ABSTRACTThe phloem-feeding Southern chinch bug,Blissus insularis, harbors a high density of the exocellular bacterial symbiontBurkholderiain the lumen of specialized midgut crypts. Here we developed an organ culture method that initially involved incubating theB. insulariscrypts in osmotically balanced insect cell culture medium. This approach enabled the crypt-inhabitingBurkholderiaspp. to make a transition to anin vitroenvironment and to be subsequently cultured in standard bacteriological media. Examinations using ribotyping and BOX-PCR fingerprinting techniques demonstrated that mostin vitro-produced bacterial cultures were identical to their crypt-inhabitingBurkholderiacounterparts. Genomic and physiological analyses of gut-symbioticBurkholderiaspp. that were isolated individually from two separateB. insularislaboratory colonies revealed that the majority of individual insects harbored a singleBurkholderiaribotype in their midgut crypts, resulting in a diverseBurkholderiacommunity within each colony. The diversity was also exhibited by the phenotypic and genotypic characteristics of theseBurkholderiacultures. Access to cultures of crypt-inhabiting bacteria provides an opportunity to investigate the interaction between symbioticBurkholderiaspp. and theB. insularishost. Furthermore, the culturing method provides an alternative strategy for establishingin vitrocultures of other fastidious insect-associated bacterial symbionts.IMPORTANCEAn organ culture method was developed to establishin vitrocultures of a fastidiousBurkholderiasymbiont associated with the midgut crypts of the Southern chinch bug,Blissus insularis. The identities of the resulting cultures were confirmed using the genomic and physiological features ofBurkholderiacultures isolated fromB. insulariscrypts, showing that host insects maintained the diversity ofBurkholderiaspp. over multiple generations. The availability of characterized gut-symbioticBurkholderiacultures provides a resource for genetic manipulation of these bacteria and for examination of the mechanisms underlying insect-bacterium symbiosis.
Variation in southern chinch bug (
Blissus insularis
) survival and damage on St. Augustinegrass germplasm
