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Oral toxicity of symbiotic bacteria Photorhabdus spp. against immature stages of insects
Journal of Asia-Pacific Entomology 14 (2011) 127–130
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Journal of Asia-Pacific Entomology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j a p e
Oral toxicity of symbiotic bacteria Photorhabdus spp. against immature stages of insects Yam Kumar Shrestha a,c, Eun-Kyung Jang a, Yeon-Su Yu b, Mijo Kwon b, Jae-Ho Shin a, Kyeong-Yeoll Lee a,⁎ a b c
College of Agriculture and Life Sciences, Kyungpook National University, Daegu, Republic of Korea Bicosys, Gajo, Republic of Korea District Agriculture Development Office, Sankhuwasava, Department of Agriculture, Nepal
a r t i c l e
i n f o
Article history: Received 25 July 2010 Revised 18 October 2010 Accepted 25 October 2010 Available online 29 October 2010 Keywords: Entomopathogenic nematodes Insecticidal toxins Oral toxicity Photorhabdus Symbiotic bacteria
a b s t r a c t The oral toxicity of 5 Photorhabdus spp. strains collected in different regions of Korea was determined in the larvae of Plodia interpunctella, Galleria mellonella, Lucilia caesar, and Culex pipiens pallens. When diet or water containing culture media containing 1 of the 5 different strains was ingested by immature insects, the first instar larvae of both G. mellonella and L. caesar and young larvae of C. pipiens pallens died within 3–5 days after treatment. However, mortality of P. interpunctella neonate larvae was slightly slower and reached 94.4%–100% within 7 days after treatment. The mortality rate of a control group given a diet containing water, the medium without cultured bacteria, or Escherichia coli culture medium was not affected. The mortality rates were 100%, 45.3%, 2.8%, and 0% for Galleria, Lucilia, Plodia, and Culex, respectively, in another control group given a culture medium of Photorhabdus luminescens ssp. laumondii (TT01). In addition, culture media containing Photorhabdus strains significantly inhibited molting of third instar Plodia larvae by as much as 88% 7 days after treatment, whereas molting inhibition was reduced by 0%, 4%, and 20% following treatments with water, E. coli, or TT01 culture media, respectively. Our results suggest that the oral administration of Photorhabdus bacterial medium was highly effective for controlling various immature insects. © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2010. Published by Elsevier B.V. All rights reserved.
Introduction Photorhabdus sp. is a gram negative bacterium that has a symbiotic relationship with entomopathogenic soil nematodes of the family Heterorhabditis (Forst et al., 1997). These bacteria produce insecticidal factors which are critical for their pathogenic activities on insects (ffrench-Constant et al., 2007). The nematodes enter the openings of an insect body, such as the mouth, spiracle, or anus. After invasion of the insect host, nematodes regurgitate bacteria, which are housed in a vesicle of their intestine, directly into the hemocoel (Ciche and Ensign, 2003). The bacteria produce a range of proteins and metabolites that kill the insect host (Bowen et al., 1998). Both nematodes and bacteria replicate in the insect cadaver (ffrench-Constant et al., 2003). Photorhabdus bacteria can be isolated from the infective juvenile nematodes that carry them or from the infected insect cadaver. They can be cultured as free living organisms in artificial media without nematodes or insect hosts under laboratory conditions (Forst et al., 1997; Daborn et al., 2001). The bacteria secrete entomopathogenic factors directly into the growth medium. Interestingly, these bacteria or their toxic factors are insecticidal when they are ingested through
⁎ Corresponding author. Fax: +82 53 950 6758. E-mail address: [email protected] (K.-Y. Lee).
the mouth and when they are injected into the hemolymph (ffrenchConstant et al., 2003). The pest control efficacy of entomopathogenic nematodes has not had much success in field applications. This is because nematodes are not very stable in many environmental conditions and because there are difficulties associated with nematode invasion into an insect body. However, in the case of Bacillus thuringiensis, the practical application of symbiotic bacteria formulations could improve control efficacy because insects can ingest bacteria via drinking or sucking and then be affected by the toxins they produce. There have been several reports of oral toxicity of symbiotic bacteria and their toxins. The toxin complex of Photorhabdus luminescens was lethal to Manduca sexta larvae by oral ingestion as well as hemocoel injection (Blackburn et al., 1998). The live cells and secreted proteins of Xenorhabdus nematophilus were orally toxic to neonatal larvae of Helicoverpa armigera (Khandelwal and Banerjee-Bhatnagar, 2003). Gerritsen et al. (2005) reported differential toxicity of Photorhabdus temperata strains against two thrips species. They found that 6 North American P. temperata isolates screened from 46 Photorhabdus were orally toxic to thrips but European strains were not. Furthermore, recombinant P. temperata strain K122 expressing the Cry genes of B. thuringiensis showed improved oral toxicity against Prays oleae (Tounsi et al., 2006). Herein, several Photorhabdus strains were collected from various regions of Korea and the oral toxicities of those strains were
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demonstrated in immature stages of the Indianmeal moth Plodia interpunctella, the grater wax moth Galleria mellonella, the greenbottle fly Lucilia caesar, and the house mosquito Culex pipiens pallens. Symbionts of entomopathogenic nematodes can be used as microbial biopesticides in agriculture (ffrench-Constant et al., 2007). In addition, the molecular information of these symbiont toxins can be used as an alternative genetic source of B. thuringiensis. This is currently the only source for transgenic crops but its use in resistance development against major pest insects has arisen (Ferré and van Rie, 2002). Materials and methods Collection, identification, and culture of symbiotic bacteria of entomopathogenic nematodes Symbiotic bacteria were isolated from entomopathogenic Heterorhabditis spp. nematodes collected from soil from various regions in Korea. Isolated bacteria were streaked onto NBTA medium agar supplemented with 0.025% (wt./vol.) bromothymol blue and 0.004% (wt./vol.) triphenyl tetrazolium chloride. Broth cultures were grown from a single primary phase colony in 5YS medium (5% yeast extract, 0.5% NaCl, 0.05% K2HPO4, 0.05% NH4H2PO4, 0.02% MgSO4.7H2O) on a shaker (180 rpm) at 28 °C. Isolated symbiotic bacteria were identified by nucleotide sequence analysis of 16S ribosomal DNA (rDNA). The universal primer set used was a forward 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and a reverse 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′). Polymerase chain reactions (PCR) were performed with genomic DNA as a template in a total volume of 50 μl containing 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.2 mM dNTPs, 0.2 pmol of each primer, and Taq DNA polymerase (Takara, Japan). PCR products were sequenced by a DNA sequencing company (SolGent, Korea) and nucleotide similarity was searched using the BLASTN program in NCBI. The nucleotide sequences were aligned using ClustalW2 in EBI (www.ebi.ac.uk/ Tools/clustalw2). Five Photorhabdus strains were identified and named J3, J4, J5, J6, and J7 (Table 1). A colony of P. luminescens ssp. laumondii TT01 (KACC 12283) was procured from the Korean Agricultural Culture Collection (KACC) and was used as a control. A colony of Escherichia coli DH5a (Stratagene, La Jolla, CA, USA) was used as another control. Insects The colonies of P. interpunctella and G. mellonella were kept at 27 ± 2 °C, 70% ± 5% relative humidity, and a 16 h light and 8 h dark (16L:8D) photoperiodic cycle. Larvae were reared in a plastic box (15 × 15 × 10 cm3) with an artificial diet consisting of wheat bran, pollen, honey, glycerin, and water (1:1:0.3:0.3:0.15, wt./vol.) (Aye et al., 2004). Larvae of C. pipiens pallens and L. caesar were collected at Kyungpook National University campus (Daegu, Korea).
