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Potential of Photorhabdus temperata K122 bioinsecticide in protecting wheat flour against Ephestia kuehniella
Journal of Stored Products Research 53 (2013) 61e66
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Journal of Stored Products Research journal homepage: www.elsevier.com/locate/jspr
Potential of Photorhabdus temperata K122 bioinsecticide in protecting wheat flour against Ephestia kuehniella Wafa Jallouli a, *, Lobna Abdelkefi-Mesrati a, Slim Tounsi a, Samir Jaoua a, b, Nabil Zouari a, b a b
Biopesticides Team (LPAP), Centre of Biotechnology of Sfax, Sfax University, P.O. Box 1177, 3018 Sfax, Tunisia Department of Biological and Environmental Sciences, College of Arts and Sciences, Qatar University, P.O. Box 2713, Doha, Qatar
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 1 March 2013
The present study reports investigations on the insecticidal activity of the entomopathogenic bacterium Photorhabdus temperata K122 against the Mediterranean flour moth Ephestia kuehniella. Cultured in the optimized medium, P. temperata K122 cells aged 32 h exhibited 51% growth inhibition at a concentration of 9 108 cells/ml. However, culture must be prolonged up to 48 h incubation in the proteose peptone medium to reach only 28.6% inhibition. At the same concentration, no adult emergence was observed in the case of larvae feeding on wheat flour treated with the whole culture of P. temperata K122 after physical lysis. Interestingly, P. temperata K122 cells in the viable but non culturable (VBNC) state retained the same toxicity level as the culturable cells. At a high concentration of 12 108 cells/ml, 100% mortality of E. kuehniella larvae could be reached. Insect mortality is due to toxaemia as confirmed by the absence of Variants small colony (Vsm) or P. temperata colonies in E. kuehniella tissue. The investigation of the histopathological effect of P. temperata toxins on the gut of infected E. kuehniella larvae showed destruction of the gut epithelium, appearance of large cavities and cellular disintegration. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Photorhabdus temperata K122 Ephestia kuehniella Toxicity Histopathological effect
1. Introduction The Mediterranean flour moth, Ephestia kuehniella Zeller is a serious pest of stored food products, especially whole and milled grains. Ephestia kuehniella attacks also all sorts of grains, dried fruits, cocoa, nuts and almonds (Jacob and Cox, 1977). Larvae cause direct damage by feeding and they also reduce product quality by their presence and by the production of frass and webbing (Johnson et al., 1997). Moreover, E. kuehniella larvae grow completely and form pupae within the same products they infest. Its close association with human foods makes it a prime target for biological control methods other than chemical pesticides. Endotoxins from Bacillus thuringiensis Berliner are the most used biopesticides (Schnepf et al., 1998). But, tolerance of the flour moth E. kuehniella can be induced by pre-exposure to a low concentration of B. thuringiensis formulation. Tolerance correlates with an elevated immune response and can be transmitted to offspring (Rahman et al., 2004). Therefore, we have focused our research on the use of the entomopathogenic bacterium Photorhabdus temperata K122 belonging to the Enterobacteriacae family (Wang and Dowds, 1993). The strain K122 of P. temperata was isolated from the
* Corresponding author. Tel./fax: þ216 74 874 446. E-mail address: [email protected] (W. Jallouli). 0022-474X/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jspr.2013.03.001
nematode Heterorhabditis downesii K122 (Stock et al., 2002). When the infective dauer juvenile (IJ) nematode larvae carrying symbiotic Photorhabdus cells enter the insect haemocoel, the cells are directly released into the open blood system of the insect larvae. The released Photorhabdus cells propagate and kill the insect host (ffrench-Constant et al., 2003) by insecticidal toxins such as the toxin complexes (Tcs) (Waterfield et al., 2001), the “makes caterpillars floppy” (Mcf) toxins (Daborn et al., 2002), the “Photorhabdus insect-related” (Pir) toxins (Waterfield et al., 2005) or/and the “Photorhabdus virulence cassettes” (PVCs) (Yang et al., 2006). Photorhabdus temperata K122 was also shown to exhibit toxicity independently from nematode larvae against the lepidopteran olive tree pest Prays oleae (L.) (Tounsi et al., 2006) and the sugarcane stalk borer Diatraea saccharalis (F.) (Carneiro et al., 2008). Moreover, P. temperata K122 was shown to be a suitable control agent when ingested by E. kuehniella larvae (Jallouli et al., 2008). Indeed, oral toxicity, assessed as the growth inhibition of E. kuehniella larvae fed with P. temperata K122 at 4 108 cells/ml, was 27%, when cultured in the optimized medium (OM) (Jallouli et al., 2008). Several studies have been already carried out to improve toxicity of P. temperata K122 by avoiding Vsm polymorphism (Jallouli et al., 2008) and addition of sodium chloride at 5 g/l (Jallouli et al., 2011). Improvement could be achieved also by control of the dissolved oxygen concentration during the fermentation process using the OM or the complex medium.
