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A note on the laboratory culture of the benthic foraminifer Cornuloculina balkwilli (MacFadyen)



Genetic studies of the Foraminifera provide valuable insights into marine speciation and biogeography, yet the discovery of vitally needed new genetic markers for this important group is being severely limited by an extreme lack of genetic data. The establishment of a laboratory culture from a single, asexually reproducing foraminifer, will be essential to provide enough pooled genetic material from these unicellular organisms, to facilitate full genome sequencing and genetic marker discovery, using next-generation sequencing techniques.


The aim of this study was to develop a simple and inexpensive method of culturing benthic foraminifera, via asexual reproduction, in a controlled laboratory environment. Individual specimens of the benthic foraminfer Cornuloculina balkwilli (MacFadyen) were placed in 7 cm plastic beakers, containing 50 ml natural seawater, filtered to 0.2 μm, and kept at 23°C, with a 12-hour light/dark cycle, and fed weekly on a mixed algal diet of Dunaliella tertiolecta and Phaeodactylum tricornutum. Asexually derived cultures were successfully established from 4 specimens of Cornuloculina balkwilli, originally added to the culture vessels as immature specimens. Many thousands of individuals were present after 6 months.


The method presented here demonstrates that only basic laboratory equipment is required to establish and maintain a thriving culture of the benthic foraminfer, C. balkwilli, from a single asexually reproducing specimen, providing an excellent source of genetic material for use in next generation sequencing. The method is easily reproducible and will greatly aid in the discovery of critically needed new genetic markers in the Foraminifera. It also highlights C. balkwilli as a good candidate species for use in the field of environmental micropaleontology.



The Foraminifera are a diverse and widespread group of marine protists that are commonly used in genetic studies of marine speciation, biogeography, population genetics, and phylogenetics [1]. There is a pressing need to identify new genetic markers in the Foraminifera, to enhance both phylogenetic analyses and population genetic studies, however, our current ability to amplify and sequence new genes is severely limited by an extreme lack of genetic data for this important group. As foraminifera are unicellular, many individuals must be pooled to provide sufficient genetic material for full genome sequencing or for marker discovery via reduced representation techniques (e.g. S-RAD or RNA-Seq). Whilst specimens may be pooled from natural populations, minor genetic variation could lead to sequence ambiguities. Thus in order to obtain many identical copies of the genome it will be essential to establish a culture system in a controlled laboratory environment, starting from a single, asexually reproducing individual.

A number of species of benthic foraminifera have been successfully maintained (kept without reproduction) or cultured (grown and reproduced for several generations) under laboratory conditions [214]. Some culturing methods require sophisticated equipment, and provide a highly tailored environment [5, 9, 10], whilst others are simple in design, but easy to set up and less time-consuming to maintain [38, 1114].

Our aim was to culture a benthic foraminfer from a single asexually reproducing individual, using a simple culturing system. A basic culture vessel would be used (such as a petri dish or beaker), with light, temperature, and salinity manipulated manually using standard laboratory equipment. A variety of species of benthic foraminifera have been previously cultured, using only simple equipment, including Allogromia laticollaris (Arnold) [3], Allogromia sp.[7], Spiroloculina hyaline (Schulze) [4], Rotaliella heterocaryotica (Grell) [6], Amphisorus hemprichii (Ehrenberg) and Amphistegina lobifera (Larsen) [8], Ammonia tepida (Cushman) [1113], and Ammonia beccarii (Linné) [13, 14]. We selected a British intertidal benthic foraminfer that had not been previously cultured in the laboratory. Cornuloculina balkwilli (MacFadyen), of the suborder Miliolina, has a calcitic, porcellaneous, white test wall, giving it a slightly opaque appearance. The test is oval in outline, compressed, with each chamber forming roughly two thirds of a whorl, and the aperture is simple and terminal [15]. It is thought to be an inner shelf marine species, which is transported into the mouths of muddy estuaries [15], and is considered to be epifaunal, frequently attaching to available substrate, but also able to move freely [16]. The specific life cycle of C. balkwilli has yet to be documented, though as in most benthic foraminifera, it is likely to be dimorphic, consisting of a regular alternation between sexual and asexual generations [6, 9, 1720]. It may even be trimorphic, involving the addition of successive asexual generations [9, 13, 2127], which would be particularly favourable in terms of growing a large culture rapidly in the laboratory.


