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

Soildwelling 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 nonsymbiotic 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. 16SrRNA gene sequence based maximum parsimony, maximum likelihood and neighbour joining phylogenetic analyses were conducted. Two nonsymbiotic 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 nonsymbiotic bacteria associated with EPNs infection.