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Indeed, toxicity reached 64.2% when two-stage oxygen supply was applied for P. temperata bioinsecticide production in the complex medium (Jallouli et al., 2012). However, there are no reports in the literature on the toxicity or histopathological effects of P. temperata K122 on E. kuehniella larvae. In the present study, we were interested to investigate the toxicity of P. temperata K122 cells, particularly when entered in the viable but non culturable (VBNC) state, the mortality elicited and the histopathological effect on the gut of infected E. kuehniella larvae. 2. Materials and methods 2.1. Strains This work was carried out with Photorhabdus temperata strain K122, Photorhabdus luminescens sp. Q167/2 (Ehlers and Niemann, 1998) offered by courtesy of Dr. Mark Blight (CNRS, GIF sur Yvette, France) and Escherichia coli strain Top 10 (Amersham, France). 2.2. Media Three media were used: Lauria-Bertani (LB) medium (Sambrook et al., 1989) with a composition of (g/l): peptone, 10; yeast extract, 5; and NaCl, 5, 2% proteose peptone medium (Fluka 29185) and the OM composed of (g/l): Na2HPO4 2H2O, 1.2; NH4Cl, 1.07; KCl, 0.35; C6H5O7Na3 2H2O, 0.5; Na2SO4, 0.28; MgCl2, 0.12; CaCl2, 0.05; NaCl, 5; FeCl3 6H2O, 0.0017; yeast extract, 10; and glucose monohydrate, 5 (Jallouli et al., 2008). Glucose stock solution (20%) was autoclaved separately and later added to the OM. The pH of the different media was adjusted to seven using HCl 1 N or NaOH 1 N before sterilization during 20 min at 121 C. 2.3. Inocula preparation The inocula were prepared as follows: A 48-h old Photorhabdus strain colony was dispersed in 3 ml of LB liquid medium and incubated overnight at 30 C. For E. coli Top 10, a 24-h old colony was dispersed in 3 ml of LB liquid medium and incubated overnight at 37 C. The culture broth was used to inoculate the culture medium at an initial optical density (OD) of 0.025 at 725 nm as previously optimized (Jallouli et al., 2008). Cultures were developed in 500 ml shake flasks containing 85 ml of the culture medium, incubated at 30 C and 37 C in a rotary shaker set at 200 rpm for Photorhabdus strains and E. coli, respectively. Shake flasks were incubated according to the culture condition for 72 h. 2.4. Biomass determination Total cell count was determined by preparing dilutions of the sample and cells were counted microscopically using a Thoma counting microscope (ZUZI) at 100-fold magnification. Biomass was determined also by measuring the OD at 725 nm with a spectrophotometer (Biorad). 2.5. Bioassays Bioassays were carried out using first-instar larvae of E. kuehniella. Ten larvae were weighed before they were transferred to a sterile Petri dish containing 1 g of wheat flour mixed with 800 ml of diluted sample at a final concentration of 9 108 cells/ml P. temperata K122 culture broth. After incubation at 26 C for 7 days, the ten-larval weight was recorded. Oral toxicity was assessed as the growth inhibition of the fed larvae with P. temperata K122, compared to growth of similar larvae number fed with the nontoxic P. luminescens Q167/2. It was calculated as follows:
Growth inhibitionð%Þ ¼ ðððGQ 167=2 GK122Þ=GQ 167=2ÞÞ 100 GQ167/2: (weight of the ten larvae fed with P. luminescens Q167/2 after 7 days) (weight of the ten larvae fed with P. luminescens Q167/2 at t ¼ 0). GK122: (weight of the ten larvae fed with P. temperata K122 after 7 days) (weight of the ten larvae fed with P. temperata K122 at t ¼ 0). The values presented in the results section are the average of the weight of 30 larvae collected from three replicates carried out with 10 larvae each. 2.6. Bacterial colonization The number of recoverable bacteria within infected tissues of E. kuehniella larvae was determined after 7 days of incubation at 26 C. Replicates of ten fed larvae with P. temperata K122 and P. luminescens Q167/2 were dissected after treatment. Each larva was surface sterilized with 70% ethanol and then bled to collect the internal organs. The gut and the fat body were homogenized in PBS using a hand-held Potter homogenizer. To determine the number of recoverable bacteria, serial dilutions of tissue homogenates were prepared with PBS and plated onto LB agar and LB agar supplemented with catalase at 2000 Units/plate to enhance the recovery of P. temperata K122 in the VBNC state (Jallouli et al., 2010). 2.7. Preparation and sectioning of insects tissues After exposure to P. temperata K122 cells at a concentration of 12 108 cells/ml for 5 days, first-instar larvae of E. kuehniella were chilled on ice for 15 min. The guts were then excised and placed in 10% formaldehyde then dehydrated by increasing ethanol concentration, rinsed with 100% toluene, and embedded in paraffin wax. Sections (5 mm) were placed in carriers loaded with a mix of 1.5% egg albumin and 3% glycerol in distilled water. For histopathological localization of toxins effect, the sections already de-paraffinated by 100% toluene were stained with hematoxylineosin as reported by Ruiz et al. (2004). 2.8. Statistical analysis All the results related to determination of bioassays and bacterial colonization were the average of three replicates of three separate experiments. They were statistically analyzed by SPSS software (version 100) using Duncan test performed after analysis of variance (ANOVA). 3. Results 3.1. Comparison of P. temperata K122 pathogenicity against E. kuehniella with P. luminescens Q167/2 and E. coli Top 10 Toxicity evaluation of P. temperata strain K122, P. luminescens strain Q167/2 and E. coli strain Top 10 was carried out by using the proteose peptone medium known to be a suitable medium for toxins production (Lining et al., 1999). By using similar cell counts of 9 108 cells/ml, it was shown that P. temperata cells exhibited a maximal growth inhibition of 28.6% after 48 h of incubation (Fig. 1). However, the strains Q167/2 and Top 10 were not toxic against E. kuehniella larvae even at high cell densities of 12 108 cells/ml. To confirm non toxicity of these strains, different culture fractions were prepared (supernatant, bacterial culture, washed bacterial cells and finally bacterial culture after physical lysis) and tested for
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Fig. 1. Comparison of P. temperata K122 pathogenicity against E. kuehniella larvae with P. luminescens Q167/2 and E. coli Top 10. (-)E. kuehniella larvae fed with P. luminescens Q167/ 2; (,) E. kuehniella larvae fed with P. temperata K122; ( ) E. kuehniella larvae fed with E. coli Top 10. Each value represents the mean S.D. from three replicates of three separate experiments (n ¼ 9).