Table 1 Tested bacteria.
Bioassays The oral toxicity of Photorhabdus strains was tested by allowing the ingestion of bacteria culture media by the larvae of 4 insect species. Neonate larvae (n = 30) of P. interpunctella and G. mellonella were reared in a 200 μl clear thin PCR test tube and allowed to ingest pollen (Actinidia arguta) containing either water or culture media including bacteria. Concentrations of culture media were diluted to between 2.7 × 107 and 5.0 × 108 cfu/ml for Photorhabdus strains and to 1.0 × 107 cfu/ml for E. coli. A mixture (1:1, wt./vol.) of pollen and culture medium was briefly dried at 35 °C and 10 μl was added to each tube. The tubes were ventilated by making a hole in their lids which then had a fine mesh pasted over them. The mortality, feeding behavior, and molting process of the test larvae were observed every day for 1 week at 25 °C. L. caesar eggs were collected from moistened dog food and were allowed to hatch in a plastic pot. Immediately after hatching, larvae were collected and washed 3 times with distilled water. The larvae (n = 50) were then allowed to feed on a mixture of dog food and culture medium (1:1, wt./vol.) in a plastic dish at 25 °C. C. pipiens pallens larvae (n = 20) were reared in transparent plastic tubes and added 1% bacterial culture medium to water. Water was used as a control. The mortality of the larvae was observed for 7 days at room conditions. Each set of experiments was done 3 times on different dates under similar environmental conditions. Statistical analysis Analysis of variance (ANOVA) and multiple mean comparisons were performed using the general linear model (GLM) by Statistical Analysis System program (SAS, 2003) version 9.1 to identify significant effects of bacterial culture medium on mortality and development of larvae. Differences among mean values were determined using DMRT at P ≤ 0.05. Data were analyzed by completely randomized design with three and five replications. Results and discussion Identification of symbiotic bacteria strains Five symbiotic bacteria strains were isolated from Heterorhabditis spp. collected from different regions in Korea and were identified using 16S rDNA sequence analysis. All strains were highly similar (N99%) to Photorhabdus sp. (Jang et al., in preparation). However, further analysis including biochemical characterization and fatty acid analysis is required for the identification of symbiotic bacteria at the species level (Tóth and Lakatos, 2008). Thus, our 5 strains were named J3, J4, J5, J6, and J7 of Photorhabdus sp.. Previously, Kang et al. (2004) reported one Korean strain of P. temperata ssp. temperata isolated from the entomopathogenic nematode, Heterorhabditis megidis, collected in Andong. Our J3 strain had an identical 16S rDNA sequence to that of the Andong strain (Jang et al., in preparation). Oral toxicity of bacterial culture medium on larvae of pest insects
Strains
Species
Concentrations (cells/ml)
Escherichia coli TT01
E. coli Photorhabdus luminescens ssp. laumondii Photorhabdus sp. Photorhabdus sp. Photorhabdus sp. Photorhabdus sp. Photorhabdus sp.
1.0 × 107 5.9 × 107
J3 J4 J5 J6 J7
The mosquito and blowfly species were identified by their morphological and behavioral characteristics.
5.0 × 108 2.3 × 108 2.7 × 107 5.0 × 108 4.3 × 108
Oral ingestion of Photorhabdus strains was highly effective for killing immature insects of all 4 tested species (Table 2). Similar effects have been reported in other lepidopteran larvae, such as M. sexta (Blackburn et al., 1998) and P. oleae (Tounsi et al., 2006), and in two species of thrips (Gerritsen et al., 2005), although oral toxicity has not been tested on various kinds of insects. We used whole culture medium that contained both bacteria and excreted toxic factors. These media may be more effective than
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Table 2 Mortalities of selected insects on the ingestion of Photorhabdus culture medium. Insects
Treatments
Mortality (% ± SE) Day 1
Plodia
Galleria
Lucilia
Culex
Water Medium Escherichia coli TT01 J3 J4 J5 J6 J7 Water Medium E. coli TT01 J3 J4 J5 J6 J7 Water Medium E. coli TT01 J3 J4 J5 J6 J7 Water E. coli TT01 J3 J4 J5 J6 J7
0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 1.4 ± 1.4b 5.6 ± 3.7ab 0.0 ± 0.0b 12.5 ± 11.0a 0.0 ± 0.0a 0.0 ± 0.0a 0.0 ± 0.0a 46.7 ± 29.0a 33.3 ± 21.3a 58.3 ± 21.3a 45.0 ± 21.8a 65.0 ± 25.6a 46.7 ± 23.3a – – – – – – – – – 0.0 ± 0.0a 0.0 ± 0.0a 0.0 ± 0.0a 41.7 ± 22.4a 16.7 ± 16.7a 28.3 ± 21.3a 41.7 ± 21.8a 31.7 ± 24.5a
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
