Paratransgenesis: The dynamics of engineered Enterobacter symbionts and Cry1Ac-producing Enterobacter for biocontrol of Helicoverpa insect pests in crop production

Paratransgenesis: The dynamics of engineered Enterobacter symbionts and Cry1Ac-producing Enterobacter for biocontrol of Helicoverpa insect pests in crop production | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Paratransgenesis: The dynamics of engineered Enterobacter symbionts and Cry1Ac-producing Enterobacter for biocontrol of Helicoverpa insect pests in crop production Abhishek Ojha doi: https://doi.org/10.1101/2025.01.01.630992 Abhishek Ojha 1 International Centre for Genetic Engineering and Biotechnology , Aruna Asaf Ali Marg, New Delhi 110067, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: ab.ojha{at}gmail.com Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract The potential application of insect gut microorganisms in paratransgenic crop pest management techniques is being investigated. The Helicoverpa gut is known to harbor the symbiont Enterobacter . However, little is known about the dynamics of Enterobacter in the Helicoverpa gut, its interaction with the phyllosphere, and the mechanisms by which it is delivered to this Lepidopteran species. To evaluate the insecticidal activity of Cry1Ac and its capacity to colonize the Helicoverpa gut and the tomato phyllosphere, the symbiont Enterobacter , which was isolated from the Helicoverpa gut, was transformed with constructs 2 ( cry1Ac - KmR -pUC18) and 3 ( KmR -pGFP). Within 48 hours and 24 hours, respectively, the Helicoverpa gut and tomato phyllosphere were effectively colonized by the GFP-producing construct 3- Enterobacter . In insect bioassays, Cry1Ac-producing constructs 2- E. coli (K12) and 2- Enterobacter were used for diet and leaf-dip testing. Helicoverpa neonates died 86-100% when fed diets supplemented with 10 -10 cells of Cry1Ac-producing construct 2- E. coli (K12), whereas 79–100% died when fed diets supplemented with 10 -10 cells of Cry1Ac-producing construct 2- Enterobacter . Furthermore, when the Cry1Ac-producing construct 2- Enterobacter (0.10 x 10 9 cells) was applied to the tomato phyllosphere, it caused 100% mortality of neonates within 192 hours. The successful vector modification, genetic transformation, and establishment of recombinant Enterobacter cells in the guts of Helicoverpa larvae and on tomato leaves lay the foundation for advancing a paratransgenic strategy. This engineered bacterium could potentially replace synthetic insecticides for managing lepidopteran pests in crops. 1. Introduction A wide variety of insects are known for their adverse impacts on agriculture. Efforts to control these insects are being questioned by the global emergence of insecticide resistance ( Robinson et al., 2004 ). Therefore, it is worthwhile to explore other approaches to complement existing insect control management ( Beard et al., 1993 ). This study demonstrates a paratransgenic strategy introducing foreign molecules into the host insects via symbionts. The plan aims to produce anti-insect gene products in mutualistic (symbiotic) microbes contained by the insects ( Chapco and Kelln, 1994 ; Tang et al., 2004 ). Host specificity plays an important role in paratransgenic strategies that help gut microbes transfer foreign (anti-insect) gene products into the insect gut. Native gut microbes are preferred as gene-delivery systems in the paratransgenic method because foreign (non-native) microbes are generally incapable of colonizing a specific gut environment ( Chapco and Kelln, 1994 ; Husseneder and Grace, 2005 ; Mehta and Murthy, 1992 ; Thimm et al., 1998 ; Veivers et al., 1982 ). Furthermore, genetically engineered microbes firmly connected with a single host insect can reduce potential impacts on non-target organisms. Moreover, the negative impacts frequently depicted when genetically engineered organisms are delivered into competing environmental conditions ( Irvin et al., 2004 ; Van-Elsas, et al., 1994 ) could potentially be mitigated by employing genes that help the formation of recombinant strains in composite