their toxicity. A 100% value for the relative weight gain was obtained after an incubation period of 7 days of E. kuehniella fed larvae, resembling control larvae fed on wheat flour treated by the sterile proteose peptone medium (data not shown). The same result was obtained after 14 days incubation of E. kuehniella larvae at 26 C. Consequently, the first instars E. kuehniella larvae fed on diet treated with bacterial culture of P. luminescens Q167/2 at 12 108 cells/ml were used as negative control. 3.2. Effect of the production medium on P. temperata K122 oral toxicity Toxin production was carried out using two different media, the proteose peptone medium and the OM ensuring P. temperata K122 nutritional requirements, at the same cell counts of 9 108 cells/ml (Fig. 2). Variance analysis showed a statistically significant (P < 0.05) difference between the toxicity of P. temperata K122 cells cultured in two media. Indeed, growth inhibition of 19.3% was reached after 32 h incubation of P. temperata K122 cells in the proteose peptone medium. In this medium, culture had to be prolonged up to 48 h to reach only 28.6% growth inhibition. In contrast, when P. temperata K122 cells were cultured in the OM, a higher toxicity of 51% was obtained after 32 h of incubation. Interestingly, entry in the VBNC state from 48 h of cultivation in both media did not affect P. temperata K122 toxicity. Indeed, P. temperata cells retained the same toxicity level of 49.5 and 28.6%, until 72 h of cultivation, when cultured in the OM and the proteose peptone medium, respectively. Toxicity evaluation of P. luminescens Q167/2 cells cultured in the OM showed no inhibition of E. kuehniella larvae growth. Consequently, further experiments were carried out using the OM for bioinsecticide production of both strains. 3.3. Origin of P. temperata K122 pathogenicity To investigate P. temperata K122 oral toxicity origin against the first instar E. kuehniella larvae, toxicity of different culture fractions
(supernatant, heated supernatant, bacterial culture, washed bacterial cells and finally bacterial culture after physical lysis) was evaluated at different incubation times. As shown in Fig. 3, the supernatant exhibited the lowest growth inhibition until 32 h of incubation, followed by the washed bacterial cells, the whole culture and finally the whole culture after physical lysis. After 32 h of incubation, toxicity of the supernatant increased significantly to reach the interesting level of 49.6% at 72 h of incubation. However, a decreased growth inhibition of insect larvae was observed by feeding E. kuehniella larvae on wheat flour treated with washed bacterial cells. Interestingly, the highest oral toxicity was obtained with the whole culture after physical lysis after the different incubation times tested. Ephestia kuehniella larvae fed on diet treated with heated supernatant exhibited almost no growth inhibition after an incubation period of 7 days, similar to those fed with P. luminescens Q167/2. However, the reproduction rate of growth-inhibited E. kuehniella larvae was very reduced in the different P. temperata K122 culture fractions tested. Interestingly, no adult emergence was observed in the case of larvae feeding on diet treated with the whole P. temperata K122 culture broth after physical lysis. However, the percentage of adults emerging from E. kuehniella larvae feeding on wheat flour treated with the whole bacterial culture of P. luminescens Q167/2 (12 108 cells/ml) varied between 93 and 98% after two months. 3.4. Effect of P. temperata K122 cells concentration on growth inhibition of E. kuehniella larvae Growth inhibition of E. kuehniella larvae exposed to varying concentrations of P. temperata K122 cells is shown in Fig. 4. By increasing P. temperata cells concentration, toxicity was improved. A profound growth inhibition was observed at a concentration of 16 108 cells/ml. Interestingly, the growth of the first-instar E. kuehniella larvae was entirely inhibited by a concentration of 12 108 cells/ml.
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Fig. 2. Effect of the production medium on P. temperata K122 oral toxicity. (-) OM; (,) proteose peptone medium. Each value represents the mean S.D. from three replicates of three separate experiments (n ¼ 9).
To further examine E. kuehniella larvae death origin, CFU counts determination was carried out in the insect tissue (gut þ fat body). After plating diluted homogenates tissue of infected E. kuehniella larvae on LB plates and LB plates supplemented with catalase at 2000 Units/plate, no colonies corresponding to P. temperata K122 or Vsm forms were detected.
3.5. Histopathological effects of P. temperata K122 in E. kuehniella larvae After 5 days of incubation at 26 C, the histopathological effect on the first-instar larvae of E. kuehniella fed on P. temperata K122 toxins treated diet was examined. As shown in Fig. 5A, oral toxicity
Fig. 3. Study of P. temperata K122 oral toxicity in different culture medium fractions. (-) Heated supernatant; (,) whole culture; ( ) supernatant; ( ) cells; ( ) whole culture after physical lysis. Each value represents the mean S.D. from three replicates of three separate experiments (n ¼ 9).
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Fig. 4. Effect of P. temperata K122 cells concentration on growth inhibition of E. kuehniella larvae. Each value represents the mean S.D. from three replicates of three separate experiments (n ¼ 9).
