The macro-epibiotic communities of sea turtles have been subject to growing interest in recent years, yet their micro-epibiotic counterparts are almost entirely unknown. Here, we provide the first evidence that diatoms are epibionts for all seven extant species of sea turtle. Using Scanning Electron Microscopy, we inspected superficial carapace or skin samples from a single representative of each turtle species. We distinguished 18 diatom taxa from these seven individuals, with each sea turtle species hosting at least two diatom taxa. We recommend that future research is undertaken to confirm whether diatom communities vary between sea turtle species and whether these diatom taxa are facultative or obligate commensals.
Citation: Robinson NJ, Majewska R, Lazo-Wasem EA, Nel R, Paladino FV, Rojas L, et al. (2016) Epibiotic Diatoms Are Universally Present on All Sea Turtle Species. PLoS ONE 11(6): e0157011. https://doi.org/10.1371/journal.pone.0157011
Editor: Senjie Lin, University of Connecticut, UNITED STATES
Received: January 4, 2016; Accepted: May 22, 2016; Published: June 3, 2016
Copyright: © 2016 Robinson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper.
Funding: This study was funded by a WCSU-AAUP grant (#241290) to Theodora Pinou. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Sea turtles often harbour complex communities of epibionts [1, 2, 3]. These epibiont communities can provide valuable insights into the hosts’ behaviour [4, 5] and health [6, 7]; however, most studies on sea turtle epibiosis have focused exclusively on macro-epibiota. To date, little is known about the prevalence, and potential ecological function, of sea turtles’ micro-epibiota.
Diatoms are often some of the earliest colonizers on any marine substrate  and it has been suggested that sea turtles should harbour epibiotic diatom communities . Moreover, numerous other studies have reported large clumps of periphytic algae growing on the carapace of several sea turtle species [2, 10]. Nevertheless, direct evidence of epibiotic diatoms on sea turtles has only recently been provided on loggerhead turtles Caretta caretta  and olive ridley Lepidochelys olivacea turtles . Consequently, we predict that epibiotic diatoms are likely present on each of the world’s seven extant sea turtle species.
In this study, we used a Scanning Electron Microscopy (SEM) to examine the carapace scutes or skin of flatback Natator depressus, green Chelonia mydas, hawksbill Eretmochelys imbricata, Kemp’s ridley Lepidochelys kempii, leatherback Dermochelys coriacea, loggerhead Caretta caretta, and olive ridley Lepidochelys olivacea turtles in search of epibiotic diatoms. Knowledge of the prevalence, characteristic, and diversity of epibiotic diatoms of sea turtles could provide the impetus for more detailed studies into the micro-epibiota of sea turtles.
Materials and Methods
Carapace scutes were opportunistically collected from a single flatback, green, hawksbill, Kemp’s ridley, loggerhead, and olive ridley turtle. Samples were collected from deceased animals that had been stored in either museum or research collections. As leatherback turtles do not have an external shell like the hard-shelled Cheloniidae, we did not collect carapace samples from leatherback turtles. Instead, we collected skin samples from the flippers of nesting turtles using at 6 mm biopsy punches. Full details on sample collection and storage see Table 1.
Prior to imaging, the leatherback skin samples were dehydrated in a graded series of hexamethyldisilazane (HMDS) of increasing concentrations until 100% of the latter. HMDS drying for SEM is generally preferred to critical point drying as it is cheaper and it less likely to distort the shape of any microbes of interest . The carapace samples were stored dry and did not need further drying for SEM.
All samples were mounted on aluminium specimen mounts and sputter-coated with carbon. SEM images were collected using a FEI XL-30 field emission gun environmental scanning electron microscope at an accelerating voltage of 10kV, and a Zeiss EM900 transmission electron microscope at 80kV with an objective aperture of 90 μm diameter. Each sample was inspected haphazardly at various magnifications to search for micro-epibionts. We attempted to identify each unique diatom to the lowest taxonomic level by consulting appropriate literature [14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25]. When it was not clear that two diatoms were different taxa, they were considered as one so as not to over-estimate the number of species recorded. As each sample had been stored for various lengths of times and under varying conditions, we only attempted to determine whether micro-epibionts were present or absent—we did not attempt to quantitatively assess the abundance of micro-epibionts. Samples were not cleaned or sonicated prior to imaging as it is expected that these processes would remove the micro-epibionts of interest. SEM work was conducted at the Department of Geology and Geophysics, Yale University.