0.0 ± 0.0e 0.0 ± 0.0e 0.0 ± 0.0e 0.0 ± 0.0e 13.9 ± 6.0d 22.2 ± 3.7cd 37.5 ± 4.2a 27.8 ± 1.4bc 31.9 ± 1.4ab 1.7 ± 1.6b 0.0 ± 0.0b 0.0 ± 0.0b 91.7 ± 1.7a 66.7 ± 8.3a 96.7 ± 3.3a 90.0 ± 10.0a 96.7 ± 03.3a 91.7 ± 08.3a 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 6.7 ± 6.6b 81.3 ± 13.1a 90.7 ± 5.8a 88.7 ± 6.3a 84.7 ± 13.4a 100 ± 0.0a 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 86.7 ± 8.3a 56.7 ± 19.2a 66.7 ± 23.3a 88.3 ± 4.4a 65.0 ± 21.8a
0.0 ± 0.0d 0.0 ± 0.0d 0.0 ± 0.0d 1.4 ± 1.4d 20.8 ± 8.6c 47.2 ± 7.7a 55.6 ± 3.7a 44.5 ± 5.0ab 33.3 ± 0.0bc 1.7 ± 1.6b 0.0 ± 0.0b 3.3 ± 1.7b 100 ± 0.0a 90.0 ± 10.0a 98.3 ± 1.7a 96.7 ± 3.a 100.0 ± 0.0a 98.0 ± 1.7a 0.0 ± 0.0c 0.0 ± 0.0c 0.0 ± 0.0c 34.7 ± 3.5b 93.3 ± 6.7a 100 ± 0.0a 94.7 ± 5.3a 90.0 ± 10.0a 100 ± 0.0a 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 95.0 ± 2.9a 93.3 ± 3.3a 96.7 ± 3.3a 96.7 ± 1.7a 98.3 ± 1.7a
0.0 ± 0.0d 0.0 ± 0.0d 0.0 ± 0.0d 1.4 ± 1.4d 31.9 ± 10.0c 65.3 ± 6.0b 80.5 ± 5.0a 62.5 ± 2.4b 43.0 ± 1.4c 1.7 ± 1.6bc 0.0 ± 0.0c 5.0 ± 2.9b 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 98.3 ± 1.7a 100 ± 0.0a 100 ± 0.0a 0.0 ± 0.0c 0.0 ± 0.0c 0.0 ± 0.0c 37.3 ± 4.8b 93.3 ± 6.6a 100 ± 0.0a 99.3 ± 0.6a 98.0 ± 2.0a 100 ± 0.0a 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 98.3 ± 1.7a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a
0.0 ± 0.0d 0.0 ± 0.0d 0.0 ± 0.0d 1.4 ± 1.4d 61.1 ± 3.7c 90.3 ± 6.0a 90.3 ± 2.7a 81.9 ± 3.7a 72.2 ± 3.7b 1.7 ± 1.6c 0.0 ± 0.0c 5.0 ± 2.9b 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 0.0 ± 0.0c 0.0 ± 0.0c 0.0 ± 0.0c 41.3 ± 8.1b 93.3 ± 6.6a 100 ± 0.0a 100 ± 0.0a 98.7 ± 1.3a 100 ± 0.0a 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 98.3 ± 1.7a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a
0.0 ± 0.0c 0.0 ± 0.0c 0.0 ± 0.0c 1.4 ± 0.0c 81.9 ± 5.6b 98.6 ± 1.4a 98.6 ± 1.4a 98.6 ± 1.4a 95.8 ± 2.4a 11.7 ± 11.6b 0.0 ± 0.0b 13.3 ± 8.8b 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 0.0 ± 0.0c 0.0 ± 0.0c 0.0 ± 0.0c 41.3 ± 8.1b 95.3 ± 4.7a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a
0.0 ± 0.0d 0.0 ± 0.0d 0.0 ± 0.0d 2.8 ± 1.4c 94.4 ± 1.4b 100 ± 0.0a 100 ± 0.0a 98.6 ± 1.4a 100 ± 0.0a 11.7 ± 11.6bc 0.0 ± 0.0c 18.3 ± 6.0b 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 0.0 ± 0.0c 0.0 ± 0.0c 0.0 ± 0.0c 45.3 ± 11.8b 95.3 ± 4.7a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a
Means within a column followed by the same small letter do not differ significantly according to DMRT at 5% level of significance. This data is mortality percentage of first instar larvae of Plodia interpunctella and Galleria mellonella, first instar maggots of Lucilia caesar, and younger larvae of Culex pipiens pallens when exposed to food test and drink test for 7 days.
extracted supernatant because bacteria may continue to produce toxins in the gut of insects after they are ingested. The concentration range of the bacterial media was between 2.7 × 107 and 5.0 × 108 cfu/ ml (Table 1). The toxicity of the J3 strain was slightly lower than the other strains, particularly in Plodia, even though its concentration is higher. Water only, culture medium only, E. coli, and P. luminescens ssp. laumondii TT01 strains were used as controls (Table 2). Diets containing either water or the culture medium without bacteria did not have any significant effects on mortality, even though a watertreated diet slightly induced mortality of Galleria. The effect of a water-treated diet on the mortality of Galleria neonate larvae is not clear but the high water content in the food may alter consumption and utilization of dietary proteins (Jindra and Sehnal, 1989). The culture medium of E. coli increased the mortality of Galleria slightly (18.3% mortality 7 days after treatment) but had no effect on other species. The effects of the TT01 culture medium varied among the 4 species. The mortality rates of Galleria, Lucilia, Plodia, and Culex were 100%, 45.3%, 2.8%, and 0%, respectively. Previous reports also indicated that oral toxicity varied in different groups of insects. The TT01 strain was not orally toxic to thrips (Gerritsen et al., 2005). This strain was also classified as an orally nontoxic group of Photorhabdus (Marokhazi et al., 2003). Oral ingestion of culture broths of various strains of P. luminescens was not lethal to fourth instar M. sexta larvae, but purified toxins of P. luminescens were orally toxic to lepidopterans (M. sexta, G. mellonella), Coleoptera (Tenebrio molitor), Hymenoptera (Monomorium pharaonis), and Dictyoptera (Blatella germanica) (Bowen and Ensign, 1998).