bacterial environments ( Hillman, 2002 ). In the gut of polyphagous insects like Helicoverpa armigera ( H. armigera ) and others, a complex and diverse bacterial population helps in the digestion of plant products ( Gayatri et al., 2012 ; Cazemier et al., 2003 ; Lemke et al., 2003 ). The gut bacterial populations that are related to H. armigera ( Gayatri et al., 2012 ), Melolontha melolontha ( Egert et al., 2005 ), and Dermolepida albohirtum ( Pittman et al., 2008 ) are typically observed across the geological allocation of these insect hosts. These commonly revealed gut microbes may play a key role in paratransgenic strategies to control insect pests in crops ( Pittman et al., 2008 ). This could be established by genetically modifying gut microbes to produce anti-insect molecules that adversely affect the insects and employing these microbes in soil infested with insect pests. Previous studies have revealed the isolation of microbes from the gut of H. armigera larvae, as well as from insects collected from different crops and various locations in India, along with an assessment of their diversity ( Gayatri et al., 2012 ). Further, we observed that Enterobacter was present in all insect samples ( Gayatri et al., 2012 ). Based on this, we assumed that if Enterobacter is capable of colonizing both the insect gut and the leaf surface, it could be a favorable element of a paratransgenic strategy to control insect pests. In this study, we investigate the re-colonization ability of genetically engineered Enterobacter (expressing GFP and KmR ) within the H. armigera gut and on the tomato phyllosphere. We also expand this study to include genetically engineered Enterobacter (expressing Cry1Ac and KmR ) to assess Cry1Ac expression within the gut of H. armigera and its associated mortality. Furthermore, we examine the lethality of the Enterobacter (expressing Cry1Ac and KmR ) population when applied to tomato phyllosphere, using H. armigera neonates in insect bioassays. Our outcomes reveal valuable insights into the potential of using Enterobacter as a paratransgenic microbe to control this serious insect pest. 2. Materials and methods 2.1. Insect larval sampling The H. armigera strain was maintained in the insectary under controlled conditions of temperature (25±5 ° C), 70±5% relative humidity (RH), and a 14 h/ 10 h L/D (light/dark) cycle using an artificial diet as described by Sachdev et al. (2014) ( Sachdev et al., 2014 ; Shorey and Hale, 1965 ). First-instar neonates were then subjected to an insect bioassay. Ten neonates, in triplicates, were used for each insect bioassay experiment. 2.2. Recombineering, transformation, and protein expression A 10 ng cry1Ac -pKK223-3 vector (Bacillus Genetic Stock Centre, Columbus, OH) was used as a template for the polymerase chain reaction (PCR, Bio-Rad, USA) to amplify the cry1Ac gene. The forward primer 5’-CCCGGGATGTGAACAGTGCCCTTACAA-3’ (which includes an NdeI restriction sit) and the reverse primer 5’-CATATGCCACGCTGTCCACGATAAATG-3’ (which includes a Cfr9I or XmaI recognition site) were employed in the PCR. A PCR was achieved in a 25 µl reaction containing 200 µM dNTPs, 5U of Taq DNA polymerase (New England BioLabs, NEB, Inc., USA), and 200 µM of each primer. The PCR conditions were as follows: 95°C for 2 minutes, followed by 35 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 45 seconds, with a final extension at 72°C for 5 minutes. A 3.0 kb DNA fragment was examined on a 1.5% TAE agarose gel ( Sambrook and Russell, 2001 ) run at 5v/cm. The gel was visualized with ethidium bromide staining, and the DNA was gel-purified using the QIAquick Gel Extraction kit (Qiagen, Inc., Germany) (data not shown) and quantified using a NanoVue Plus spectrometer (data not shown). The purified PCR product was digested with NdeI and Cfr9I and ligated into the pUC18 vector to achieve construct 1 ( cry1Ac -pUC18). DH5α competent Escherichia coli ( E. coli ) cells were transformed with 10 ng of construct 1. The transformants were selected on agar plates containing ampicillin (100 µg/ml) and subjected to plasmid purification. The colonies were further