of P. temperata K122 resulted in extensive damage of the gut epithelium, since large cavities appeared in the latter and only a disorganized layer of cell membranes remained. Moreover, cellular debris appeared in the gut lumen. In contrast, the midgut of E. kuehniella treated with the non-toxic P. luminescens Q167/2 showed uniform morphology and well-defined epithelial cells (Fig. 5B). 4. Discussion In this study, toxicity of P. temperata K122 against E. kuehniella larvae was investigated. Indeed, P. temperata K122 is shown to be highly virulent to E. kuehniella larvae when used at a concentration of 12 108 cells/ml since 100% mortality was recorded after 32 h of incubation in the OM. When a lower concentration was used (9 108 cells/ml), growth inhibition of feeding first-instar E. kuehniella larvae was 51%. However, when the proteose peptone medium was used for P. temperata K122 toxins production, 48 h incubation produced only 28.6% growth inhibition at the same cell concentration. Similarly, Bowen and Ensign (1998) have reported that
maximal toxicity level of P. luminescens W14, Hm and NC-19, against Manduca sexta (L.) larvae was reached at 48 he72 h incubation in the proteose peptone medium. Consequently, the OM not only ensured P. temperata K122 nutritional requirements and overcoming of Vsm polymorphism, but was also adequate for P. temperata K122 toxins production. Monitoring oral toxicity during different incubation times of P. temperata K122 cells cultured in the OM, revealed that oral toxicity was not affected by existence of VBNC state. Indeed, although almost all P. temperata K122 cells at age 48 h had entered the VBNC state, they retained the same toxicity level than those at 32 h. Absence of P. temperata K122 or Vsm colonies in infected E. kuehniella tissue clearly evidenced the involvement of toxaemia in E. kuehniella mortality. The same mechanism was reported to be the origin of P. oleae larval mortality when infected by P. temperata K122 (Tounsi et al., 2006). Mortality of this olive moth can occur even in absence of tca or tcd genes products, missing in P. temperata K122, and necessary for oral toxicity against M. sexta (Waterfield et al., 2001). Moreover, Gerritsen et al. (2005) showed that only
Fig. 5. Histopathological effects of Photorhabdus strains on the gut of E. kuehniella larvae. (A) Histopathological effect of P. temperata K122; (B) Histopathological effect of P. luminescens Q167/2. Lu, lumen; Am, apical membrane; Bm, basal membrane; V, vacuole formation. Magnification: 40. Arrows indicate vesicle formation.
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the North American P. temperata strains were orally toxic to thrips. These results suggest that oral toxicity depends on both the nature of toxins produced by P. temperata strains and the insect host. The heat lability of P. temperata K122 supernatant indicates that oral toxicity is associated with protein. Insecticidal activity against E. kuehniella is probably caused by toxins like the Tcc toxins (Waterfield et al., 2001), or/and Mcf toxins (Daborn et al., 2002). Toxin secretion begins in the late exponential growth phase (24 h) and reaches an interesting level in the stationary growth phase (48 h). On the other hand, reduction of adult emergence in different culture fractions tested at a concentration of 9 108 cells/ml clearly showed that even if E. kuehniella larvae do not take up enough toxins to get 100% mortality, toxins could affect the reproduction rate and give a reduced population in the next generation. Consequently, combined effect of growth inhibition and reduction of fecundity might be enough for the control of E. kuehniella larvae in the stored products at this lower concentration. This result is on accordance with those found by Gerritsen et al. (2005) demonstrating that P. temperata toxin has a double effect for the control of Frankliniella occidentalis (Pergande), not only killing adult thrips but also reducing the next generations by reducing the reproduction rate. Investigation of the histopathological effects on E. kuehniella larvae showed clearly that P. temperata toxins attack the midgut of E. kuehniella larvae, causing disruption of epithelial cells and packed debris in the lumen. This histopathological effect resembles that described for other toxins that are active in the gut (Blackburn et al., 1998, 2005). This indicated that P. temperata K122 toxins are as potent as the delta endotoxins (Rouis et al., 2007) and vegetative insecticidal protein (Vip) toxins (Abdelkefi-Mesrati et al., 2011) of B. thuringiensis. Therefore, P. temperata K122 might provide a useful alternative to the deployment of B. thuringiensis toxins especially as resistance to the latter has been reported in several insect field populations (Huang et al., 1999; Liu et al., 1999). Acknowledgments This work was supported by grants from the “Ministère de l’Enseignement Supérieur et de la Recherche Scientifique”. References Abdelkefi-Mesrati, L., Boukedi, H., Dammak-Karray, M., Sellami-Boudawara, T., Jaoua, S., Tounsi, S., 2011. Study of the Bacillus thuringiensis Vip3Aa16 histopathological effects and determination of its putative binding proteins in the midgut of Spodoptera littoralis. Journal of Invertebrate Pathology 106, 250e254. Blackburn, M., Golubeva, E., Bowen, D., ffrench-Constant, R.H., 1998. A novel insecticidal toxin from Photorhabdus luminescens, Toxin complex a (Tca), and its histopathological effects on the midgut of Manduca sexta. Applied and Environmental Microbiology 64, 3036e3041. Blackburn, M.B., Domek, J.M., Gelman, D.B., Hu, J.S., 2005. The broadly insecticidal Photorhabdus luminescens toxin complex a (Tca): activity against the Colorado potato beetle, Leptinotarsa decemlineata, and sweet potato whitefly, Bemisia tabaci. Journal of Insect Science 5, 1e11. Bowen, D.J., Ensign, C.J., 1998. Characterization of a high molecular weight insecticidal protein complex produced by the entomopathogenic bacterium Photorhabdus luminescens. Applied and Environmental Microbiology 64, 3029e3035. Carneiro, C.N.B., DaMatta, R.A., Samuels, R.I., Silva, C.P., 2008. Effects of entomopathogenic bacterium Photorhabdus temperata infection on the intestinal microbiota of the sugarcane stalk borer Diatraea saccharalis (Lepidoptera: Crambidae). Journal of Invertebrate Pathology 99, 87e91.