Results and Discussion
Diatoms were present on every sea turtle species (Figs 1 & 2) and from sea turtles from distinct ocean basins (Atlantic, Pacific, and Indian Ocean). We were able to divide all observed diatoms into 18 unique taxa (Table 2). We were only able to identify a single diatom taxon to the species level (Melosira sol), all others were only identified to genus level. All diatoms were pennate, with the exception of Melosira sol. Adnate forms (Amphora spp., Cocconeis sp., Diploneis sp.) constituted 56% of all identified taxa and erect (Achnanthes sp., Poulinea spp.) and motile diatoms (Navicula sp., Nitzschia sp.) constituted 22% and 11%, respectively. The growth form of Tursiocola should be considered as uncertain. According to [20, 26], Tursiocola spp. has been observed in cetaceans with one end embedded in the epidermis. However, recent observations of live diatoms collected from manatee skin suggest that some Tursiocola spp. are highly motile (TA Frankovich, personal communication).
Flatback turtle: A = Achnanthes sp., B & C = Poulinea sp. 1; Green turtle: D = Cocconeis sp., E = Amphora sp. 1, F = Broken pieces of Amphora sp. and Navicula sp., G = broken pieces of Amphora sp.; Hawksbill turtle: H = Amphora sp. 2, I = Amphora sp. 3, J & K = Poulinea sp. 2; Kemp’s ridley turtle: L = Melosira sol, M = Poulinea sp. 1, N = (i) Achnanthes sp. & (ii) Poulinea sp. 1, O = Poulinea sp. 1. All scale bars are 10 μm.
Leatherback turtle: A = Navicula sp., B, C, and D = Tursiocola sp.; Loggerhead turtle: E: Amphora sp. 4, F = Amphora sp. 5, G = Amphora sp. 6, H = Amphora sp. 7, I = Diploneis sp. and other adnate unknown diatom; Olive ridley turtle: J = Nitzschia sp., K = broken pieces of Achnanthes sp (upper left) and possibly other diatom species (arrow), L = Achnanthes sp., M = Poulinea sp. 3, N = Amphora sp. 5, O = Achnanthes sp. All scale bars are 10 μm.
When compared to descriptions of known diatom taxa, many of the 18 diatom taxa seen in this study differed in important aspects of their morphology. For example, the diatom shown in Fig 2I could not be satisfactorily assigned to any existing genus. So far, only one Poulinea sp. has been described ) and, due to differences in the central area, shape, and number of areolae, we believe that the taxa in this study to do not belong to this species. A detailed taxonomic analysis of sea turtle diatoms would therefore be a productive avenue for future research.
Many diatom taxa were only observed on a single host; however, three diatom species were found on multiple host species. Achnanthes sp. was found on flatback, Kemp’s ridley, and olive ridley turtles. Amphora sp. 5 was found on loggerhead and olive ridley turtles, and Poulinea sp. 1 was found on flatback and Kemp’s ridley turtles. Even with the limited sample size used in this study, the presence of comparable diatom taxa on different host species from different localities suggests that that diatom assemblages on sea turtles may be very similar in structure and composition regardless of the hosts’ species or geographic location.
Epibiosis in the marine environment is primarily facultative in nature  and this is probably the case with the majority of diatoms documented here. As such the survival of these epibiotic diatoms may not depend upon settling on a sea turtle host and they may be present in the micro-plankton or on a variety of substrates. Indeed, taxa such as Melirosa sol and Nitzchia sp. are often living in the plankton and may have been present in the surrounding water at the time that the sea turtle tissue sample was collected. Nevertheless, there is also evidence that some diatom taxa observed in our study are obligate sea turtle epibionts. The diatom genera, Poulinea and Chelonicola, have only recently been described from olive ridley carapaces and have not been observed elsewhere . Tursiocola spp. may also be an obligate epibiont, even though it is not exclusive to sea turtles and has been observed on cetaceans , freshwater and marine turtles [11, 27 respectively] and the West Indian manatee Trichechus manatus .