determined by restriction enzyme digestion and PCR. The cry1Ac gene in the recombinant plasmid was sequenced using the Sanger method (data not shown). Further, a 1.5 kb kanamycin-resistance ( KmR ) gene cassette (from pET-28a) was amplified, as described by Chandra et al. (2008) ( Chandra et al., 2008 ; Banai et al., 1985 ), using the forward primer 5’-CGATCGGATAAACCCAGCGAACCATTTGAG-3’, which includes a PvuI recognition site, and the reverse primer 5’-CGATCGCCACGCTGTCCACGATAAATG-3’, which also includes a PvuI restriction site. The PCR product was determined on a TAE 1.0% agarose gel ( Sambrook and Russell, 2001 ), and gel-purified using the QIAquick Gel Extraction kit, and quantified (data not shown). The purified PCR product was digested with PvuI and cloned into construct 1 and pGFP (ClonTech, Takara Inc.) to achieve construct 2 ( cry1Ac-KmR -pUC18) and construct 3 ( KmR -pGFP), respectively. Separate E. coli cells were used to transform constructs 2 and 3. The transformants were selected on ampicillin (50 µg/ml) containing agar plates and subjected to plasmid purification. The colonies were then determined by restriction enzyme digestion and PCR. Finally, these constructs (2 and 3) were transformed into both Enterobacter aerogenes and E. coli K12, isolated from the gut of H. armigera ( Gayatri et al., 2012 ). The recombinant construct 3 ( KmR -pGFP) was expressed by a transformed cell of Enterobacter aerogenes and E. coli K12 on LA/ KmR agar and in LB/ KmR broth, incubated overnight at 37°C. 2.3. Protein gel blot analysis The examination of Cry1Ac from recombinant Enterobacter aerogenes (construct 2) and E. coli K12 (construct 2) was achieved by applying the Western blot test. The cell pellet was suspended in the extraction buffer ( Ojha et al., 2021 ). The whole-cell lysate was denatured by heating for 10 min at 99°C in 1x SDS-PAGE loading dye. Fifty micrograms (50 µg) of protein from the lysate were separated on a 7.5% SDS-PAGE gel, and the resolved proteins were transferred to a nitrocellulose membrane (0.4µ pore size) (GE Healthcare) by blotting at 180 mA for 45 minutes. The membrane was incubated for 1 h (room temperature, RT) with rabbit anti-Cry1Ac antibody (1:5,000 dilution). It was then incubated for 1 h at RT with goat anti-rabbit IgG-ALP (alkaline phosphatase) antibody (1:3,000 dilution). Finally, color development was achieved using 10 ml of BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) substrate solution (Genei Laboratories Pvt Ltd, Bangalore, India). The reaction produced a bluish-grey to black precipitate of Cry1Ac on the membrane. 2.4. Feeding of H. armigera larvae with GFP bacteria To achieve the colonizing potential of E. aerogenes- (construct 3) and E. coli K12-(construct 3) in the gut of H. armigera , a total of 60 first-instar larvae (neonates) were fed a sterile artificial diet supplemented with antibiotics (sterile antibiotic diet) ( Brummel et al., 2004 ) for 48 hrs (2 days). Each larva was fed small cubes (1 g) of the sterile antibiotic diet inoculated with 100 µl/cm 2 of an overnight-grown bacterial culture of E. aerogenes- (construct 3) for 48 hrs. After that, the neonates were transferred to a normal artificial diet. The control group consisted of neonates continuously fed an artificial diet supplemented with E. aerogenes (native bacteria, isolated from H. armigera ). The neonates’ fecal samples were then homogenized in sterile Luria Broth (LB, HiMedia Laboratories Pvt Ltd). A total of 100 μl from the 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , and 10 -6 dilution of GFP-expressing bacteria was applied on LB agar plates containing kanamycin (20 μg/ml). The plates were incubated at 37°C for 24 hrs to examine GFP expression by E . aerogenes- (construct 3). A similar experiment was performed to examine GFP expression in E. coli K12-(construct 3) on LB agar plates containing kanamycin (20 μg/ml). The control group for this experiment consisted of neonates continuously fed an artificial diet supplemented with E. coli K12. 