive ribosomal pro-tein S3a (RPS3a, as positive internal expression gene) andAMPs(attacin,gloverin,lebocin,lysozyme,andpeptidogly-canrecognitionpeptide,PGRP),specificinternalgeneprim-erswereusedinapolymerasechainreaction(PCR).Inafinalvolume of 50  l l, 1   buffer (200 mM Tris–HCl at pH 8.4,500 mMKCl),5  l loftemplate(cDNA),3  l l1.5 mMMgCl 2 ,1  l l of 800  l M of dNTP’s, and 10 pmol of each primer weremixed with 1 U of DNA taq polymerase (Bioline). RPS3awas amplified using the primers RPS3a-1-AGGCACCGTCTAGTTCACC-, RPS3a-2-GCCAGCGAGACTTCAAAAAC-)(BorovskyandWuyts,2001).Attacinwasampli-fied using the primers ATA1-CAAATTGATTTTGGGATTGG and ATA2-CACTTATTACCAAAAGACCGGC,for an expected product of 750 bp (GenBank: U46130; Sug-iyama et al., 1995; Kang et al., 1996b). Gloverin was ampli-fied using the primers GLO1-GAATCGTTCACCATGCAGTC, and GLO2-TCCTCATTTTAACCATACACGAAA, for an expected product of 808 bp (GenBank:AF233590; Lundstrom et al., 2002). Lebocin was amplifiedusing the primers LEB1-TCTGGTGTTGTGTGTGCTCTC, and LEB2-GGACAAAAACAGAAAAGTGCAA,for an expected product of 857 bp (GenBank: AF233589;YamakawaandTanaka,1999).Lysozymewasisolatedfrom T. ni   (Kang et al., 1996a) using the primers LISHv-ATTCGCTAACCAGTGGTCGT, and LISHv2-GGTACAGTGCCTTTTTAATTTGC, and these primers were used todetect an expected product of 925 bp (GenBank: U50551;Spies et al., 1986). PGRP was amplified using the primersPGRP1-GACTGTGAGTGGAGATTGCG, and PGRP2-TTTTGGTCTATTTCACCATTTACG, to obtain a605 bp product (GenBank: AF076481).Preliminary tests were performed to select the amplifica-tion cycle number for each sample. AMP transcripts wereamplified for 25 cycles for whole body, 35 cycles for mid-gut, and 40 cycles for hemolymph in a thermocycler(Touchgene Techne, Cambridge, England). Each cyclehad a denaturing step of 94  C/1 min, an annealing stepof 55  C/1 min, and an extension step of 72  C/2 min anda final extension of 72  C/7 min. Ten microliters of theamplified sample was analyzed in a 1.5% agarose gel andethidium bromide stain under UV light using a UVPtrans-illuminator (VWR, USA). Optical densities of theDNA bands were detected using a UVP spectrometer (Bio-Spectrum Imaging Systems, UVP, Inc. Upland, CA). Foreach transcript, the RPS3 detection was used as internal 220  P. Tamez-Guerra et al./Journal of Invertebrate Pathology 98 (2008) 218–227   control. If inconclusive results were obtained in any repli-cate, samples were retested using more amplification cycles(up to 40). 3. Results To detect genes that encode antimicrobial peptides(AMPs), mRNA was isolated from larval and pupal wholebody and 4th instar larval hemolymph and midgut, andwas used as a template for cDNA amplification by RT-PCR. The products of typical amplification reactions withUS and GTOX are shown in Fig. 1. Constitutive RPS3gene was used as internal positive control and was detectedin all samples.When all of the samples were combined from all devel-opmental stages, the most frequently detected AMP geneswere lysozyme and PGRP, found in 80.5% and 70.2% of the samples, respectively (Fig. 2A). Gloverin was foundin 33.0% of samples, whereas attacin and lebocin were inless than 20.0% of all samples.The expression of AMP genes (attacin, gloverin, lebocin,lysozyme, or PGRP) differed within the various develop-mental stages of the  T. ni   strains (Fig. 2B). However, lyso-zyme and PGRP were the most frequently detected AMPgenes in all but 2nd instar larvae. Attacin was not detectedin the midgut tissue of any strain, and was found in only4.17% of 2nd instar and hemolymph samples, 16.0 and8.70% of 3rd and 4th instar larvae, respectively, but GLOVERIN 2nd 3rd 4th. PupaeHEM MG LEBOCIN LYSOZYMEInstarsTissuePGRPATTACIN CTLEXPCTLEXP GTOX CTLEXPCTLEXPCTLEXPCTLEXPCTLEXPCTLEXP USGTOXUSGTOXUSGTOXUSGTOXUS CTLEXPCTLEXP RPS3 GTOXUS CTLEXPCTLEXP StrainsAMP Fig. 1. Comparision of relative expression of antimicrobial peptide genes (attacin, gloverin, lebocin, lysozyme, and PGRP) in two  T. ni   strains (GTOX andUS) in different larval instars and tissue samples by RT-PCR. CTL, unexposed control; EXP, exposed to Bt-XT (500 IU/mL) until larvae