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Applied and Environmental Microbiology, September 2006, p. 6417-6418, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00519-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Virulence of Serratia Strains against Costelytra zealandica

Binglin Tan, Trevor A. Jackson,* and Mark R. H. Hurst

Biocontrol Technologies, AgResearch, Canterbury Agricultural and Science Centre, P.O. Box 60, Lincoln, New Zealand

Received 3 March 2006/ Accepted 11 July 2006


   ABSTRACT
Top
 Abstract
Introduction
 References
 
Strains of Serratia spp. showed a high level of virulence when injected into the hemocoel of larvae Costelytra zealandica, with Serratia entomophila, S. plymuthica, and S. marcescens showing significantly higher virulence than S. proteamaculans. Toxicity was independent of the amber disease-causing plasmid pADAP, suggesting a generalized Serratia toxin.


   INTRODUCTION
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 Abstract
 Introduction
References
 
The genus Serratia (Enterobacteriaceae) contains insect pathogenic strains (5) which are usually considered to be opportunistic or facultative pathogens, as they are often avirulent to insects when present in the digestive tract but are lethal upon entering insect hemocoel following injury or stress (3). In contrast, strains of Serratia entomophila and S. proteamaculans containing the pADAP plasmid consistently produce amber disease after ingestion by the New Zealand grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae) (7, 9). Amber disease is a gut-colonizing disease caused by a cluster of three genes termed sep (S. entomophila pathogenicity) genes on the pADAP plasmid, with the bacteria invading the hemocoel only after a long period of chronic infection (4, 7, 10). The sep proteins show significant similarity to the toxin complexes produced by bacterial entomopathogens Photorhabdus luminescens and Xenorhabdus nematophila, which are usually nematode vectored, leading to death of the nematode by septicemia, but can also be orally active (1, 13, 14). Thus, while oral activity of amber disease-causing strains has been well characterized (9, 10), the effects of coelomic delivery were not known. In this study, we examined virulence and pathology of pADAP-bearing and pADAP-free Serratia strains against C. zealandica larvae, estimated the intracoelomic lethal doses of the bacteria, and examined the resultant pathology.

The bacterial strains bioassayed in this study are listed in Table 1. Bacteria were produced in an overnight shake culture in Luria-Bertani (LB) broth at 37°C for Escherichia coli and 30°C for Serratia (7). Cell densities of bacteria were estimated from CFU after dilution plating with phosphate-buffered saline (PBS) onto LB agar plates.


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  		TABLE 1. Estimated LD50s for intracoelomic delivery of Serratia strains with and without the pADAP plasmid and E. coli against C. zealandica larvae

 
pADAP-bearing strains S. entomophila 154+, S. proteamaculans 2742, and S. proteamaculans 142 and pADAP-free strains S. plymuthica 590, S. marcescens 363, and E. coli DH10B (11) were injected into healthy grass grub larvae. Two microliters of diluted suspension of bacterial culture was injected into the hemolymph through the larval head capsule by using a 1-ml Hamilton syringe with a 30.5-gauge needle (8) to achieve doses ranging from 100 to 106 cells per insect. Larvae for uninfected controls were injected with equal volumes of PBS. The symptoms of toxicity were recorded and the mortalities monitored over time postinjection. The experiment was repeated on three occasions in a completely randomized design. The experimental unit was a group of eight larvae. Inoculated larvae were individually placed in wells of 24-well tissue culture plates, held in the dark in an incubator at 15°C, and observed until 14 days postinjection. Data were analyzed using Genstat 7.1 software (Genstat UK) with treatment mortalities corrected for control mortalities and median lethal doses estimated by probit analysis.