2.5. Colonization assay with Enterobacter on the tomato phyllosphere The tomato ( Solanum lycopersicum ) plants used in this investigation were maintained in the greenhouse at the International Center for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India. The tomato leaves (20-25 days old) were cleaned (five times) with sterile water. E . aerogenes -(construct 3) cells pellets (overnight grown) were washed (thrice) with 20 ml PBS, pH 7.4 ( Ojha et al., 2014 ). The leaves were then sprayed with a suspension of E . aerogenes- (construct 3) at a concentration of 8 x 10 8 cells/ ml. The plants were incubated in an enclosed plastic tent on the laboratory bench for 6 hrs at 22°C. Afterward, the plants were transferred to a growth chamber and incubated at 28°C and 95% relative humidity (RH, range 75-99%) with a 12-hour photoperiod for an additional 3 days. Three triplicate leaves from plants were harvested at 24, 96, and 192 hrs. These leaves were washed, five times, with PBS, pH 7.4. The individual leaves were then placed in 15 ml bacterial culture tubes containing 10 ml of LB medium supplemented with 50 µg/ml kanamycin, and incubated for 2 hours at 37°C with shaking at 200 rpm. Fifty microliters of each culture were plated on LB agar plates containing 20 µg/ml kanamycin, and the plates were incubated for 15-17 hrs (overnight) at 37°C. Each experiment was conducted in triplicate and repeated three times. 2.6. Diet incorporates bioassays The diet inclusion method was used to perform the bioassay. Enterobacter- (construct 2) was incorporated into the artificial diet at concentrations of 10 5 , 10 6 , 10 7 , 10 8 , and 10 9 bacterial cells/cm 2 of diet. Thirty neonates were used for each of the five concentrations of Enetrobacter- (construct 2) cells, as well as for the control group, which consisted of neonates on a diet supplemented with 10 9 Enetrobacter -(construct 3) cells/cm 2 of diet. Enterobacter cells were serially diluted in PBS (10 mM, pH 7.4), and (100 µl) of each dilution was added to the semi-synthetic diet in each well of a 12-well plate. The plates were then dried for 1 hour in a laminar airflow workbench. After that, 10 neonates were placed in each well. The lids of all diet plates were perforated with six ventilation holes (2.5 mm diameter). Insect mortality was checked at 24, 48, and 72 hrs. Mortality rates were corrected using Abbott’s formula (Abbott, 1925). In parallel, a Similar experiment was performed with E. coli K12-(construct 2) cells at concentrations of 10 5 , 10 6 , 10 7 , 10 8 , and 10 9 bacterial cells/cm 2 of diet, with a control group consisting of neonates on a diet supplemented with 10 9 E. coli K12 - (construct 3) cells/cm 2 of diet. All bioassay experiments were carried out under controlled environment conditions in the insectary at 25±5 ° C, 70±5% relative humidity, with a 14 h:10 h L/D cycle. 2.7. Leaf dip bioassay Laboratory investigations using a leaf dip bioassay approach were executed against the cotton bollworm. Tomato plants (20-25 days old) were used to collect the leaves. The harvested leaves were rinsed twice with sterile water for 10 minutes to eliminate any potential contamination. Enterobacter, Enterobacter- (construct 3), and Enterobacter- (construct 2) cultures were grown overnight (16-18 hrs, at 37°C, 200 rpm), and the cell pellets were rinsed three times with sterile PBS (pH 7.4). The bacterial cells were then resuspended in 1 ml of PBS, and the optical density was recorded at A 600 nm. For treatment, 100 µl (0.15 x 10 9 cells/ml) of Enterobacter , 100 µl (0.15 x 10 9 cells/ml) of Enterobacter- (construct 3), and 100 µl (0.10 x 10 9 cells/ml) of Enterobacter- (construct 2) were spread onto the surface of the leaves, which were then dried for 1 h in a laminar airflow workbench. The treated leaves were placed onto 0.8% solidified agarose (10 ml) in Petri dishes. Ten neonates were allowed to feed on each bacterial-treated leaf. This experiment was conducted with four replicates. Insect mortality rates were examined at 24, 48, 72, 96, and 192 hrs. The mortality rates were corrected using Abbott’s formula ( Abbott, 1925 ). 