Oral toxicity of bacterial culture media gradually increased during the 7 days after treatment. However, the lethal susceptibility was different among the 4 species. Complete mortality of Lucilia appeared as early as 2 days after treatment with the J7 strain (Table 2). At 3 days after treatment with TT01 and J6, and J4 strains, Galleria and Lucilia were completely dead, respectively. Culex larvae were dead at 4 days after treatment (except when treated with the J3 strain), but the complete mortality of Plodia appeared 7 days after treatment with the J4, J5, and J7 strains. Our results showed that relative lethal susceptibility of toxins was various in different species. Further studies are required to understand the mechanism of differential toxicity of Photorhabdus toxins to various species. Eleftherianos et al. (2008) reported that the immune competence to Photorhabdus toxins was varied even within a single larval stage of M. sexta. Developmental inhibition of the third instar larvae of P. interpunctella To determine the effect of the ingestion of bacterial symbiont culture media on larval developmental rate, third instar larvae of P. interpunctella were allowed to ingest a diet containing various culture media. When 0-day-old third instar larvae ingested a diet containing water, E. coli culture medium, or TT01 culture medium, the molting rates to fourth or fifth instar larvae were 100%, 96.0%, and 80.0%, respectively, by 7 days after treatment (Table 3). When larvae ingested a diet containing the culture medium of Photorhabdus strains, the molting rates were significantly lower (12.0%–16.0%) by 7 days after treatment. Furthermore, larvae fed a diet containing Photorhabdus culture media had significantly smaller bodies than the
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Table 3 Developmental inhibition of 3rd instar larvae of Plodia interpunctella by the ingestion of bacteria culture media. Treatments
Water Escherichia coli TT01 J3 J4 J5 J6 J7
No. of larvae used
Molting rates into fourth and fifth instar larvae (% ± SE) Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
20 20 20 20 20 20 20 20
0 0 0 0 0 0 0 0
10.0 ± 6.9a 6.0 ± 6.0a 3.0 ± 2.0a 2.0 ± 2.0a 1.0 ± 1.0a 6.0 ± 6.0a 2.0 ± 2.0a 2.0 ± 2.0a
44.0 ± 18.0a 35.0 ± 16.8ab 14.0 ± 7.5bc 3.0 ± 2.0c 1.0 ± 1.0c 7.0 ± 7.0c 3.0 ± 3.0c 3.0 ± 3.0c
86.0 ± 8.3a 67.0 ± 16.0ab 34.0 ± 14.4bc 9.0 ± 3.3c 16.0 ± 12.4c 22.0 ± 12.5c 17.0 ± 12.3c 18.0 ± 15.6c
96.0 ± 2.4a⁎ 87.0 ± 7.2ab 58.0 ± 14.3b 11.0 ± 5.1c 17.0 ± 12.2c 21.0 ± 10.6c 18.0 ± 11.8c 19.0 ± 15.5c
99.0 ± 1.0a⁎ 96.0 ± 1.9a⁎ 74.0 ± 8.0b 11.0 ± 5.1c 16.0 ± 11.2c 18.0 ± 9.7c 16.0 ± 9.8c 8.0 ± 5.2c
100 ± 0.0a⁎ 96.0 ± 1.9a⁎ 80.0 ± 7.6a⁎ 12.0 ± 4.9b 15.0 ± 10.2b 16.0 ± 8.6b 13.0 ± 8.1b 12.0 ± 8.7b
Means within a column followed by the same small letter do not differ significantly according to DMRT at 5% level of significance. ⁎ Percentages of both fourth and fifth instar larvae are added together.
control larvae (Fig. 1). A similar result was seen in M. sexta larvae following oral ingestion and hemocoel injection of isolated toxins (Blackburn et al., 1998). In addition, these toxins damaged midgut epithelium of M. sexta larvae. Interestingly, most larvae offered a diet containing Photorhabdus culture media stayed away from the diet and did not eat during the 7 days after treatment. Observations of starvation behavior made during the current research may be due to a physiological damage of the digestive system. Several toxins that are active via oral administration have been identified. Toxin complexes (Tc's) of Photorhabdus are active via oral administration as well as hemocoel injection (ffrench-Constant et al., 2003). However, the oral toxicity mechanism of Photorhabdus bacteria and their toxins remains unsolved. A culture medium of Photorhabdus bacteria contains various active factors, including proteins and metabolite. Further investigation is needed to determine the orally active factors of this bacterial medium. In conclusion, the 5 isolated strains of Photorhabdus bacteria from entomopathogenic nematodes had high oral toxicity against immature insects. Neonate larvae died as early as 2 days after treatment. Older larvae were highly inhibited in growth and development which eventually caused death. This suggests that the oral administration of symbiotic bacteria or their toxins as microbial biopesticides would be of great value for insect pest control in agriculture.
Fig. 1. Inhibition of Plodia interpunctella larval development by the ingestion of bacteria culture media. The 3rd instar larvae were fed a diet including culture medium for 7 days. 1 indicates no culture medium; 2, Escherichia coli culture medium; 3, Photorhabdus luminescens ssp. laumondii TT01 culture medium; 4–8, culture medium of Photorhabdus temperata ssp. temperata strains J3, J4, J5, J6, and J7, respectively.
Acknowledgment This work was supported by the Agriculture and Forestry R&D Program of the Ministry for Food, Agriculture, Forestry, and Fisheries of Korea. References Aye, T.T., Lee, K.-Y., Kwon, Y.-J., 2004. Cloning of immune protein hemolin cDNA from the Indianmeal moth, Plodia interpunctella, and its high induction during development and by bacterial challenge. Entomol. Res. 34, 269–275. Blackburn, M., ffrench-Constant, R.H., Bowen, D., Golubeva, E., 1998. A novel insecticidal toxin from Photorhabdus luminescens, toxin complex a (Tca), and its histopathological effects on the midgut of Manduca sexta. Appl. Environ. Microbiol. 64, 3036–3041. Bowen, D.J., Ensign, J.C., 1998. Purification and characterization of a high-molecularweight insecticidal protein complex produced by the entomopathogenic bacterium Photorhabdus luminescens. Appl. Environ. Microbiol. 64, 3029–3035. Bowen, D., Rocheleau, T.A., Blackburn, M., Andreev, O., Golubeva, E., Bhartia, R., ffrenchConstant, R.H., 1998. Insecticidal toxins from the bacterium Photorhabdus luminescens. Science 280, 2129–2132. Ciche, T.A., Ensign, J.C., 2003. For the insect pathogen Photorhabdus luminescens, which end of a nematode is out? Appl. Environ. Microbiol. 