To assess the ability of bacteria to survive and grow within the insect hemocoel, we compared the S. entomophila A1MO2(pADK6) E. coli DH10B and labeled by green fluorescent protein using plasmid pGFPuvsm (6). Bacteria were delivered as described above, with doses of approximately 101 to 105 cells larva–1 for E. coli DH10B and approximately 101 to 103 cells larva–1 for the S. entomophila strain containing pADK6. Larvae were bled up to 48 h postinjection when 20 µl of hemolymph was removed from each larva and dilution plated with PBS onto LB agar plates containing the appropriate antibiotics (6). Resultant CFU were assessed by visualization of green fluorescent protein-based fluorescence after exposure on a UV transilluminator. The experiment was carried out with six larvae for each dose rate of each strain, and three larvae assessed at each time point for all dose rates of each strain. Pathology was monitored by examination of hemolymph extracts by phase-contrast microscopy and examination of thin sections of embedded tissue fixed from moribund larvae.

All Serratia strains caused mortality after injection into the larvae. Larvae inoculated with doses of ≥104 cells larva–1 of Serratia strains began to die within 24 h postinjection, while larvae inoculated with doses of ≤103 cells larva–1 tended to die after 48 h postinjection. After six days, S. entomophila 154+ and 154–, S. plymuthica 590, and S. marcescens 363 produced the highest mortality, with estimated 50% lethal doses (LD50s) that were significantly (P<0.05) lower than those of S. proteamaculans 2746 and 142, which were in turn significantly (P<0.05) lower than those of controls, including E. coli DH10B (Table 1). Disease symptoms included lethargy and a darkening of the larvae, but gut clearance, a primary symptom of amber disease, was not a symptom here. Following intracoelomic inoculation of the pathogenic S. entomophila strain containing pADK6, the bacteria grew rapidly within the hemolymph, with the CFUs reaching 109 cells per ml of hemolymph in 48 h. In contrast, after injection of E. coli DH10B, no CFUs were recovered, indicating that cells were rapidly cleared from the hemolymph shortly after inoculation. Microscopic visualization of hemolymph revealed strong bacterial growth accompanied by vacuolization of the hemocytes, leading to cell lysis. Infection was accompanied by a severe disruption of the midgut epithelium in moribund larvae.

These experiments show that the Serratia strains tested had a high virulence against larvae of the New Zealand grass grub, C. zealandica, when injected directly into the hemocoel and can be considered potential pathogens by the definition of Bucher (2). The effects of Serratia sp. inoculation were clearly distinct from those produced by E. coli DH10B, which did not cause mortality even when applied at a concentration of 105 cells larva–1. Serratia-induced mortality could be produced with inoculation of very low numbers of cells, and in this regard, our results coincide with those of Lysenko (12) for intracoelomic inoculation of S. marcescens into caterpillars. Microscopic examination of larvae through the disease process indicates that Serratia virulence is related to the ability of the bacteria to avoid or overcome the insect host's defenses and proliferate in the hemocoel producing cytotoxic effects against both hemocytes and gut epithelial tissue.

This rapid pathology accompanied by cytotoxic effects is clearly distinct from the chronic amber disease produced by strains of S. entomophila and S. proteamaculans (9, 10). No relationship was found between the presence of the amber disease-causing plasmid pADAP and virulence in these experiments. Indeed, the matched strains S. entomophila 154+ and 154– showed similar virulences, while the plasmid-bearing S. proteamaculans strains showed the lowest virulence of the Serratia strains tested. Furthermore, the characteristic amber disease symptoms of cessation of feeding and gut clearance (9, 10) were not observed in these experiments. Thus, the pathogenicities of these strains of Serratia to grass grub larvae following coelomic injection appear to be related to a generalized Serratia toxin rather than caused by the toxin complex/Sep-type gene complex found on the pADAP plasmid.


   ACKNOWLEDGMENTS
 
We are grateful to the members of the Biocontrol Technologies Team, AgResearch, for their excellent technical assistance and David Saville for data analysis.

This research was funded by grant C10X0313 of the New Economy Research Fund, administered by the New Zealand Foundation for Research, Science and Technology.