Daborn, P.J., Waterfield, N., Silva, C.P., Au, C.P.Y., Sharma, S., ffrench-Constant, R.H., 2002. A single Photorhabdus gene, makes caterpillar floppy (mcf), allows Escherichia coli to persist within and kill insects. Proceedings of the National Academy of Sciences of the United States of America 99, 10742e10747. Ehlers, R.U., Niemann, I., 1998. Molecular identification of Photorhabdus luminescens strains by amplification of specific fragments of the 16S ribosomal DNA. Systematic and Applied Microbiology 21, 509e519. ffrench-Constant, R.H., Waterfield, N., Daborn, P., Joyce, S., Bennet, H., Au, C., Dowling, A., Boundy, S., Reynolds, S., Clarke, D., 2003. Photorhabdus: towards a functional genomic analysis of a symbiont and pathogen. FEMS Microbiology Review 26, 433e456. Gerritsen, L.J.M., Georgieva, J., Wiegers, G.L., 2005. Oral toxicity of Photorhabdus toxins against thrips species. Journal of Invertebrate Pathology 88, 207e211. 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Potential of Photorhabdus temperata K122 bioinsecticide in protecting wheat flour against Ephestia kuehniella Wafa Jallouli a, *, Lobna Abdelkefi-Mesrati a, Slim Tounsi a, Samir Jaoua a, b, Nabil Zouari a, b a b
Biopesticides Team (LPAP), Centre of Biotechnology of Sfax, Sfax University, P.O. Box 1177, 3018 Sfax, Tunisia Department of Biological and Environmental Sciences, College of Arts and Sciences, Qatar University, P.O. Box 2713, Doha, Qatar
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 1 March 2013
The present study reports investigations on the insecticidal activity of the entomopathogenic bacterium Photorhabdus temperata K122 against the Mediterranean flour moth Ephestia kuehniella. Cultured in the optimized medium, P. temperata K122 cells aged 32 h exhibited 51% growth inhibition at a concentration of 9 108 cells/ml. However, culture must be prolonged up to 48 h incubation in the proteose peptone medium to reach only 28.6% inhibition. At the same concentration, no adult emergence was observed in the case of larvae feeding on wheat flour treated with the whole culture of P. temperata K122 after physical lysis. Interestingly, P. temperata K122 cells in the viable but non culturable (VBNC) state retained the same toxicity level as the culturable cells. At a high concentration of 12 108 cells/ml, 100% mortality of E. kuehniella larvae could be reached. Insect mortality is due to toxaemia as confirmed by the absence of Variants small colony (Vsm) or P. temperata colonies in E. kuehniella tissue. The investigation of the histopathological effect of P. temperata toxins on the gut of infected E. kuehniella larvae showed destruction of the gut epithelium, appearance of large cavities and cellular disintegration. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Photorhabdus temperata K122 Ephestia kuehniella Toxicity Histopathological effect
1. Introduction The Mediterranean flour moth, Ephestia kuehniella Zeller is a serious pest of stored food products, especially whole and milled grains. Ephestia kuehniella attacks also all sorts of grains, dried fruits, cocoa, nuts and almonds (Jacob and Cox, 1977). Larvae cause direct damage by feeding and they also reduce product quality by their presence and by the production of frass and webbing (Johnson et al., 1997). Moreover, E. kuehniella larvae grow completely and form pupae within the same products they infest. Its close association with human foods makes it a prime target for biological control methods other than chemical pesticides. Endotoxins from Bacillus thuringiensis Berliner are the most used biopesticides (Schnepf et al., 1998). But, tolerance of the flour moth E. kuehniella can be induced by pre-exposure to a low concentration of B. thuringiensis formulation. Tolerance correlates with an elevated immune response and can be transmitted to offspring (Rahman et al., 2004). Therefore, we have focused our research on the use of the entomopathogenic bacterium Photorhabdus temperata K122 belonging to the Enterobacteriacae family (Wang and Dowds, 1993). The strain K122 of P. temperata was isolated from the
* Corresponding author. Tel./fax: þ216 74 874 446. E-mail address: [email protected] (W. Jallouli). 0022-474X/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jspr.2013.03.001
nematode Heterorhabditis downesii K122 (Stock et al., 2002). When the infective dauer juvenile (IJ) nematode larvae carrying symbiotic Photorhabdus cells enter the insect haemocoel, the cells are directly released into the open blood system of the insect larvae. The released Photorhabdus cells propagate and kill the insect host (ffrench-Constant et al., 2003) by insecticidal toxins such as the toxin complexes (Tcs) (Waterfield et al., 2001), the “makes caterpillars floppy” (Mcf) toxins (Daborn et al., 2002), the “Photorhabdus insect-related” (Pir) toxins (Waterfield et al., 2005) or/and the “Photorhabdus virulence cassettes” (PVCs) (Yang et al., 2006). Photorhabdus temperata K122 was also shown to exhibit toxicity independently from nematode larvae against the lepidopteran olive tree pest Prays oleae (L.) (Tounsi et al., 2006) and the sugarcane stalk borer Diatraea saccharalis (F.) (Carneiro et al., 2008). Moreover, P. temperata K122 was shown to be a suitable control agent when ingested by E. kuehniella larvae (Jallouli et al., 2008). Indeed, oral toxicity, assessed as the growth inhibition of E. kuehniella larvae fed with P. temperata K122 at 4 108 cells/ml, was 27%, when cultured in the optimized medium (OM) (Jallouli et al., 2008). Several studies have been already carried out to improve toxicity of P. temperata K122 by avoiding Vsm polymorphism (Jallouli et al., 2008) and addition of sodium chloride at 5 g/l (Jallouli et al., 2011). Improvement could be achieved also by control of the dissolved oxygen concentration during the fermentation process using the OM or the complex medium.