In addition to epibiotic diatoms, other micro-organisms were also discovered by SEM. Although we could not unequivocally identify these organisms we presume them to be a hystrichosphere from a dinoflagellate (Fig 3A) and an encrusting foraminifera (Fig 3B). Furthermore, evidence of a diatom valve (Fig 3C) and a coccolithophore (Fig 3D) were also found on the foraminifera in Fig 3B. Interestingly, these micro-organisms were all found on the carapace sample from a loggerhead turtle. Loggerhead turtles generally host the most diverse macro-epibiont communities  and thus it stands to reason that loggerhead turtles also host the most diverse micro-epibiont communities.
The samples used in this study were either donated by colleagues (green, hawksbill, Kemp’s ridley, loggerhead, and olive ridley samples), borrowed from The Bishop Museum, Honolulu, Hawaii (flatback samples), or collected directly (leatherback samples). Flatback samples were provided by Molly Hagemann from the Bishop Museum. Greg Watkins-Colwell facilitated the shipment of flatback samples to the Peabody Museum of Natural History. Green, hawksbill, and olive ridley scute samples were provided to JDZ by George Balazs, NOAA Pacific Islands Fisheries Science Center, Honolulu, Hawaii. Kemp’s ridley turtle scute samples were provided by Thane Wibbels, University of Alabama. Loggerhead material from Florida was provided to JDZ through the assistance of DuBose Griffin, Sea Turtle Stranding Coordinator for the South Carolina Department of Natural Resources. Permits for collecting skin samples from leatherback turtles were granted by the Department of Environmental Affairs, South Africa (#RES2013/10). Leatherback turtles were imported into the US under CITES permits (#12U589757A/9). We would like to thank Zhenting Jiang for providing essential assistance in the use of the SEM.
Conceived and designed the experiments: NJR TP. Performed the experiments: NJR LR. Analyzed the data: NJR RM. Contributed reagents/materials/analysis tools: EAL-W JDZ. Wrote the paper: NJR RM JZ. Facilitated permitting and field work in South Africa: RN FVP.
- 1. Frick M, Williams K, Robinson M. Epibionts associated with nesting loggerhead sea turtles (Caretta caretta) in Georgia. Herpetological Review 1998;29:211–214.
- 2. Lazo-Wasem EA, Pinou T, Peña de Niz A, Feuerstein A. Epibionts associated with the nesting marine turtle Lepidochelys olivacea and Chelonia mydas in Jalisco, Mexico: a review and field guide. Bull Peabody Mus Nat Hist 2011;52(2):221–240.
- 3. Robinson NJ, Lazo-Wasem EA, Paladino FV, Zardus JD, Pinou T. In press. Assortative epibiosis of leatherback, olive ridley, and green sea turtles in the Eastern Tropical Pacific. Journal of the Marine Biological Association of the United Kingdom.
- 4. Reich KJ, Bjorndal KA, Frick MG, Witherington BE, Johnson C, Bolton AB. Polymodal foraging in adult female loggerheads (Caretta caretta). Mar Biol 2010;157(1):113–121.
- 5. Pfaller JB, Alfaro-Shigueto J, Balazs G, Ishihara T, Kopitsky K, Mangel JC, et al. Hitchhikers reveal cryptic host behavior: new insights from the association between Planes major and sea turtles in the Pacific Ocean. Mar Biol 2014;161:2167–2178.
- 6. Greenblatt RJ, Work TM, Balazs GH, Sutton CA, Casey RN, Casey JW. The Ozobranchus leech is a candidate mechanical vector for the fibropapilloma-associated turtle herpesvirus found latently infecting skin tumors on Hawaiian green turtles (Chelonia mydas). Virol 2004;1(1):101–110.
- 7. Bunkley-Williams L, Williams EH Jr, Horrocks JA, Horta HC, Mignucci-Giannoni AA, Poponi AC. New leeches and diseases for the hawksbill sea turtle and the West Indies. Comp Parasitol 2008;75(2):263–270.
- 8. Wahl M, Mark O. The predominantly facultative nature of epibiosis: experimental and observational evidence. Mar Ecol Prog Ser 1999;187:59–66
- 9. Frick MG, Pfaller JB. Sea turtle epibiosis. In: Wyneken J, Lohmann KJ, Musick JA, editors. The Biology of Sea Turtles Vol. 3. Boca Raton: CRC Press; 2013. pp. 399–426.
