Culturing and Characterization of Gut Symbiont Burkholderia spp. from the Southern Chinch Bug, Blissus insularis (Hemiptera: Blissidae)

The phloem-feeding Southern chinch bug,Blissus insularis , harbors a high density of the exocellular bacterial symbiontBurkholderia in the lumen of specialized midgut crypts. Here we developed an organ culture method that initially involved incubating theB. insularis crypts in osmotically balanced insect cell culture medium. This approach enabled the crypt-inhabitingBurkholderia spp. to make a transition to anin vitro environment 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-inhabitingBurkholderia counterparts. Genomic and physiological analyses of gut-symbioticBurkholderia spp. that were isolated individually from two separateB. insularis laboratory colonies revealed that the majority of individual insects harbored a singleBurkholderia ribotype in their midgut crypts, resulting in a diverseBurkholderia community within each colony. The diversity was also exhibited by the phenotypic and genotypic characteristics of theseBurkholderia cultures. Access to cultures of crypt-inhabiting bacteria provides an opportunity to investigate the interaction between symbioticBurkholderia spp. and theB. insularis host. Furthermore, the culturing method provides an alternative strategy for establishingin vitro cultures of other fastidious insect-associated bacterial symbionts.IMPORTANCE An organ culture method was developed to establishin vitro cultures of a fastidiousBurkholderia symbiont 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 ofBurkholderia cultures isolated fromB. insularis crypts, showing that host insects maintained the diversity ofBurkholderia spp. over multiple generations. The availability of characterized gut-symbioticBurkholderia cultures provides a resource for genetic manipulation of these bacteria and for examination of the mechanisms underlying insect-bacterium symbiosis.