69, 1890–1897. Daborn, P.J., Waterfield, N., Blight, M.A., ffrench-Constant, R.H., 2001. Measuring virulence factor expression by the pathogenic bacterium Photorhabdus luminescens in culture and during insect infection. J. Bacteriol. 183, 5834–5839. Eleftherianos, I., Baldwin, H., ffrench-Constant, R.H., Reynolds, S.E., 2008. Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus. J. Insect Physiol. 54, 309–318. Ferré, J., van Rie, J., 2002. Biochemistry and genetics of insect resistance to Bacillus thuringiensis. Annu. Rev. Entomol. 47, 501–533. ffrench-Constant, R., Waterfield, N., Daborn, P., Joyce, S., Bennett, H., Dowling, A.C., Boundy, A.S., Reynolds, S., Clarke, D., 2003. Photorhabdus: towards a functional genomics analysis of a symbiont and pathogen. FEMS Microbiol. Rev. 26, 433–456. ffrench-Constant, R.H., Dowling, A., Waterfield, N.R., 2007. Insecticidal toxins from Photorhabdus bacteria and their potential use in agriculture. ToxiconReview 49, 436–451. Forst, S., Dowds, B., Boemare, N., Stackebrandt, E., 1997. Xenorhabdus and Photorhabdus spp.: bugs that kill bugs. Annu. Rev. Microbiol. 51, 47–72. Gerritsen, L.J.M., Georgieva, J., Wiegers, G.L., 2005. Oral toxicity of Photorhabdus toxins against thrips species. J. Invertebr. Pathol. 88, 207–211. Jindra, M., Sehnal, F., 1989. Larval growth, food consumption, and utilization of dietary protein and energy in Galleria mellonella. J. Insect Physiol. 35, 719–724. Kang, S., Han, S., Kim, Y., 2004. Identification of an entomopathogenic bacterium, Photorhabdus temperata subsp. temperata in Korea. J. Asia Pac. Entomol. 7 (3), 331–337. Khandelwal, P., Banerjee-Bhatnagar, N., 2003. Insecticidal activity associated with the outer membrane vesicles of Xenorhabdus nematophilus. Appl. Environ. Microbiol. 69, 2032–2037. Marokhazi, J., Waterfield, N., LeGoff, G., Feil, E., Stabler, R., Hinds, J., Fodor, A., ffrenchConstant, R.H., 2003. Using a DNA microarray to investigate the distribution of insect virulence factors in strains of Photorhabdus bacteria. J. Bacteriol. 185, 4648–4656. SAS Institute, 2003. SAS Version 9.1. SAS Institute Inc, Cary. NC, USA. Tóth, T., Lakatos, T., 2008. Photorhabdus temperata subsp. cinerea subsp. nov., isolated from Heterorhabditis nematodes. Int. J. Syst. Evol. Microbiol. 58, 2579–2581. Tounsi, S., Aoun, A.E., Blight, M., Rebai, A., Jaoua, S., 2006. Evidence of oral toxicity of Photorhabdus temperata strain K122 against Prays oleae and its improvement by heterologous expression of Bacillus thuringiensis cry1Aa and cry1Ia genes. J. Invertebr. Pathol. 91, 131–135.
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Journal of Asia-Pacific Entomology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j a p e
Oral toxicity of symbiotic bacteria Photorhabdus spp. against immature stages of insects Yam Kumar Shrestha a,c, Eun-Kyung Jang a, Yeon-Su Yu b, Mijo Kwon b, Jae-Ho Shin a, Kyeong-Yeoll Lee a,⁎ a b c
College of Agriculture and Life Sciences, Kyungpook National University, Daegu, Republic of Korea Bicosys, Gajo, Republic of Korea District Agriculture Development Office, Sankhuwasava, Department of Agriculture, Nepal
a r t i c l e
i n f o
Article history: Received 25 July 2010 Revised 18 October 2010 Accepted 25 October 2010 Available online 29 October 2010 Keywords: Entomopathogenic nematodes Insecticidal toxins Oral toxicity Photorhabdus Symbiotic bacteria
a b s t r a c t The oral toxicity of 5 Photorhabdus spp. strains collected in different regions of Korea was determined in the larvae of Plodia interpunctella, Galleria mellonella, Lucilia caesar, and Culex pipiens pallens. When diet or water containing culture media containing 1 of the 5 different strains was ingested by immature insects, the first instar larvae of both G. mellonella and L. caesar and young larvae of C. pipiens pallens died within 3–5 days after treatment. However, mortality of P. interpunctella neonate larvae was slightly slower and reached 94.4%–100% within 7 days after treatment. The mortality rate of a control group given a diet containing water, the medium without cultured bacteria, or Escherichia coli culture medium was not affected. The mortality rates were 100%, 45.3%, 2.8%, and 0% for Galleria, Lucilia, Plodia, and Culex, respectively, in another control group given a culture medium of Photorhabdus luminescens ssp. laumondii (TT01). In addition, culture media containing Photorhabdus strains significantly inhibited molting of third instar Plodia larvae by as much as 88% 7 days after treatment, whereas molting inhibition was reduced by 0%, 4%, and 20% following treatments with water, E. coli, or TT01 culture media, respectively. Our results suggest that the oral administration of Photorhabdus bacterial medium was highly effective for controlling various immature insects. © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2010. Published by Elsevier B.V. All rights reserved.
Introduction Photorhabdus sp. is a gram negative bacterium that has a symbiotic relationship with entomopathogenic soil nematodes of the family Heterorhabditis (Forst et al., 1997). These bacteria produce insecticidal factors which are critical for their pathogenic activities on insects (ffrench-Constant et al., 2007). The nematodes enter the openings of an insect body, such as the mouth, spiracle, or anus. After invasion of the insect host, nematodes regurgitate bacteria, which are housed in a vesicle of their intestine, directly into the hemocoel (Ciche and Ensign, 2003). The bacteria produce a range of proteins and metabolites that kill the insect host (Bowen et al., 1998). Both nematodes and bacteria replicate in the insect cadaver (ffrench-Constant et al., 2003). Photorhabdus bacteria can be isolated from the infective juvenile nematodes that carry them or from the infected insect cadaver. They can be cultured as free living organisms in artificial media without nematodes or insect hosts under laboratory conditions (Forst et al., 1997; Daborn et al., 2001). The bacteria secrete entomopathogenic factors directly into the growth medium. Interestingly, these bacteria or their toxic factors are insecticidal when they are ingested through
⁎ Corresponding author. Fax: +82 53 950 6758. E-mail address: [email protected] (K.-Y. Lee).