3. Results The green fluorescent protein (GFP)-encoding DNA plasmid (pGFP) revealed good expression in E . coli K12 and Enterobacter aerogenes on ampicillin-containing agar plates. These results also showed that only a small number of untransformed Enterobacter cells were present on the ampicillin-containing agar plates, showing that Enterobacter cells were resistant to ampicillin (data not shown). Previous reports have demonstrated that Enterobacter strains are susceptible to kanamycin ( Davin-Regli, 2019 ). Following this, Enterobacter aerogenes cells (carrying the KmR gene) were tested on kanamycin-(30 mg/ml)-containing agar plates. No growth of Enterobacter aerogenes was observed on the kanamycin-containing agar plates (data not shown), suggesting that Enterobacter aerogenes was susceptible to kanamycin. 3.1. Colonization of Enterobacter within Helicoverpa gut and on phyllosphere of tomato A KmR gene was introduced into the pGFP plasmid, resulting in a 4689 bp construct, referred to as construct 3 ( KmR -pGFP) ( Fig. S1 ). This construct was then transformed into Enterobacter , and pure GFP-expressing cells were obtained (data not shown). This investigation aims to develop a biological expression system for the production of heterologous proteins in Enterobacter , utilizing the central transcription enzyme (RNA polymerase, RNAP) and promoters found in natural isolates of the Enterobacterales order. The construct proved useful in colonization studies. The number of transformants of GFP expressing E . aerogenes was successfully achieved on kanamycin-containing agar plates. Fluorescence from the transformants could be easily visualized using an optical fluorescence microscope and a UV transilluminator. Under normal conditions, all transformants revealed uniform green fluorescence, as observed microscopically and with the UV transilluminator. The fluorescence intensity and optical density scales displayed typical GFP production in E . aerogenes- (construct 3), with an excitation peak at 488 nm and an emission peak at 513 nm (data not shown). Neonates were exposed to an artificial diet containing antibiotics for two days. On the third day, the neonates were transferred to an artificial diet containing Enterobacter- (construct 3) for two consecutive days. On the fifth day, the neonates were maintained on a diet without Enterobacter- (construct 3). Fecal pellets were collected and examined at 24-hrs intervals for the next 7 days to detect GFP-expressing E . aerogenes . The bacteria were observed to be capable of stable gut colonization for 7 consecutive days (up to the 5 th -instar stage), suggesting a native colonization characteristic in the gut of H. armigera ( Table 1a ). The colonization potential of GFP-expressing E . aerogenes on the surface of tomato leaves was also determined. GFP-expressing E . aerogenes was sprayed on the leaves and the number of colonies (50, 110, and 255) was counted at 24, 96, and 192 hrs, respectively ( Table 1b ). These results revealed that E . aerogenes can effectively colonize both the insect gut and the surface of the leaves. View this table: View inline View popup Download powerpoint Table 1 Colonization potential of GFP-expressing Enterobacter in the gut of insects and on the phyllosphere. (a) Colonization of the intestinal tract of the polyphagous insect H. armigera by the GFP-expressing gut microbe E. aerogenes. (b) Colonization of tomato leaves by the GFP-expressing gut microbe E. aerogenes. 3.2. Cloning and expression of cry1Ac and kanamycin genes To achieve construct 2 ( cry1Ac - KmR -pUC18), we amplified 3.0 kb cry1Ac and 1.489 kb KmR amplicons (data not shown). The 3.0 kb cry1Ac gene was cloned into the pUC18 vector ( Fig. 1a ), in-frame with the first methionine of the beta-galactosidase gene in the open reading frame (ORF), resulting in a 5435 bp construct (construct 1: cry1Ac -pUC18) (data not shown, Fig. 1b ). Download figure Open in new tab Fig. 1 A diagrammatic representation of the structure of the modified pUC18 plasmid vector. The pUC18 vector (a) was modified incorporating the cry1Ac (3000 bp, shown in cyan) gene (b) and the kanamycin resistance gene ( KmR 1489 bp, shown in purple), along with the promoter, to produce construct 2 ( cry1Ac - KmR -pUC18) (c). After then, a 1.489 kb KmR DNA fragment was cloned into construct 1 to produce a 6924 bp construct 2 ( cry1Ac - KmR -pUC18) ( Fig. 1c ). The resulting construct 2 was delivered into Enterobacter and E. coli K12. Cry1Ac expression was revealed in overnight-cultured Enterobacter -(construct 2) and E. coli K12-(construct 2) cells under the control of the lac promoter ( Fig. S2 ). The expressed ∼133 kDa Cry1Ac protein was verified by western blotting using anti-Cry1Ac antibody ( Fig. S2 ). 