Microhabitats within venomous cone snails contain diverse actinobacteria

Abstract

Actinomycetes can be symbionts in diverse organisms, including both plants and animals. Some actinomycetes benefit their host by producing small molecule secondary metabolites; the resulting symbioses are often developmentally complex. Actinomycetes associated with three cone snails were studied. Cone snails are venomous tropical marine gastropods which have been extensively examined because of their production of peptide-based neurological toxins, but no microbiological studies have been reported on these organisms. A microhabitat approach was used in which dissected tissue from each snail was treated as an individual sample in order to explore bacteria in the tissues separately. Our results revealed a diverse, novel, and highly culturable cone snail-associated actinomycete community, with some isolates showing promising bioactivity in a neurological assay. This suggests that cone snails may represent an underexplored reservoir of novel actinomycetes of potential interest for drug discovery.

FIG. 1.

Radial cladogram showing the evolutionary relationships between C. rolani, C. pulicarius, and C. tribblei (, ; N. Puillandre, M. Watkins, and B. M. Olivera, unpublished data). The tree was generated using mitochondrial 16S sequences from the Olivera database. This figure shows that snails used in this study occupy distinct branches of the Conus radiation.

FIG. 2.

Neighbor-joining phylogenetic tree of 16S rRNA gene sequences from uncultivated and cultivated actinobacteria. Dots indicate cultivated isolates, while capital letters indicate sequences obtained from the body of C. pulicarius (CPB), C. rolani (CRB), and C. tribblei (CTB) and hepatopancreas of C. tribblei (CTHP) and C. pulicarius (CPHP). Colors indicate source snails: black (C. tribblei), blue (C. pulicarius), and red (C. rolani). Reference strains from GenBank are marked by an asterisk. f and p indicate branches that were also found using the Fitch-Margoliash or maximum parsimony methods, respectively. The numbers at the nodes are percentages indicating the levels of bootstrap support, based on a neighbor-joining analysis of 1,000 resampled data sets. Only values of >50% are shown. Bar, 0.01 substitutions per nucleotide position. All actinobacteria identified fall into the actinomycete group.

FIG. 3.

Epifluorescence micrograph section of Conus pulicarius tissue visualized by FISH. (A) Body region hybridized with fluorescein-labeled non-EUB338. (B) Body region hybridized with fluorescein-labeled EUB338 and Cy3-labeled HGC69a. (C) Hepatopancreas region hybridized with fluorescein-labeled non-EUB338. (D) Hepatopancreas region hybridized with fluorescein-labeled EUB338 and Cy3-labeled HGC69a.

FIG. 4.

Dorsal root ganglion assay of CT8 extract. The initial KCl injection and first wash provided the control. Once the cells return to their original state, a series of injections follow, starting with injection of the CT8 extract. In panel C, it can be seen that some cells respond with an influx of calcium directly upon injection of the extract, while others attenuate the response until a second KCl injection (B and C). Other samples show no response to the compound (A).