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Hydrothermal Vent-Endemic Shrimp Episymbiont Diversity and Distribution on the Mid-Atlantic and Central Indian Ridges
Knee, McKiness, Cavanaugh, and Shank

Abstract
The caridean hydrothermal vent shrimp Rimicaris aff. exoculata appears to be the only vent species that has successfully maintained populations between the Mid-Atlantic Ridge and the Central Indian Ridge1,2. R. exoculata at the Snake Pit vent site on the Mid-Atlantic Ridge has been found to host a monoculture (single phylotype) of epsilon-Proteobacteria on the internal walls of its branchial chamber, generating hypotheses of an intimate association between these organisms3. Found in dense clusters of 40,000 individuals per m3 at Mid-Atlantic Ridge sites4, the discovery of R. aff. exoculata in the Indian Ocean raises fundamental questions as to the cospeciation of this specific shrimp host and epibiont. To examine host-symbiont specificity, we compared 16S ribosomal DNA (rDNA) epibiont sequences described previously from Snake Pit3 with epibiont sequences recently collected from shrimp inhabiting three geographically separated vent sites: Rainbow (36˚14’N, 33˚54’W, 2300 m deep) and Snake Pit (23˚22’N, 44˚57’W, 3600 m deep) on the Mid-Atlantic Ridge, and the Kairei vent field on the Central Indian Ridge (25˚19’S, 70˚02’E, 2450 m deep); these vent sites span a total distance of ca. 17,000 km along the global mid-ocean ridge system1. Contrary to the former observation of a single epibiont phylotype3, multiple e-Proteobacteria phylotypes were recovered from the shrimp’s inner branchial carapace and recognized through PCR, cloning, automated sequencing, and subsequent alignment analyses. A total of 65 cloned sequences (21 from Snake Pit, 22 from Rainbow, 22 from Kairei), obtained from epibiotic scrapings of five adult shrimp per site, has yielded a minimum of 18 phylotypes. Preliminary analysis revealed a sequence divergence range of 3-15% when compared to the previously known phylotype3. Work to ground-truth these findings with in-situ hybridization on Rimicaris epibionts is ongoing. The discovery of a diverse collection of Mid-Atlantic Ridge shrimp epibionts may yield novel insights into the ecology of symbiotic bacterial assemblages and their cospeciation with metazoan hosts found at hydrothermal vents.

Background
Hydrothermal vents provide ideal systems for biogeographic and evolutionary studies, as they are ephemeral oases of life, distinct in time and space yet organized along the axes of the global mid-ocean ridges. We currently lack an understanding of vent species distribution processes between the Atlantic and Indian Oceans, as patterns of dispersal through the southern Atlantic have not been generated. However, the presence of the caridean vent shrimp Rimicaris aff. exoculata at hydrothermal vent fields along the Central Indian Ridge1,2 (CIR) suggests an extant link between those sites and the northern Mid-Atlantic Ridge (MAR), where R. exoculata maintains equally dominant populations. Like many other hydrothermal vent fauna5,6,7, R. exoculata from the Snake Pit vent site (23˚22’N, 44˚57’W) on the MAR hosts a culture of epibiotic epsilon-Proteobacteria; yet unlike the bacterial associations with these other metazoans, the shrimp epibiont found on the internal walls of its host’s branchial chamber is a monotypic phylotype3. The presence of Rimicaris sp. in densities up to 40,000 individuals per m3 at other vent sites4 raises fundamental questions about the maintenance of this intimate host-epibiont association along the 17,000km spanning the MAR and CIR.

Symbiotic bacteria associated with dominant vent fauna require mechanisms of transmission and dispersal to maintain their symbioses in time and space. Such symbionts are known to be dispersed with their host either via vertical transmission from parent to offspring (e.g. mussels, clams)8, or via horizontal transmission, acquired from the environment (e.g. vestimentiferans)9. The hypothesized association of a single sulfur-oxidizing e-Proteobacteria phylotype with the vent-endemic shrimp R. exoculata3, coupled with the recently discovered expansive geographic distribution of the shrimp species1, suggests that—despite the bacteria’s environmental ubiquity10,11,12—the proposed monoculture of epibionts (for Atlantic Rimicaris) may be dispersing great distances to maintain host-symbiont specificity and support the trophic requirements of their shrimp hosts13. Thus, the characterization of Rimicaris epibionts at additional sites in the Atlantic and Indian Oceans is essential in understanding the processes that generate the observed Snake Pit monoculture and other potential patterns of epibiont exclusivity or community structure.