3.3. Diet and Leaf-Dip Incorporation Insect Bioassay A bioassay experiment was performed with a total of 420 neonates and overnight-grown bacterial cells [ Enterobacter -(construct 2), Enterobacter -(construct 3), E. coli K12-(construct 2), and E. coli K12-(construct 3)] ( Table 2 ). Five concentrations of Cry1Ac-expressing E. coli K12 cells (10 5 , 10 6 , 10 7 , 10 8 , and 10 9 cells) were individually tested, and all concentrations caused mortality in the neonates. Among the concentrations, 10 6 , 10 7 , 10 8 , and 10 9 E. coli K12 (Cry1Ac-expressing) cells displayed the highest (100%, corrected) mortality, while 10 5 cells (Cry1Ac-expressing E. coli K12 cells) showed the lowest (86%, corrected) mortality ( Table 3 ). In contrast, 10 9 cells of GFP-expressing Enterobacter and GFP-expressing E. coli K12, which served as negative controls, revealed zero (0%, corrected) mortality ( Table 2a ). Similarly, as a positive control, five concentrations of Cry1Ac-expressing Enterobacter -(construct 2) cells (10 5 , 10 6 , 10 7 , 10 8 , and 10 9 cells) were individually tested, and all concentrations caused mortality. Among these, 10 8 and 10 9 Enterobacter (Cry1Ac-expressing) cells showed the highest (100%, corrected) mortality, while 10 5 cells (Cry1Ac-expressing Enterobacter ) resulted in the lowest (79%, corrected) mortality ( Table 2b ). These outcomes suggest that the gut microbes can be modified to transfer proteins into the hosts. View this table: View inline View popup Download powerpoint Table 2 Diet incorporates insect bioassays. (a) Cry1Ac-producing E. coli K12 (construct 2) and (b) Enterobacter (construct 2) were used in the diet incorporating insect bioassay. (a) E. coli K12 (construct 3) and (b) Enterobacter (construct 3) were used as a control in this bioassay. Note: Abbott’s formula was used to calculate the corrected percentage (%) mortality: (= M observed - M control / 100 - M control ) x100. Where KmR denotes the Kanamycin resistance gene with promoter, and M represents mortality. Construct 2 refers to cry1Ac - KmR -pUC18, and Construct 3 refers to KmR -pGFP. To examine the potency of Enterobacter -(construct 2) in managing H . armigera populations on tomato leaves, we applied Enterobacter (0.15 x 10 9 cells), Enterobacter -(construct 3) (0.15 x 10 9 cells), and Enterobacter -(construct 2) (0.15 x 10 9 cells) to the surface of detached tomato leaves. A total of 120 neonates (10 neonates per leaf, 4 replicates) were released onto the leaves ( Table 3 ). The results showed 100% (corrected) mortality of neonates on Enterobacter -(construct 2)-treated leaves, compared to the controls ( Table 3 ). View this table: View inline View popup Download powerpoint Table 3 Leaf dip bioassay. Cry1Ac-producing Enterobacter (construct 2) was used to perform the leaf dip bioassay. Wild-type Enterobacter and Enterobacter (construct 3) were used as controls in this bioassay. Note: Abbott’s formula was used to calculate the corrected percentage (%) mortality: (= M observed - M control / 100 - M control ) x100. Where KmR denotes the Kanamycin resistance gene with promoter, and M represents mortality. Construct 2 refers to cry1Ac - KmR - pUC18, and Construct 3 refers to KmR -pGFP. 4. Discussions Microorganisms are found in almost every environmental niche in nature. The H . armigera larva gut contains a diverse bacterial population, including both transient species and microbes that are firmly connected with the host. Previous investigations have shown that resident bacteria are ideal candidates for biological gene delivery systems ( Pittman et al., 2008 ; Durvasula et al., 1997 ; Kuzina et al., 2002 ). Therefore, this study aimed to deliver recombinant Enterobacter expressing Cry1Ac, which can pass through the gut of Helicoverpa larvae and withstand the harsh conditions of the gut environment. This study highlights the strong relationship between Enterobacter and its host insect, offering the potential for the development of novel insect control approaches in pest management. We have previously reported ( Gayatri et al., 2012 ) diverse microbes from H. armigera . This study establishes Enterobacter as a key colonizer of the H. armigera gut. This bacterium was found in neonates, regardless of the crop variety or the locality from which they were sampled. In a previous study, Enterobacter was also detected in other insect species, further highlighting the significance of its interactions with these pests. This investigation reveals an important relationship between Enterobacter and its insect host, providing insights for the development of a promising new strategy for insect control. These outcomes confirm that, based on the principle of paratransgenesis, the symbiotic bacterium could be a promising tool for controlling H. armigera . A previous investigation described the feasibility of using Enterobacter from the insect gut to produce toxins ( Kuzina et al., 2002 ). This investigation applied a shuttle vector to genetically modify Enterobacter and observed poor production of Cyt1A in the bacterium. Additionally, similar to B. thuringiensis and E. coli, inclusion bodies (IBs) were detected, although there were notable differences in the IBs expressed in Enterobacter . However, while the Enterobacter community could perform as a delivery system, its effectiveness was limited, highlighting that the plasmid DNA designed to transfer the toxin did not function efficiently in Enterobacter . Microbes used in such strategies must be easily acquired by the insect during feeding and capable of colonizing the insect gut thereafter ( Watanabe et al., 2000 ). Therefore, it was necessary to investigate the colonization potential of Enterobacter in the gut of Helicoverpa and on the phyllosphere. Enterobacter is similar to E . coli and both of them are associated with the Enterobacteriaceae family. E. coli is currently the best-known experimental organism for genetic manipulation and serves as the workhorse for heterologous protein expression systems ( Madigan et al., 2000 ; Cronan, 2014 ). Investigations have shown that many of these expression systems are designed for protein production, particularly in engineered E . coli strains, such as those with T7 promoters or mutations. The promoters and polymerases mentioned above are not present in native Enterobacter, making its application in a paratransgenic strategy unsuitable for producing genetically modified (GM) crops containing cry genes, which have become a significant agricultural innovation in modern farming. However, Bt cotton crops producing toxins have been established and have confirmed their potential in controlling the insect pest H . armigera ( Abbas, 2018 ). There are practical challenges in producing GM Cry1Ac or related proteins in a wide variety of crops grown in the field, particularly in developing countries like India. Therefore, the adoption of paratransgenic Enterobacter strains could provide a more effective insect control strategy in the field. In this context, our outcomes show that recombinant Enterobacter containing the cry1Ac gene is capable of sustaining and colonizing healthy leaves, ultimately killing the neonates of Helicoverpa . Enterobacter aerogenes isolated from the gut of H. armigera larvae (the cotton bollworm), was engineered for use in a paratransgenic strategy. The results revealed that this engineered microbe can effectively colonize within the gut of H. armigera larvae and the leaves of tomato plants. Furthermore, the introduction of a modified plasmid carrying the cry1Ac gene under the control of the lac promoter enabled the effective production of Cry1Ac Enterobacter . When applied to H. armigera larvae, this engineered