the mouth and when they are injected into the hemolymph (ffrenchConstant et al., 2003). The pest control efficacy of entomopathogenic nematodes has not had much success in field applications. This is because nematodes are not very stable in many environmental conditions and because there are difficulties associated with nematode invasion into an insect body. However, in the case of Bacillus thuringiensis, the practical application of symbiotic bacteria formulations could improve control efficacy because insects can ingest bacteria via drinking or sucking and then be affected by the toxins they produce. There have been several reports of oral toxicity of symbiotic bacteria and their toxins. The toxin complex of Photorhabdus luminescens was lethal to Manduca sexta larvae by oral ingestion as well as hemocoel injection (Blackburn et al., 1998). The live cells and secreted proteins of Xenorhabdus nematophilus were orally toxic to neonatal larvae of Helicoverpa armigera (Khandelwal and Banerjee-Bhatnagar, 2003). Gerritsen et al. (2005) reported differential toxicity of Photorhabdus temperata strains against two thrips species. They found that 6 North American P. temperata isolates screened from 46 Photorhabdus were orally toxic to thrips but European strains were not. Furthermore, recombinant P. temperata strain K122 expressing the Cry genes of B. thuringiensis showed improved oral toxicity against Prays oleae (Tounsi et al., 2006). Herein, several Photorhabdus strains were collected from various regions of Korea and the oral toxicities of those strains were
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demonstrated in immature stages of the Indianmeal moth Plodia interpunctella, the grater wax moth Galleria mellonella, the greenbottle fly Lucilia caesar, and the house mosquito Culex pipiens pallens. Symbionts of entomopathogenic nematodes can be used as microbial biopesticides in agriculture (ffrench-Constant et al., 2007). In addition, the molecular information of these symbiont toxins can be used as an alternative genetic source of B. thuringiensis. This is currently the only source for transgenic crops but its use in resistance development against major pest insects has arisen (Ferré and van Rie, 2002). Materials and methods Collection, identification, and culture of symbiotic bacteria of entomopathogenic nematodes Symbiotic bacteria were isolated from entomopathogenic Heterorhabditis spp. nematodes collected from soil from various regions in Korea. Isolated bacteria were streaked onto NBTA medium agar supplemented with 0.025% (wt./vol.) bromothymol blue and 0.004% (wt./vol.) triphenyl tetrazolium chloride. Broth cultures were grown from a single primary phase colony in 5YS medium (5% yeast extract, 0.5% NaCl, 0.05% K2HPO4, 0.05% NH4H2PO4, 0.02% MgSO4.7H2O) on a shaker (180 rpm) at 28 °C. Isolated symbiotic bacteria were identified by nucleotide sequence analysis of 16S ribosomal DNA (rDNA). The universal primer set used was a forward 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and a reverse 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′). Polymerase chain reactions (PCR) were performed with genomic DNA as a template in a total volume of 50 μl containing 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.2 mM dNTPs, 0.2 pmol of each primer, and Taq DNA polymerase (Takara, Japan). PCR products were sequenced by a DNA sequencing company (SolGent, Korea) and nucleotide similarity was searched using the BLASTN program in NCBI. The nucleotide sequences were aligned using ClustalW2 in EBI (www.ebi.ac.uk/ Tools/clustalw2). Five Photorhabdus strains were identified and named J3, J4, J5, J6, and J7 (Table 1). A colony of P. luminescens ssp. laumondii TT01 (KACC 12283) was procured from the Korean Agricultural Culture Collection (KACC) and was used as a control. A colony of Escherichia coli DH5a (Stratagene, La Jolla, CA, USA) was used as another control. Insects The colonies of P. interpunctella and G. mellonella were kept at 27 ± 2 °C, 70% ± 5% relative humidity, and a 16 h light and 8 h dark (16L:8D) photoperiodic cycle. Larvae were reared in a plastic box (15 × 15 × 10 cm3) with an artificial diet consisting of wheat bran, pollen, honey, glycerin, and water (1:1:0.3:0.3:0.15, wt./vol.) (Aye et al., 2004). Larvae of C. pipiens pallens and L. caesar were collected at Kyungpook National University campus (Daegu, Korea).
Table 1 Tested bacteria.
Bioassays The oral toxicity of Photorhabdus strains was tested by allowing the ingestion of bacteria culture media by the larvae of 4 insect species. Neonate larvae (n = 30) of P. interpunctella and G. mellonella were reared in a 200 μl clear thin PCR test tube and allowed to ingest pollen (Actinidia arguta) containing either water or culture media including bacteria. Concentrations of culture media were diluted to between 2.7 × 107 and 5.0 × 108 cfu/ml for Photorhabdus strains and to 1.0 × 107 cfu/ml for E. coli. A mixture (1:1, wt./vol.) of pollen and culture medium was briefly dried at 35 °C and 10 μl was added to each tube. The tubes were ventilated by making a hole in their lids which then had a fine mesh pasted over them. The mortality, feeding behavior, and molting process of the test larvae were observed every day for 1 week at 25 °C. L. caesar eggs were collected from moistened dog food and were allowed to hatch in a plastic pot. Immediately after hatching, larvae were collected and washed 3 times with distilled water. The larvae (n = 50) were then allowed to feed on a mixture of dog food and culture medium (1:1, wt./vol.) in a plastic dish at 25 °C. C. pipiens pallens larvae (n = 20) were reared in transparent plastic tubes and added 1% bacterial culture medium to water. Water was used as a control. The mortality of the larvae was observed for 7 days at room conditions. Each set of experiments was done 3 times on different dates under similar environmental conditions. Statistical analysis Analysis of variance (ANOVA) and multiple mean comparisons were performed using the general linear model (GLM) by Statistical Analysis System program (SAS, 2003) version 9.1 to identify significant effects of bacterial culture medium on mortality and development of larvae. Differences among mean values were determined using DMRT at P ≤ 0.05. Data were analyzed by completely randomized design with three and five replications. Results and discussion Identification of symbiotic bacteria strains Five symbiotic bacteria strains were isolated from Heterorhabditis spp. collected from different regions in Korea and were identified using 16S rDNA sequence analysis. All strains were highly similar (N99%) to Photorhabdus sp. (Jang et al., in preparation). However, further analysis including biochemical characterization and fatty acid analysis is required for the identification of symbiotic bacteria at the species level (Tóth and Lakatos, 2008). Thus, our 5 strains were named J3, J4, J5, J6, and J7 of Photorhabdus sp.. Previously, Kang et al. (2004) reported one Korean strain of P. temperata ssp. temperata isolated from the entomopathogenic nematode, Heterorhabditis megidis, collected in Andong. Our J3 strain had an identical 16S rDNA sequence to that of the Andong strain (Jang et al., in preparation). Oral toxicity of bacterial culture medium on larvae of pest insects
Strains
Species
Concentrations (cells/ml)
Escherichia coli TT01
E. coli Photorhabdus luminescens ssp. laumondii Photorhabdus sp. Photorhabdus sp. Photorhabdus sp. Photorhabdus sp. Photorhabdus sp.
1.0 × 107 5.9 × 107
J3 J4 J5 J6 J7
The mosquito and blowfly species were identified by their morphological and behavioral characteristics.