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Indeed, toxicity reached 64.2% when two-stage oxygen supply was applied for P. temperata bioinsecticide production in the complex medium (Jallouli et al., 2012). However, there are no reports in the literature on the toxicity or histopathological effects of P. temperata K122 on E. kuehniella larvae. In the present study, we were interested to investigate the toxicity of P. temperata K122 cells, particularly when entered in the viable but non culturable (VBNC) state, the mortality elicited and the histopathological effect on the gut of infected E. kuehniella larvae. 2. Materials and methods 2.1. Strains This work was carried out with Photorhabdus temperata strain K122, Photorhabdus luminescens sp. Q167/2 (Ehlers and Niemann, 1998) offered by courtesy of Dr. Mark Blight (CNRS, GIF sur Yvette, France) and Escherichia coli strain Top 10 (Amersham, France). 2.2. Media Three media were used: Lauria-Bertani (LB) medium (Sambrook et al., 1989) with a composition of (g/l): peptone, 10; yeast extract, 5; and NaCl, 5, 2% proteose peptone medium (Fluka 29185) and the OM composed of (g/l): Na2HPO4 2H2O, 1.2; NH4Cl, 1.07; KCl, 0.35; C6H5O7Na3 2H2O, 0.5; Na2SO4, 0.28; MgCl2, 0.12; CaCl2, 0.05; NaCl, 5; FeCl3 6H2O, 0.0017; yeast extract, 10; and glucose monohydrate, 5 (Jallouli et al., 2008). Glucose stock solution (20%) was autoclaved separately and later added to the OM. The pH of the different media was adjusted to seven using HCl 1 N or NaOH 1 N before sterilization during 20 min at 121 C. 2.3. Inocula preparation The inocula were prepared as follows: A 48-h old Photorhabdus strain colony was dispersed in 3 ml of LB liquid medium and incubated overnight at 30 C. For E. coli Top 10, a 24-h old colony was dispersed in 3 ml of LB liquid medium and incubated overnight at 37 C. The culture broth was used to inoculate the culture medium at an initial optical density (OD) of 0.025 at 725 nm as previously optimized (Jallouli et al., 2008). Cultures were developed in 500 ml shake flasks containing 85 ml of the culture medium, incubated at 30 C and 37 C in a rotary shaker set at 200 rpm for Photorhabdus strains and E. coli, respectively. Shake flasks were incubated according to the culture condition for 72 h. 2.4. Biomass determination Total cell count was determined by preparing dilutions of the sample and cells were counted microscopically using a Thoma counting microscope (ZUZI) at 100-fold magnification. Biomass was determined also by measuring the OD at 725 nm with a spectrophotometer (Biorad). 2.5. Bioassays Bioassays were carried out using first-instar larvae of E. kuehniella. Ten larvae were weighed before they were transferred to a sterile Petri dish containing 1 g of wheat flour mixed with 800 ml of diluted sample at a final concentration of 9 108 cells/ml P. temperata K122 culture broth. After incubation at 26 C for 7 days, the ten-larval weight was recorded. Oral toxicity was assessed as the growth inhibition of the fed larvae with P. temperata K122, compared to growth of similar larvae number fed with the nontoxic P. luminescens Q167/2. It was calculated as follows:
Growth inhibitionð%Þ ¼ ðððGQ 167=2 GK122Þ=GQ 167=2ÞÞ 100 GQ167/2: (weight of the ten larvae fed with P. luminescens Q167/2 after 7 days) (weight of the ten larvae fed with P. luminescens Q167/2 at t ¼ 0). GK122: (weight of the ten larvae fed with P. temperata K122 after 7 days) (weight of the ten larvae fed with P. temperata K122 at t ¼ 0). The values presented in the results section are the average of the weight of 30 larvae collected from three replicates carried out with 10 larvae each. 2.6. Bacterial colonization The number of recoverable bacteria within infected tissues of E. kuehniella larvae was determined after 7 days of incubation at 26 C. Replicates of ten fed larvae with P. temperata K122 and P. luminescens Q167/2 were dissected after treatment. Each larva was surface sterilized with 70% ethanol and then bled to collect the internal organs. The gut and the fat body were homogenized in PBS using a hand-held Potter homogenizer. To determine the number of recoverable bacteria, serial dilutions of tissue homogenates were prepared with PBS and plated onto LB agar and LB agar supplemented with catalase at 2000 Units/plate to enhance the recovery of P. temperata K122 in the VBNC state (Jallouli et al., 2010). 2.7. Preparation and sectioning of insects tissues After exposure to P. temperata K122 cells at a concentration of 12 108 cells/ml for 5 days, first-instar larvae of E. kuehniella were chilled on ice for 15 min. The guts were then excised and placed in 10% formaldehyde then dehydrated by increasing ethanol concentration, rinsed with 100% toluene, and embedded in paraffin wax. Sections (5 mm) were placed in carriers loaded with a mix of 1.5% egg albumin and 3% glycerol in distilled water. For histopathological localization of toxins effect, the sections already de-paraffinated by 100% toluene were stained with hematoxylineosin as reported by Ruiz et al. (2004). 2.8. Statistical analysis All the results related to determination of bioassays and bacterial colonization were the average of three replicates of three separate experiments. They were statistically analyzed by SPSS software (version 100) using Duncan test performed after analysis of variance (ANOVA). 3. Results 3.1. Comparison of P. temperata K122 pathogenicity against E. kuehniella with P. luminescens Q167/2 and E. coli Top 10 Toxicity evaluation of P. temperata strain K122, P. luminescens strain Q167/2 and E. coli strain Top 10 was carried out by using the proteose peptone medium known to be a suitable medium for toxins production (Lining et al., 1999). By using similar cell counts of 9 108 cells/ml, it was shown that P. temperata cells exhibited a maximal growth inhibition of 28.6% after 48 h of incubation (Fig. 1). However, the strains Q167/2 and Top 10 were not toxic against E. kuehniella larvae even at high cell densities of 12 108 cells/ml. To confirm non toxicity of these strains, different culture fractions were prepared (supernatant, bacterial culture, washed bacterial cells and finally bacterial culture after physical lysis) and tested for
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Fig. 1. Comparison of P. temperata K122 pathogenicity against E. kuehniella larvae with P. luminescens Q167/2 and E. coli Top 10. (-)E. kuehniella larvae fed with P. luminescens Q167/ 2; (,) E. kuehniella larvae fed with P. temperata K122; ( ) E. kuehniella larvae fed with E. coli Top 10. Each value represents the mean S.D. from three replicates of three separate experiments (n ¼ 9).