reached theindicated instar and analyzed 20 h after exposure; HL, hemolymph; MG, midgut. P. Tamez-Guerra et al./Journal of Invertebrate Pathology 98 (2008) 218–227   221  45.8% of the pupae samples. Similar to attacin, gloverinwas not detected in any midgut sample, but was found in19.4%, 40.0%, 30.0%, and 38.7% of 2nd, 3rd, and 4th instarand hemolymph samples, respectively, and in 71.0% of pupae samples. Lebocin was detected in 71.4% of 2ndinstar larvae, 13.0% of pupae, but not in any of the otherdevelopmental stages. Lysozyme was detected in more than90.0% of the samples, except for 2nd instar larvae, of which37.5% expressed the gene. PGRP also was highly expressedin most samples, detected in 33.3% of the hemolymph sam-ples, 47.6%, 90.0%, and 95.0% of 2nd, 3rd, and 4th instar,respectively, 85.0% of pupa, and 65.4% of the midgutsamples.Regardless of the strain, lysozyme and PGRP were themore prevalent AMPs found in  T. ni   (Fig. 2C). In general,AMPs were detected more in Mexican strains that werepreviously exposed to Bt in the field and in the laboratory(SALX and GTOX) compared with a laboratory strain(NL), but expression was similar to that of the USAstrains (US and USX). Surprisingly, the US laboratorystrain had a similar AMP expression pattern comparedto that of the Mexican field strain selected with Bt-XT,GTOX. The relative expression patterns of lysozymeand PGRP were the same in all of the strains, with lyso-zyme followed closely by PGRP as the most frequentlyencountered AMP genes. However, gloverin also wasprevalent, expressed in 36.1–58.3% of US, USX, andGTOX strains.In comparisons of the relative expression of AMP genesin laboratory  T. ni   strains (NL + US, lab) and thoseexposed to Bt-XT (USX, SALX, and GTOX) in all treat-ments and samples tested, the expression of all AMPswas increased in the Bt-XT exposed strains, especially forgloverin, lebocin, and PGRP (Fig. 2D). The smallestincrease was observed with lysozyme, with only a 1.05-foldincrease in the Bt-exposed  T. ni   strains. 3.1. Attacin expression The attacin transcript was not detected in any develop-mental stage or tissue of untreated controls of   T. ni   strains(Tables 2 and 3, Fig. 1). Attacin gene expression generally increased in response to Bt-XT or Btt exposure, and mostpositive samples were from US, USX, and GTOX strains.The highest percentage of attacin expression was 66.7% inthe GTOX strain exposed to 500 IU of Bt-XT and analyzedafter 20 h. Expression of the attacin gene transcript wasfound in two treatments: first, exposed to Bt-XT in onegiven instar and tested after 20 h or as neonate using higherdoses of Bt-XT (US, USX, and GTOX); or second,exposed to a lower dose of Bt-XT dose as a neonate (NLand SALX). Attacin gene expression was found in less than 020406080100US USX NL SALX GTOX Trichoplusia ni strains    R   T  -   P   C   R   D  e   t  e  c   t   i  o  n   (   %   ) attacingloverinlebocinlysozymePGRP AMP 020406080100attacin gloverin lebocin lysozyme PGRP Antimicrobial peptides    R   T  -   P   C   R   D  e   t  e  c   t   i  o  n   (   %   ) labBt-exp T. ni   strains 020406080100attacin gloverin lebocin lysozyme PGRP Antimicrobial peptides    R   T  -   P   C   R   D  e   t  e  c   t   i  o  n   (   %   ) 0204060801002nd 3rd 4th MG HL Pupa T. ni instars    R   T  -   P   C   R   D  e   t  e  c   t   i  o  n   (   %   ) attacingloverinlebocinlysozymePGRP AMP Fig. 2. Expression of antimicrobial peptide (AMP) genes in  T. ni   strains, as detected by RT-PCR and including all samples from all instars tested. (A)Percentage of total positive samples expressing AMP; (B) percentage of AMP detection in different  T. ni   instars; (C) comparison of AMP detection among T. ni   strains; (D) percentage of AMP detection among control and treated samples. Lab = NL + US strains; Bt-exp, previously exposed to  Bacillusthuringiensis  (XenTari  , Bt-XT) for more than three generations in the laboratory (a combination of responses from USX + SALX + GTOX).222  P. Tamez-Guerra et al./Journal of Invertebrate Pathology 98 (2008) 218–227 
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