5.0 × 108 2.3 × 108 2.7 × 107 5.0 × 108 4.3 × 108
Oral ingestion of Photorhabdus strains was highly effective for killing immature insects of all 4 tested species (Table 2). Similar effects have been reported in other lepidopteran larvae, such as M. sexta (Blackburn et al., 1998) and P. oleae (Tounsi et al., 2006), and in two species of thrips (Gerritsen et al., 2005), although oral toxicity has not been tested on various kinds of insects. We used whole culture medium that contained both bacteria and excreted toxic factors. These media may be more effective than
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Table 2 Mortalities of selected insects on the ingestion of Photorhabdus culture medium. Insects
Treatments
Mortality (% ± SE) Day 1
Plodia
Galleria
Lucilia
Culex
Water Medium Escherichia coli TT01 J3 J4 J5 J6 J7 Water Medium E. coli TT01 J3 J4 J5 J6 J7 Water Medium E. coli TT01 J3 J4 J5 J6 J7 Water E. coli TT01 J3 J4 J5 J6 J7
0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 1.4 ± 1.4b 5.6 ± 3.7ab 0.0 ± 0.0b 12.5 ± 11.0a 0.0 ± 0.0a 0.0 ± 0.0a 0.0 ± 0.0a 46.7 ± 29.0a 33.3 ± 21.3a 58.3 ± 21.3a 45.0 ± 21.8a 65.0 ± 25.6a 46.7 ± 23.3a – – – – – – – – – 0.0 ± 0.0a 0.0 ± 0.0a 0.0 ± 0.0a 41.7 ± 22.4a 16.7 ± 16.7a 28.3 ± 21.3a 41.7 ± 21.8a 31.7 ± 24.5a
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
0.0 ± 0.0e 0.0 ± 0.0e 0.0 ± 0.0e 0.0 ± 0.0e 13.9 ± 6.0d 22.2 ± 3.7cd 37.5 ± 4.2a 27.8 ± 1.4bc 31.9 ± 1.4ab 1.7 ± 1.6b 0.0 ± 0.0b 0.0 ± 0.0b 91.7 ± 1.7a 66.7 ± 8.3a 96.7 ± 3.3a 90.0 ± 10.0a 96.7 ± 03.3a 91.7 ± 08.3a 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 6.7 ± 6.6b 81.3 ± 13.1a 90.7 ± 5.8a 88.7 ± 6.3a 84.7 ± 13.4a 100 ± 0.0a 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 86.7 ± 8.3a 56.7 ± 19.2a 66.7 ± 23.3a 88.3 ± 4.4a 65.0 ± 21.8a
0.0 ± 0.0d 0.0 ± 0.0d 0.0 ± 0.0d 1.4 ± 1.4d 20.8 ± 8.6c 47.2 ± 7.7a 55.6 ± 3.7a 44.5 ± 5.0ab 33.3 ± 0.0bc 1.7 ± 1.6b 0.0 ± 0.0b 3.3 ± 1.7b 100 ± 0.0a 90.0 ± 10.0a 98.3 ± 1.7a 96.7 ± 3.a 100.0 ± 0.0a 98.0 ± 1.7a 0.0 ± 0.0c 0.0 ± 0.0c 0.0 ± 0.0c 34.7 ± 3.5b 93.3 ± 6.7a 100 ± 0.0a 94.7 ± 5.3a 90.0 ± 10.0a 100 ± 0.0a 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 95.0 ± 2.9a 93.3 ± 3.3a 96.7 ± 3.3a 96.7 ± 1.7a 98.3 ± 1.7a
0.0 ± 0.0d 0.0 ± 0.0d 0.0 ± 0.0d 1.4 ± 1.4d 31.9 ± 10.0c 65.3 ± 6.0b 80.5 ± 5.0a 62.5 ± 2.4b 43.0 ± 1.4c 1.7 ± 1.6bc 0.0 ± 0.0c 5.0 ± 2.9b 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 98.3 ± 1.7a 100 ± 0.0a 100 ± 0.0a 0.0 ± 0.0c 0.0 ± 0.0c 0.0 ± 0.0c 37.3 ± 4.8b 93.3 ± 6.6a 100 ± 0.0a 99.3 ± 0.6a 98.0 ± 2.0a 100 ± 0.0a 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 98.3 ± 1.7a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a
0.0 ± 0.0d 0.0 ± 0.0d 0.0 ± 0.0d 1.4 ± 1.4d 61.1 ± 3.7c 90.3 ± 6.0a 90.3 ± 2.7a 81.9 ± 3.7a 72.2 ± 3.7b 1.7 ± 1.6c 0.0 ± 0.0c 5.0 ± 2.9b 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 0.0 ± 0.0c 0.0 ± 0.0c 0.0 ± 0.0c 41.3 ± 8.1b 93.3 ± 6.6a 100 ± 0.0a 100 ± 0.0a 98.7 ± 1.3a 100 ± 0.0a 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 98.3 ± 1.7a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a
0.0 ± 0.0c 0.0 ± 0.0c 0.0 ± 0.0c 1.4 ± 0.0c 81.9 ± 5.6b 98.6 ± 1.4a 98.6 ± 1.4a 98.6 ± 1.4a 95.8 ± 2.4a 11.7 ± 11.6b 0.0 ± 0.0b 13.3 ± 8.8b 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 0.0 ± 0.0c 0.0 ± 0.0c 0.0 ± 0.0c 41.3 ± 8.1b 95.3 ± 4.7a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a
0.0 ± 0.0d 0.0 ± 0.0d 0.0 ± 0.0d 2.8 ± 1.4c 94.4 ± 1.4b 100 ± 0.0a 100 ± 0.0a 98.6 ± 1.4a 100 ± 0.0a 11.7 ± 11.6bc 0.0 ± 0.0c 18.3 ± 6.0b 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 0.0 ± 0.0c 0.0 ± 0.0c 0.0 ± 0.0c 45.3 ± 11.8b 95.3 ± 4.7a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a
Means within a column followed by the same small letter do not differ significantly according to DMRT at 5% level of significance. This data is mortality percentage of first instar larvae of Plodia interpunctella and Galleria mellonella, first instar maggots of Lucilia caesar, and younger larvae of Culex pipiens pallens when exposed to food test and drink test for 7 days.