their toxicity. A 100% value for the relative weight gain was obtained after an incubation period of 7 days of E. kuehniella fed larvae, resembling control larvae fed on wheat flour treated by the sterile proteose peptone medium (data not shown). The same result was obtained after 14 days incubation of E. kuehniella larvae at 26 C. Consequently, the first instars E. kuehniella larvae fed on diet treated with bacterial culture of P. luminescens Q167/2 at 12 108 cells/ml were used as negative control. 3.2. Effect of the production medium on P. temperata K122 oral toxicity Toxin production was carried out using two different media, the proteose peptone medium and the OM ensuring P. temperata K122 nutritional requirements, at the same cell counts of 9 108 cells/ml (Fig. 2). Variance analysis showed a statistically significant (P < 0.05) difference between the toxicity of P. temperata K122 cells cultured in two media. Indeed, growth inhibition of 19.3% was reached after 32 h incubation of P. temperata K122 cells in the proteose peptone medium. In this medium, culture had to be prolonged up to 48 h to reach only 28.6% growth inhibition. In contrast, when P. temperata K122 cells were cultured in the OM, a higher toxicity of 51% was obtained after 32 h of incubation. Interestingly, entry in the VBNC state from 48 h of cultivation in both media did not affect P. temperata K122 toxicity. Indeed, P. temperata cells retained the same toxicity level of 49.5 and 28.6%, until 72 h of cultivation, when cultured in the OM and the proteose peptone medium, respectively. Toxicity evaluation of P. luminescens Q167/2 cells cultured in the OM showed no inhibition of E. kuehniella larvae growth. Consequently, further experiments were carried out using the OM for bioinsecticide production of both strains. 3.3. Origin of P. temperata K122 pathogenicity To investigate P. temperata K122 oral toxicity origin against the first instar E. kuehniella larvae, toxicity of different culture fractions
(supernatant, heated supernatant, bacterial culture, washed bacterial cells and finally bacterial culture after physical lysis) was evaluated at different incubation times. As shown in Fig. 3, the supernatant exhibited the lowest growth inhibition until 32 h of incubation, followed by the washed bacterial cells, the whole culture and finally the whole culture after physical lysis. After 32 h of incubation, toxicity of the supernatant increased significantly to reach the interesting level of 49.6% at 72 h of incubation. However, a decreased growth inhibition of insect larvae was observed by feeding E. kuehniella larvae on wheat flour treated with washed bacterial cells. Interestingly, the highest oral toxicity was obtained with the whole culture after physical lysis after the different incubation times tested. Ephestia kuehniella larvae fed on diet treated with heated supernatant exhibited almost no growth inhibition after an incubation period of 7 days, similar to those fed with P. luminescens Q167/2. However, the reproduction rate of growth-inhibited E. kuehniella larvae was very reduced in the different P. temperata K122 culture fractions tested. Interestingly, no adult emergence was observed in the case of larvae feeding on diet treated with the whole P. temperata K122 culture broth after physical lysis. However, the percentage of adults emerging from E. kuehniella larvae feeding on wheat flour treated with the whole bacterial culture of P. luminescens Q167/2 (12 108 cells/ml) varied between 93 and 98% after two months. 3.4. Effect of P. temperata K122 cells concentration on growth inhibition of E. kuehniella larvae Growth inhibition of E. kuehniella larvae exposed to varying concentrations of P. temperata K122 cells is shown in Fig. 4. By increasing P. temperata cells concentration, toxicity was improved. A profound growth inhibition was observed at a concentration of 16 108 cells/ml. Interestingly, the growth of the first-instar E. kuehniella larvae was entirely inhibited by a concentration of 12 108 cells/ml.
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Fig. 2. Effect of the production medium on P. temperata K122 oral toxicity. (-) OM; (,) proteose peptone medium. Each value represents the mean S.D. from three replicates of three separate experiments (n ¼ 9).
To further examine E. kuehniella larvae death origin, CFU counts determination was carried out in the insect tissue (gut þ fat body). After plating diluted homogenates tissue of infected E. kuehniella larvae on LB plates and LB plates supplemented with catalase at 2000 Units/plate, no colonies corresponding to P. temperata K122 or Vsm forms were detected.
3.5. Histopathological effects of P. temperata K122 in E. kuehniella larvae After 5 days of incubation at 26 C, the histopathological effect on the first-instar larvae of E. kuehniella fed on P. temperata K122 toxins treated diet was examined. As shown in Fig. 5A, oral toxicity
Fig. 3. Study of P. temperata K122 oral toxicity in different culture medium fractions. (-) Heated supernatant; (,) whole culture; ( ) supernatant; ( ) cells; ( ) whole culture after physical lysis. Each value represents the mean S.D. from three replicates of three separate experiments (n ¼ 9).