Objectives
  1. Characterize Mid-Atlantic Ridge and Indian Ocean Rimicaris microbial epibiont community composition using 16S rDNA as a phylogenetic marker;
  2. Compare host-epibiont specificity between the known phylotype of e-Proteobacteria from Snake Pit vent site and same shrimp species elsewhere on the MAR (Rainbow site) and on the Central Indian Ridge (Kairei site);
  3. Identify patterns of epibiont diversity and distribution within and among sites and host individuals;
  4. Provide insights into the patterns and mechanisms that maintain potentially intimate symbiont-host associations at hydrothermal vents.
Summary

Direct PCR sequences (5 Rainbow, 5 Snake Pit, and 2 Kairei) resulted in spectropherograms too ambigous to base-call and were thereafter ignored, as they revealed several overlapping bacterial phylotypes within the epibiont extractions.

Of the 65 epibiont clones sequenced with M13f, M13r, and 519r oligonucleotides, 54 e-Proteobacteria consensus sequences--representing samples from five shrimp per site--were able to be aligned. BLAST14 searches within the e-Proteobacteria allied these epibionts into 17 phylotype clusters, including phylotypes isolated from other vent metazoan-associated bacteria and from sediment samples (Fig. 4 and Table 1). These sequences spanned various regions of the 16S rDNA, with 27 near full length (>1400 bp), 14 partially lacking middle nucleotide data (1000-1399 bp), 8 half-length (700-999 bp), and 5 single-end sequence runs (400-499 bp). The 80-300 nucleotide region (numbering for this study alignment only) of the GCG SeqLab15 alignment displays some of the pattern diversity represented by the epibionts (Fig. 3 below). Only 2 clones were exact sequence matches (SE8a-2, SE8a-3).

Remaining sequence data were either ambiguous or matched more closely with the d-Proteobacteria; the latter finding requires further investigation.

Preliminary Conclusions
  1. Bacterial epibiont assemblages on the Rimicaris internal branchial chamber walls are apparently more diverse than previously proposed3 over the connecting 17,000 km of mid-ocean ridges.
  2. Contrary to the former observation of a single epibiont phylotype3, multiple e-Proteobacteria phylotypes were recovered from the shrimp’s inner branchial carapace.
  3. Host-epibiont association appears not to be phylotype-specific, with epibiont affinities to Proteobacteria in the Pacific, Atlantic, and Indian Oceans, including from several vent metazoan-bacteria symbioses and environmental sediment samples.
  4. However, particular clades within the e-Proteobacteria persist in association with shrimp hosts independent of vent location, weakening the hypothesis of cospeciation.
Future Goals
  1. Continue testing for chimeric sequences within data, and incorporate secondary structure more intimately within epibiont sequence alignment.
  2. Develop fluorescent in situ hybridization probes for cloned e-Proteobacteria 16S rDNA in order to identify true epibiotic phylotypes on Rimicaris exoculata and R. aff. exoculata specimens from Rainbow, Snake Pit, and Kairei.
  3. While epibionts from this study do not exactly match sequences in the database, comparisons with unpublished environmental samples from hydrothermal vents will continue.
  4. Host shrimp used in this study were identified as adults, however their age and proximity to molt may contribute to a temporal establishment of epibiont diversity on the shrimp inner branchial chamber carapace. Future work will examine host specificity in Rimicaris juveniles from multiple sites.
  5. Surveying R. exoculata epibionts at currently unknown hydrothermal vent sites along the Southern MAR and Southwest Indian Ridge would further our understanding of host-symbiont association gradients between the Atlantic and Indian Oceans.
Acknowledgements

For the invaluable support provided by the Cavanaugh Lab, especially thanks to Tara Harmer, Meredith Fisher, and Irene Newton. For the mentoring of my Shank Lab peers, notably to Rob Jennings and Amy Baco-Taylor, with helpful suggestions from Kate Buckman; to Karl Krueger, Jed Goldstone, and Steve Thompson for bioinformatics guidance; and to Antoine Pagé of the Reysenbach Lab for additional 16S sequence alignment data. Support to T.M.S. was provided through the Ocean Life Institute (WHOI) and NSF (OCE0136871, OCE9712358), and to C.M.C. through grants from the NSF Biological Oceanography program.

References
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