extracted supernatant because bacteria may continue to produce toxins in the gut of insects after they are ingested. The concentration range of the bacterial media was between 2.7 × 107 and 5.0 × 108 cfu/ ml (Table 1). The toxicity of the J3 strain was slightly lower than the other strains, particularly in Plodia, even though its concentration is higher. Water only, culture medium only, E. coli, and P. luminescens ssp. laumondii TT01 strains were used as controls (Table 2). Diets containing either water or the culture medium without bacteria did not have any significant effects on mortality, even though a watertreated diet slightly induced mortality of Galleria. The effect of a water-treated diet on the mortality of Galleria neonate larvae is not clear but the high water content in the food may alter consumption and utilization of dietary proteins (Jindra and Sehnal, 1989). The culture medium of E. coli increased the mortality of Galleria slightly (18.3% mortality 7 days after treatment) but had no effect on other species. The effects of the TT01 culture medium varied among the 4 species. The mortality rates of Galleria, Lucilia, Plodia, and Culex were 100%, 45.3%, 2.8%, and 0%, respectively. Previous reports also indicated that oral toxicity varied in different groups of insects. The TT01 strain was not orally toxic to thrips (Gerritsen et al., 2005). This strain was also classified as an orally nontoxic group of Photorhabdus (Marokhazi et al., 2003). Oral ingestion of culture broths of various strains of P. luminescens was not lethal to fourth instar M. sexta larvae, but purified toxins of P. luminescens were orally toxic to lepidopterans (M. sexta, G. mellonella), Coleoptera (Tenebrio molitor), Hymenoptera (Monomorium pharaonis), and Dictyoptera (Blatella germanica) (Bowen and Ensign, 1998).
Oral toxicity of bacterial culture media gradually increased during the 7 days after treatment. However, the lethal susceptibility was different among the 4 species. Complete mortality of Lucilia appeared as early as 2 days after treatment with the J7 strain (Table 2). At 3 days after treatment with TT01 and J6, and J4 strains, Galleria and Lucilia were completely dead, respectively. Culex larvae were dead at 4 days after treatment (except when treated with the J3 strain), but the complete mortality of Plodia appeared 7 days after treatment with the J4, J5, and J7 strains. Our results showed that relative lethal susceptibility of toxins was various in different species. Further studies are required to understand the mechanism of differential toxicity of Photorhabdus toxins to various species. Eleftherianos et al. (2008) reported that the immune competence to Photorhabdus toxins was varied even within a single larval stage of M. sexta. Developmental inhibition of the third instar larvae of P. interpunctella To determine the effect of the ingestion of bacterial symbiont culture media on larval developmental rate, third instar larvae of P. interpunctella were allowed to ingest a diet containing various culture media. When 0-day-old third instar larvae ingested a diet containing water, E. coli culture medium, or TT01 culture medium, the molting rates to fourth or fifth instar larvae were 100%, 96.0%, and 80.0%, respectively, by 7 days after treatment (Table 3). When larvae ingested a diet containing the culture medium of Photorhabdus strains, the molting rates were significantly lower (12.0%–16.0%) by 7 days after treatment. Furthermore, larvae fed a diet containing Photorhabdus culture media had significantly smaller bodies than the
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Table 3 Developmental inhibition of 3rd instar larvae of Plodia interpunctella by the ingestion of bacteria culture media. Treatments
Water Escherichia coli TT01 J3 J4 J5 J6 J7
No. of larvae used
Molting rates into fourth and fifth instar larvae (% ± SE) Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
20 20 20 20 20 20 20 20
0 0 0 0 0 0 0 0
10.0 ± 6.9a 6.0 ± 6.0a 3.0 ± 2.0a 2.0 ± 2.0a 1.0 ± 1.0a 6.0 ± 6.0a 2.0 ± 2.0a 2.0 ± 2.0a
44.0 ± 18.0a 35.0 ± 16.8ab 14.0 ± 7.5bc 3.0 ± 2.0c 1.0 ± 1.0c 7.0 ± 7.0c 3.0 ± 3.0c 3.0 ± 3.0c
86.0 ± 8.3a 67.0 ± 16.0ab 34.0 ± 14.4bc 9.0 ± 3.3c 16.0 ± 12.4c 22.0 ± 12.5c 17.0 ± 12.3c 18.0 ± 15.6c
96.0 ± 2.4a⁎ 87.0 ± 7.2ab 58.0 ± 14.3b 11.0 ± 5.1c 17.0 ± 12.2c 21.0 ± 10.6c 18.0 ± 11.8c 19.0 ± 15.5c
99.0 ± 1.0a⁎ 96.0 ± 1.9a⁎ 74.0 ± 8.0b 11.0 ± 5.1c 16.0 ± 11.2c 18.0 ± 9.7c 16.0 ± 9.8c 8.0 ± 5.2c
100 ± 0.0a⁎ 96.0 ± 1.9a⁎ 80.0 ± 7.6a⁎ 12.0 ± 4.9b 15.0 ± 10.2b 16.0 ± 8.6b 13.0 ± 8.1b 12.0 ± 8.7b
Means within a column followed by the same small letter do not differ significantly according to DMRT at 5% level of significance. ⁎ Percentages of both fourth and fifth instar larvae are added together.
control larvae (Fig. 1). A similar result was seen in M. sexta larvae following oral ingestion and hemocoel injection of isolated toxins (Blackburn et al., 1998). In addition, these toxins damaged midgut epithelium of M. sexta larvae. Interestingly, most larvae offered a diet containing Photorhabdus culture media stayed away from the diet and did not eat during the 7 days after treatment. Observations of starvation behavior made during the current research may be due to a physiological damage of the digestive system. Several toxins that are active via oral administration have been identified. Toxin complexes (Tc's) of Photorhabdus are active via oral administration as well as hemocoel injection (ffrench-Constant et al., 2003). However, the oral toxicity mechanism of Photorhabdus bacteria and their toxins remains unsolved. A culture medium of Photorhabdus bacteria contains various active factors, including proteins and metabolite. Further investigation is needed to determine the orally active factors of this bacterial medium. In conclusion, the 5 isolated strains of Photorhabdus bacteria from entomopathogenic nematodes had high oral toxicity against immature insects. Neonate larvae died as early as 2 days after treatment. Older larvae were highly inhibited in growth and development which eventually caused death. This suggests that the oral administration of symbiotic bacteria or their toxins as microbial biopesticides would be of great value for insect pest control in agriculture.
Fig. 1. Inhibition of Plodia interpunctella larval development by the ingestion of bacteria culture media. The 3rd instar larvae were fed a diet including culture medium for 7 days. 1 indicates no culture medium; 2, Escherichia coli culture medium; 3, Photorhabdus luminescens ssp. laumondii TT01 culture medium; 4–8, culture medium of Photorhabdus temperata ssp. temperata strains J3, J4, J5, J6, and J7, respectively.
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