- 10. Sentíes A, Espinoza-Avalos J, Zurita JC. Epizoic algae of nesting sea turtles Caretta caretta (L.) and Chelonia mydas (L.) from the Mexican Caribbean. Bull Mar Sci 1999;64(1):185–188.
- 11. Frankovich TA, Sullivan MJ, Stacy MI. Tursiocola denysii sp. nov. (Bacillariophyta) from the neck skin of loggerhead sea turtles (Caretta caretta). Phytotaxa 2015a;234(3):227–236.
- 12. Majewska R, Santoro M, Bolaños F, Chaves G, De Stafano M. Diatoms and other epibionts associated with olive ridley (Lepidochelys olivacea) sea turtles from the Pacific Coast of Costa Rica. PLoS ONE 2015a;10(6):e0130351.
- 13. Hazrin-Chong NH, Manefield M. An alternative SEM drying method using hexamethyldisilazane (HMDS) for microbial cell attachment studies on sub-bituminous coal. J Microbiol Methods 2012;90(2):96–99. pmid:22561094
- 14. Grunow A (1860) Ueber neue oder ungenügend gekannte Algen. Erste Folge, Diatomeen, Familie Naviculaceen. Verhandlungen der Kaiserlich-Königlichen Zoologisch-Botanischen Gesellschaft in Wien 10: 503–582.
- 15. Frenguelli J, Orlando HA (1958) Diatomeas y Silicoflagelados del Sector Antartico Sudamericano. Instituto Antartico Argentino Publicacion No. 5, Buenos Aires. 191 pp.
- 16. Hustedt F. Diatomeen aus der Antarktis und dem Südatlantik. In: Deutsche Antarktische Expedition 1938/39 1958; 2:103–191.
- 17. Holmes RW (1985) The morphology of diatoms epizoic on Cetaceans and their transfer from Cocconeis to two new genera, Bennettella and Epipellis. Brit Phycol J 20: 43–57.
- 18. Giffen MH (1970) New and interesting marine and littoral diatoms from Sea Point, near Cape Town, South Africa. Botanica Marina 13: 87–99.
- 19. Round FE, Crawford RM, Mann DG (1990) The diatoms: Biology and Morphology of the Genera. Cambridge University Press, Cambridge, 747 pp.
- 20. Holmes RW, Nagasawa S, Takano H (1993) The morphology and geographic distribution of epidermal diatoms of the Dall's porpoise (Phocoenoides dalli True) in the Northern Pacific Ocean. Bulletin of the National Science Museum, Series B (Botany), Tokyo, 19: 1–18
- 21. Denys L (1997) Morphology and taxonomy of epizoic diatoms (Epiphalaina and Tursiocola) on a sperm whale (Physeter macrocephalus) stranded on the coast of Belgium. Diatom Research 12: 1–18.
- 22. Hasle GR, Syvertsen EE (1997) Marine diatoms. In: Tomas C.R. (Ed.) Identifying Marine Phytoplankton. Academic Press, San Diego, pp. 5–385.
- 23. Witkowski A, Lange-Bertalot H, Metzeltin D (2000) Diatom flora of marine coasts I. In: Lange-Bertalot H (ed) Iconographia Diatomologica. Annotated Diatom Micrographs. Diversity-Taxonomy-Identification. Vol. 7. A.R.G. Gantner Verlag K.G., Ruggell, pp. 1–925.
- 24. Frankovich TA, Sullivan MJ, Stacy MI. Three new species of Tursiocola (Bacillariophyta) from the skin of the West Indian manatee (Trichechus manatus). Phytotaxa 2015b;204(1):33–48.
- 25. Majewska R, Kociolek JP, Thomas EW, De Stefano M, Santoro M, Bolaños F, et al. Chelonicola and Poulinea, two new gomphonemoid diatom genera (Bacillariophyta) living on marine turtles from Costa Rica. Phytotaxa 2015b;233(3):236–250.
- 26. Holmes RW, Nagasawa S, Nemoto T (1989) Epidermal diatoms on the Dall’s porpoise landed at Otsuchi, Iwate, Japan. Otsuchi Marine Research Centre Reports, 15, 15–20.
- 27. Wetzel CE, Van de Vijver B, Cox EJ, De C. Bicudo D, Ector L. Tursiocola podocnemicola sp. nov., a new epizoic freshwater diatom species from the Rio Negro in the Brazilian Amazon Basin. Diatom Res 2012;27(1):1–8.