Non-symbiotic association of Citrobacter freundii and Staphylococcus succinus with the entomopathogenic nematode Steinernema feltiae

Authors

Department of Plant Protection, Faculty of Agriculture, Maragheh University

Abstract

Soil-dwelling nematodes from Steinernematidae family are obligate parasites of insects and usually referred to as entomopathogenic nematodes (EPNs). These nematodes are symbiotically associated with entomopathogenic bacteria Xenorhabdus spp. The bacterial symbionts are carried monoxenically in a special vesicle in the infective juveniles (IJs). In the present study we report the isolation of two species of non-symbiotic bacteria from infected insect cadavers by the EPN, Steinernema feltiae. Galleria mellonella L. larvae were exposed to surface sterilized infective juveniles of S. feltiae and transferred to sterile Petri dishes for a further 24 hours. Hemolymph was collected and streaked onto both MacConkey and NBTA agar. Bacteria were identified using biochemical and phylogenetic analysis. 16S-rRNA gene sequence based maximum parsimony, maximum likelihood and neighbour joining phylogenetic analyses were conducted. Two non-symbiotic species including Citrobacter freundii and Staphylococcus succinus were identified and reported to be associated with S. feltiae. Our results provide further evidence for the existence of non-symbiotic bacteria associated with EPNs infection. 