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Fig. 4. Effect of P. temperata K122 cells concentration on growth inhibition of E. kuehniella larvae. Each value represents the mean S.D. from three replicates of three separate experiments (n ¼ 9).
of P. temperata K122 resulted in extensive damage of the gut epithelium, since large cavities appeared in the latter and only a disorganized layer of cell membranes remained. Moreover, cellular debris appeared in the gut lumen. In contrast, the midgut of E. kuehniella treated with the non-toxic P. luminescens Q167/2 showed uniform morphology and well-defined epithelial cells (Fig. 5B). 4. Discussion In this study, toxicity of P. temperata K122 against E. kuehniella larvae was investigated. Indeed, P. temperata K122 is shown to be highly virulent to E. kuehniella larvae when used at a concentration of 12 108 cells/ml since 100% mortality was recorded after 32 h of incubation in the OM. When a lower concentration was used (9 108 cells/ml), growth inhibition of feeding first-instar E. kuehniella larvae was 51%. However, when the proteose peptone medium was used for P. temperata K122 toxins production, 48 h incubation produced only 28.6% growth inhibition at the same cell concentration. Similarly, Bowen and Ensign (1998) have reported that
maximal toxicity level of P. luminescens W14, Hm and NC-19, against Manduca sexta (L.) larvae was reached at 48 he72 h incubation in the proteose peptone medium. Consequently, the OM not only ensured P. temperata K122 nutritional requirements and overcoming of Vsm polymorphism, but was also adequate for P. temperata K122 toxins production. Monitoring oral toxicity during different incubation times of P. temperata K122 cells cultured in the OM, revealed that oral toxicity was not affected by existence of VBNC state. Indeed, although almost all P. temperata K122 cells at age 48 h had entered the VBNC state, they retained the same toxicity level than those at 32 h. Absence of P. temperata K122 or Vsm colonies in infected E. kuehniella tissue clearly evidenced the involvement of toxaemia in E. kuehniella mortality. The same mechanism was reported to be the origin of P. oleae larval mortality when infected by P. temperata K122 (Tounsi et al., 2006). Mortality of this olive moth can occur even in absence of tca or tcd genes products, missing in P. temperata K122, and necessary for oral toxicity against M. sexta (Waterfield et al., 2001). Moreover, Gerritsen et al. (2005) showed that only
Fig. 5. Histopathological effects of Photorhabdus strains on the gut of E. kuehniella larvae. (A) Histopathological effect of P. temperata K122; (B) Histopathological effect of P. luminescens Q167/2. Lu, lumen; Am, apical membrane; Bm, basal membrane; V, vacuole formation. Magnification: 40. Arrows indicate vesicle formation.
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the North American P. temperata strains were orally toxic to thrips. These results suggest that oral toxicity depends on both the nature of toxins produced by P. temperata strains and the insect host. The heat lability of P. temperata K122 supernatant indicates that oral toxicity is associated with protein. Insecticidal activity against E. kuehniella is probably caused by toxins like the Tcc toxins (Waterfield et al., 2001), or/and Mcf toxins (Daborn et al., 2002). Toxin secretion begins in the late exponential growth phase (24 h) and reaches an interesting level in the stationary growth phase (48 h). On the other hand, reduction of adult emergence in different culture fractions tested at a concentration of 9 108 cells/ml clearly showed that even if E. kuehniella larvae do not take up enough toxins to get 100% mortality, toxins could affect the reproduction rate and give a reduced population in the next generation. Consequently, combined effect of growth inhibition and reduction of fecundity might be enough for the control of E. kuehniella larvae in the stored products at this lower concentration. This result is on accordance with those found by Gerritsen et al. (2005) demonstrating that P. temperata toxin has a double effect for the control of Frankliniella occidentalis (Pergande), not only killing adult thrips but also reducing the next generations by reducing the reproduction rate. Investigation of the histopathological effects on E. kuehniella larvae showed clearly that P. temperata toxins attack the midgut of E. kuehniella larvae, causing disruption of epithelial cells and packed debris in the lumen. This histopathological effect resembles that described for other toxins that are active in the gut (Blackburn et al., 1998, 2005). This indicated that P. temperata K122 toxins are as potent as the delta endotoxins (Rouis et al., 2007) and vegetative insecticidal protein (Vip) toxins (Abdelkefi-Mesrati et al., 2011) of B. thuringiensis. Therefore, P. temperata K122 might provide a useful alternative to the deployment of B. thuringiensis toxins especially as resistance to the latter has been reported in several insect field populations (Huang et al., 1999; Liu et al., 1999). Acknowledgments This work was supported by grants from the “Ministère de l’Enseignement Supérieur et de la Recherche Scientifique”. References Abdelkefi-Mesrati, L., Boukedi, H., Dammak-Karray, M., Sellami-Boudawara, T., Jaoua, S., Tounsi, S., 2011. Study of the Bacillus thuringiensis Vip3Aa16 histopathological effects and determination of its putative binding proteins in the midgut of Spodoptera littoralis. Journal of Invertebrate Pathology 106, 250e254. Blackburn, M., Golubeva, E., Bowen, D., ffrench-Constant, R.H., 1998. A novel insecticidal toxin from Photorhabdus luminescens, Toxin complex a (Tca), and its histopathological effects on the midgut of Manduca sexta. Applied and Environmental Microbiology 64, 3036e3041. Blackburn, M.B., Domek, J.M., Gelman, D.B., Hu, J.S., 2005. The broadly insecticidal Photorhabdus luminescens toxin complex a (Tca): activity against the Colorado potato beetle, Leptinotarsa decemlineata, and sweet potato whitefly, Bemisia tabaci. Journal of Insect Science 5, 1e11. Bowen, D.J., Ensign, C.J., 1998. Characterization of a high molecular weight insecticidal protein complex produced by the entomopathogenic bacterium Photorhabdus luminescens. Applied and Environmental Microbiology 64, 3029e3035. Carneiro, C.N.B., DaMatta, R.A., Samuels, R.I., Silva, C.P., 2008. Effects of entomopathogenic bacterium Photorhabdus temperata infection on the intestinal microbiota of the sugarcane stalk borer Diatraea saccharalis (Lepidoptera: Crambidae). Journal of Invertebrate Pathology 99, 87e91.
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