   FOOTNOTES
 
* Corresponding author. Mailing address: AgResearch, P.O. Box 60, Lincoln, New Zealand. Phone: 64 3 3259984. Fax: 64 3 3259946. E-mail: trevor.jackson{at}agresearch.co.nz. Back


   REFERENCES
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  1. Bowen, D., A. R. Thomas, M. Blackburn, O. Andreev, E. Golubeva, R. Bhartia, and R. H. Ffrench-Constant. 1998. Insecticidal toxins from the bacterium Photorhabdus luminescens. Science 280:2129-2132.[Abstract/Free Full Text]
  2. Bucher, G. E. 1960. Potential bacterial pathogens of insects and their characteristics. J. Insect Pathol. 2:172-195.
  3. Bucher, G. E. 1963. Nonsporulating bacterial pathogens, p. 117-147. In E. A. Steinhaus (ed.), Insect pathology: an advanced treatise. Academic Press, New York, N.Y.
  4. Glare, T. R., G. E. Corbett, and A. J. Sadler. 1993. Association of a large plasmid with amber disease of the New Zealand grass grub, Costelytra zealandica, caused by Serratia entomophila and Serratia proteamaculans. J. Invertebr. Pathol. 62:165-170.[CrossRef]
  5. Grimont, P. A. D., and F. Grimont. 1978. The genus Serratia. Annu. Rev. Microbiol. 32:221-248.[CrossRef][Medline]
  6. Hurst, M. R. H., and T. A. Jackson. 2002. Use of the green fluorescent protein to monitor the fate of Serratia entomophila causing amber disease in the New Zealand grass grub, Costelytra zealandica. J. Microbiol. Methods 50:1-8.[CrossRef][Medline]
  7. Hurst, M. R. H., T. R. Glare, T. A. Jackson, and C. W. Ronson. 2000. Plasmid-located pathogenicity determination of Serratia entomophila, the causal agent of amber diseases of grass grub, showing similarity to the insecticidal toxins of Photorhabdus luminescens. J. Bacteriol. 182:5127-5138.[Abstract/Free Full Text]
  8. Jackson, T. A., and D. J. Saville. 2000. Bioassay of replicating bacteria against soil-dwelling insect pests, p. 73-94. In A. Navon and K. R. S. Ascher (ed.), Bioassays of entomopathogenic microbes and nematodes. CABI Publishing, New York, N.Y.
  9. Jackson, T. A., A. M. Huger, and T. R. Glare. 1993. Pathology of amber disease in the New Zealand grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae). J. Invertebr. Pathol. 61:123-130.[CrossRef]
  10. Jackson, T. A., D. G. Boucias, and J. O. Thaler. 2001. Pathobiology of amber disease, caused by Serratia spp., in the New Zealand grass grub, Costelytra zealandica. J. Invertebr. Pathol. 78:232-243.[CrossRef][Medline]
  11. Lorow, D., and J. Jessee. 1990. Max efficiency DH10B: a host for cloning methylated DNA. Focus 12:19.[Medline]
  12. Lysenko, O. 1981. Principles of pathogenesis of insect bacterial diseases as exemplified by the nonproreforming bacteria, p. 163-188. In E. W. Davidson (ed.), Pathogenesis of invertebrate microbial diseases. Allanhead, Osmun, Montclair, N.J.
  13. Morgan, J. A., M. Sergeant, D. Ellis, M. Ousley, and P. Jarrett. 2001. Sequence analysis of insecticidal genes from Xenorhabdus nematophila PMF1296. Appl. Environ. Microbiol. 67:2062-2069.[Abstract/Free Full Text]
  14. Waterfield, N., A. Dowling, S. Sharma, P. J. Darbon, U. Potter, and R. H. Ffrench-Constant. 2001. Oral toxicity of Photorhabdus luminescens W14 toxin complexes in Escherichia coli. Appl. Environ. Microbiol. 67:5017-5024.[Abstract/Free Full Text]

Applied and Environmental Microbiology, September 2006, p. 6417-6418, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00519-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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