Keywords


Aguillera, M. M. (1993) Bacterial symbionts of Steinernema scapterisciJournal of  Invertebrate Pathology 62, 68-72.
 Aguillera, M. M., & Smart, G. C. (1993) Development, reproduction, and pathogenicity of Steinernema scapterisci in monoxenic culture with different species of bacteria. Journal of Invertebrate Pathology 62, 289-294.
Akhurst, R. J., & Boemare, N. E. (1990) Biology and taxonomy of Xenorhabdus. pp. 75–92 in R. Gaugler & H. K. Kaya (Eds.) Entomopathogenic Nematodes in Biological Control . Boca Raton, FL: CRC Press.
Bedding, R. A., & Akhurst, R. J. (1975) A simple technique for the detection of insect parasitic rhabditid nematodes in soil. Nematologica 21, 109-110.
Boemare, N. E., Akhurst, R. J., & Mourant, R. G. (1993) DNA relatedness between Xenorhabdus spp. (Enterobacteriaceae), symbiotic bacteria of entomopathogenic nematodes, and a proposal to transfer Xenorhabdus luminescens to a new genus, Photorhabdus gen. nov. International Journal of Systematic Bacteriology 43
249-255.
Bonifassi, E., Fischer-Le Saux, M., Boemare, N., Lanois, A., Laumond, C., & Smart, G. (1999) Gnotobiological study of infective juveniles and symbionts of Steinernema scapterisci: A model to clarify the concept of the natural occurrence of monoxenic associations in entomopathogenic nematodes. Journal of  Invertebrate Pathology 74(2), 164-172. doi: 10.1006/jipa.1999.4866
Easom, C. A., Joyce, S. A., & Clarke, D. J. (2010) Identification of genes involved in the mutualistic colonization of the nematode Heterorhabditis bacteriophora by the bacterium Photorhabdus luminescensBMC Microbiology 10(45), 10. doi: http://dx.doi.org/10.1155/2015/570243
Ehlers, R.-U., Stoessel, S., & Wyss, U. (1990) The influence of phase variants of Xenorhabdus spp. and Escherichia coli (Enterobacteriaceae) on the propagation of entomopathogenic nematodes of the genera Steinernema and HeterorhabditisRevue de Nématologie 13, 417-424.
Eidt, D. C., & Thurston, G. S. (1995) Physical deterrents to infection by entomopathogenic nematodes in wireworms (Coleoptera: Elateridae) and other soil insects. Canadian Entomology 127, 423-429.
Eivazian Kary, N., Niknam, G., Griffin, C. T., Mohammadi, S. A., & Moghaddam, M. (2009) A survey of entomopathogenic nematodes of the families Steinernematidae and Heterorhabditidae (Nematoda: Rhabditida) in the north-west of Iran. Nematology 11(1), 107-116. doi: 10.1163/156854108X398453
Forst, S., & Clarke, D. (2002) Bacteria-Nematode Symbiosis. pp. 57-77 in R. Gaugler (Ed.) Entomopathogenic Nematology. Wallingford, UK: CABI Publishing.
Gaugler, R., & Han, R. (2002) Production technology. pp. 289-310 in R. Gaugler (Ed.), Entomopathogenic nematology. CABI International
Gouge, D. H., & Snyder, J. L. (2006) Temporal association of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) and bacteria. Journal of Invertebrate Pathology 91(3), 147-157. doi: 10.1016/j.jip.2005.12.003
Han, R. C., Wouts, W. M., & Li, L. (1998) Development of Heterorhabditis spp. strains as characteristics of possible Xenorhabdus luminescens subspecies. Revue de Nématologie 13(411-415).
Harth, E., Romero, J., Torres, R., & Espejo, R. T. (2007) Intragenomic heterogeneity and intergenomic recombination among Vibrio parahaemolyticus 16S rRNA genes. Microbiology 153(Pt 8), 2640-2647. doi: 10.1099/mic.0.2007/009175-0
Hominick, W. M., Briscoe, B. R., del Pino, F. G., Heng, J., Hunt, D. J., Kozodoy, E., Mracek, Z., Nguyen, K. B., Reid, A. P., Spiridonov, S. E., Stock, P., Sturhand, D., Waturu, C & Yoshida, M. (1997) Biosystematics of entomopathogenic nematodes: current status, protocols and definitions. J Helminthol 71(4), 271-298.
Kaya, H. K., & Gaugler, R. (1993). Entomopathogenic nematodes. Annual Review of Entomology 38, 181-206.
Koppenhoffer, A. M., Fuzy, E. M., Crocker, R., Gelernter, W., & Polavarapu, S. (2004) Pathogenicity of Steinernema scarabaei, Heterorhabditis bacteriophora and S. glaseri to twelve white grub species. Biocontrol Science and Technology 14, 87-92.
Krieg, N. R., & Holt, J. G. (1984) Bergey's Manual of Systematic Bacteriology (Vol. 1). Baltimore, USA: Williams and Wilkins.
Lysenko, O., & Weiser, J. (1974) Bacteria associated with the nematode Neoaplectana carpocapsae and the pathogenicity of this complex for Galleria mellonella larvae. Journal of Invertebrate Pathology 24, 332-336.
Mracek, Z. (1977) Steinernema kraussei, a parasite of the body cavity of the sawfly, Cephaleia abietis, in Czechoslovakia Journal of  Invertebrate Pathology, 30, 87-94.
Park, H. W., Kim, Y. O., Ha, J. S., Youn, S. H., Kim, H. H., Bilgrami, A. L., & Shin, C. S. (2011) Effects of associated bacteria on the pathogenicity and reproduction of the insect-parasitic nematode Rhabditis blumi (Nematoda: Rhabditida). Canadian Journal of Microbiology 57(9), 750-758.
Patel, J. B. (2001) 16S rRNA gene sequencing for bacterial pathogen identification in the clinical laboratory. Molecular Diagnostics 6, 313-321.
Quiroz-Castañeda, R. E, Mendoza-Mejía, A., Obregón-Barboza, V., Martínez-Ocampo, F., Hernández-Mendoza, A., Martínez-Garduño, F., Guillén-Solís, G., Sánchez-Rodríguez, F., Peña-Chora, G., Ortíz-Hernández, L., Gaytán-Colín, P., Dantán-González, E. (2015) Identification of a new Alcaligenes faecalis strain MOR02 and assessment of its toxicity and pathogenicity to insects. Biomed Research International 570243. doi: 10.1155/2015/570243
Ramjeet, M., Cox, A. D., Hancock, M. A., Mourez, M., Labrie, J., Gottschalk, M., & Jacques, M. (2008) Mutation in the LPS outer core biosynthesis gene, galU, affects LPS interaction with the RTX toxins ApxI and ApxII and cytolytic activity of Actinobacillus pleuropneumoniae serotype 1. Molecular Microbiology 70(1), 221-235.
Razia, M., Karthik Raja, R., Padmanaban, K., Chellapandi, P., & Sivaramakrishnan, S. (2011) 16S rDNA-Based Phylogeny of Non-Symbiotic Bacteria of Entomopathogenic Nematodes from Infected Insect Cadavers. Genomics Proteomics Bioinformatics 9(3), 104-112. doi: 10.1016/S1672-0229(11)60013-2
Rehfuss, M., & Urban, J. (2005) Alcaligenes faecalis subsp. phenolicus subsp. nov. a phenol-degrading, denitrifying bacterium isolated from a graywater bioprocessor. Systematic and  Applied  Microbiology 28(5), 421-429. doi: 10.1016/j.syapm.2005.03.003
Teyssier, C., Marchandin, H., Simeon De Buochberg, M., Ramuz, M., & Jumas-Bilak, E. (2003) Atypical 16S rRNA gene copies in Ochrobactrum intermedium strains reveal a large genomic rearrangement by recombination between rrn copies. Journal of  Bacteriology 185(9), 2901-2909.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., & Higgins, D. G. (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25(24), 4876-4882.
Whalen, J. G., Mully, T. W., & English, J. C. (2007) Spontaneous Citrobacter freundii Infection in an Immunocompetent Patient. Archives of Dermatology 143(1), 124-125.
Yadav, S., Shokal, U., Forst, S., & Eleftherianos, I. (2015) An improved method for generating axenic entomopathogenic nematodes. BMC Res Notes 8(1), 461. doi: 10.1186/s13104-015-1443-y