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


1 گروه گیاهپزشکی، دانشکده کشاورزی، دانشگاه شهید مدنی آذربایجان

2 گروه گیاهپزشکی، دانشکده کشاورزی، دانشگاه مراغه


نماتودهای خاکزی متعلق به خانواده Steinernematidae (Nematoda: Rhabditida) پارازیت های اجباری و کشنده حشرات بوده که معمولا تحت عنوان نماتودهای بیمارگر حشرات (Entomopathogenic nematodes) EPNs، از آن­ها نام برده می شود.  این گروه از نماتودها به طور طبیعی با باکتری های جنس Xenorhabdus رابطه همزیستی داشته و نماتود مرحله سوم آلوده کننده یا Infective Juvenile، این باکتری ها را در یک وزیکول اختصاصی در روده­ی خود حمل می کنند. در این مطالعه وجود دو گونه از باکتری های غیر همزیست از لاشه حشرات آلوده به نماتود بیمارگر حشرات Steinernema feltiae گزارش می گردد. شناسایی باکتری های مذکور بر اساس آزمون های بیوشیمیایی و تجزیه و تحلیل نسب شناختی ترادف ژن rRNA-s16 انجام گرفت و در نهایت دو گونه باکتری به عنوان Citrobacter freundiiو Staphylococcus succinus شناسایی شدند. مطالعه اخیر تاییدی بیشتر بر وجود ارتباط بین باکتری های غیر همزیست با نماتودهای بیمارگر حشرات و توانایی نماتودها در انتقال آن­ها می باشد.


عنوان مقاله [English]

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

نویسندگان [English]

  • Naser Eivazian Kary 1
  • Zhila Alizadeh 2
2 Department of Plant Protection, Faculty of Agriculture, Maragheh University
چکیده [English]

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. 

کلیدواژه‌ها [English]

  • Entomopathogenic nematode
  • Steinernema feltiae
  • Citrobacter freundii
  • Staphylococcus succinus

Aguillera, M. M. (1993) Bacterial symbionts of Steinernema scapterisci. Journal 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,

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 luminescens. BMC Microbiology 10(45), 10. doi:

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 Heterorhabditis. Revue 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