microbe induced mortality. These outcomes highlight the potential of using Cry1Ac as a paratransgenic agent for the effective control of lepidopterous insect pests. Declarations Ethics approval and consent to participate Not applicable. Consent to publish Not applicable. Availability of data and materials All data are provided in the main body of the manuscript; materials are available from the authors. All data are included as supplementary files (named as Figure S1 and S2). Competing interests The authors declare that they have no competing interests. Funding The author acknowledges the Department of Biotechnology (Ministry of Science and Technology), India and the National Agricultural Innovation Project, Indian Council of Agricultural Research, India, for financial support to this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Information Insect Resistance Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India. Supplementary materials Fig. S1 | Diagrammatic representation of the modification of the plasmid vector pGFP. The pGFP vector was modified by inserting a kanamycin resistance gene with a promoter ( KmR , 1489 bp in purple) to produce construct 3 ( KmR -pGFP). Fig. S2 | Cry1Ac protein immunoblotting analysis. Cry1Ac (∼133 kDa) expression was determined using an anti-Cry1Ac antibody. (1) Molecular weight markers. (2) E. coli K12 crude protein (control, without any plasmid DNA), cells collected from the stationary phase of growth (overnight culture). (3) E. coli K12 (construct 2) crude protein, cells collected from the log phase of growth. (4) E. coli K12 (construct 2) crude protein, cells collected from the stationary phase of growth (overnight culture). (5) Enterobacter (construct 2) crude protein, cells collected from the log phase of growth. (6) Enterobacter (construct 2) crude protein, cells collected from the stationary phase of growth (overnight culture). (7) Enterobacter crude protein (control, without any plasmid DNA), cells collected from the stationary phase of growth (overnight culture). (8) Purified recombinant Cry1Ac protein (positive control). Note: Construct 2 refers to cry1Ac - KmR -pUC18 for the expression of Cry1Ac protein. Acknowledgments The author thanks Raj K. Bhatnagar (International Centre for Genetic Engineering and Biotechnology, ICGEB, India) and Rajagopal Raman (ICGEB, New Delhi, India) for providing access to infrastructure, laboratory assistance, and financial support. Currently, Rajagopal Raman is working at the Department of Zoology, at the University of Delhi, Delhi, India. The author is also thankful to Natarajan Gayatri Priya and Vipin Singh Rana for providing laboratory help for this study at the University of Delhi, Delhi), India. References 1. ↵ Abbas , M.S.T ., 2018 . Genetically engineered (modified) crops ( Bacillus thuringiensis crops) and the world controversy on their safety . 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Share Paratransgenesis: The dynamics of engineered Enterobacter symbionts and Cry1Ac-producing Enterobacter for biocontrol of Helicoverpa insect pests in crop production Abhishek Ojha bioRxiv 2025.01.01.630992; doi: https://doi.org/10.1101/2025.01.01.630992 Share This Article: Copy Citation Tools Paratransgenesis: The dynamics of engineered Enterobacter symbionts and Cry1Ac-producing Enterobacter for biocontrol of Helicoverpa insect pests in crop production Abhishek Ojha bioRxiv 2025.01.01.630992; doi: https://doi.org/10.1101/2025.01.01.630992 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7640) Biochemistry (17706) Bioengineering (13902) Bioinformatics (41978) Biophysics (21465) Cancer Biology (18611) Cell Biology (25528) Clinical Trials (138) Developmental Biology (13387) Ecology (19920) Epidemiology (2067) Evolutionary Biology (24332) Genetics (15615) Genomics (22519) Immunology (17747) Microbiology (40424) Molecular Biology (17194) Neuroscience (88662) Paleontology (667) Pathology (2838) Pharmacology and Toxicology (4827) Physiology (7650) Plant Biology (15160) Scientific Communication and Education (2046) Synthetic Biology (4302) Systems Biology (9826) Zoology (2271)