Collection of specimens

Live specimens of the benthic foraminfer Cornuloculina balkwilli (MacFadyen) were collected in May 2009, from tidal mudflats at Mow Creek, Brancaster Staithe, in Norfolk, UK (Figure 1). Samples were collected at low tide, from a 6 m2 site located approximately 1 – 2 m from the low-tide line. A thin layer of surface sediment was collected by hand (to a depth of ~ 1 cm), from areas where a greenish layer of algae (a food source for the foraminifera) was observed. The sediment was washed through a 106 μm sieve, with seawater collected from the same location, to concentrate the foraminifera and remove smaller particulate matter. Seawater to be used for the laboratory maintenance of the foraminifers was collected from the same location at high tide.

Figure 1
figure 1

Sampling location (indicated by a grey star) from which live specimens of the benthic foraminifer, Cornuloculina balkwilli, were collected. The site is a tidal mudflat located in Mow Creek, at Brancaster Staithe, in North Norfolk, United Kingdom.

Laboratory culture

In the laboratory, 10 healthy specimens of Cornuloculina balkwilli (indicated by their bright orange cytoplasm and evidence of movement) were picked from the sediment, 5 of which were mature adults (~ 330 μm) and 5 of which were juveniles (~ 160 μm). Individual specimens were cleaned using a paintbrush (to remove bacteria and algae from the shell), before being placed in separate 7 cm plastic beakers containing 50 ml natural seawater, filtered to 0.2 μm as in [11, 28], and covered with a loose lid to minimize evaporation. Using a 0.2 μm filter was necessary to prevent tiny gametes of the foraminifera (potentially carried in the seawater) from transferring through to the culture vessels, an important precaution if you wish to be certain that only asexual reproduction has occurred. Samples were kept at 23°C (ambient laboratory temperature), with lighting of 2 × 20w for twelve hours per day, regulated by an automated timer. Water salinity was maintained at between 27 – 30‰, within the natural range of the estuary collection site, with two thirds of the water volume replaced every 2 weeks. Specimens were fed weekly with a combination of the algal species Dunaliella tertiolecta (500 μl liquid culture) and Phaeodactylum tricornutum (5 drops liquid culture; Sciento, Manchester). Observations were carried out under a Nikon SMZ1500 light microscope after 3 and 6 months, and photographs taken using Nikon DXM1200F camera.

Results and discussion

The mature adult specimens of Cornuloculina balkwilli used in this study measured approximately 330 μm in size, with a proloculus of ~37 μm (Figure 2A). Observations of the mature adult specimens, taken after 3 months in the culture vessels, revealed that 3 out of 5 had died without reproducing, whilst the remaining 2 appeared healthy, but had also failed to reproduce. After 6 months, even these remaining 2 specimens had died, again with no evidence of reproduction.

Figure 2
figure 2

Light microscope photographs of the benthic foraminifer Cornuloculina balkwilli in culture. A, Adult specimen; B, Many different sized specimens from the culture population, indicating multiple reproductive events; C, large juvenile produced in culture; D, Juvenile specimens attached to an algal substrate.

Observations of the specimens originally added as juveniles, however, revealed evidence of reproduction from a single individual. The offspring observed were most likely produced via asexual reproduction, commonly seen in the foraminifera as part of a dimorphic or trimorphic life-cycle [17], although autogamy (self-fertilisation), which has been observed in some foraminifera [17], cannot be ruled out without further investigations.

After 3 months, thriving populations had established in 4 out of 5 culture vessels (the remaining specimen had died). Hundreds of individuals of many different sizes were present in the 4 successful cultures, indicating multiple reproductive events (Figure 2B). The smallest juveniles were approximately 60 – 70 μm in size, whilst some had grown up to ~220 μm (Figure 2C). The specimens had attached in large numbers to an algal substrate that had formed in the culture vessels (presumably from the Dunaliella tertiolecta and Phaeodactylum tricornutum added as food) (Figure 2D), and were heavily crowded, making it impossible to determine how many young were produced per brood. After 6 months the 4 successful cultures were all still thriving and reproducing, each containing many thousands of individuals.

It is interesting to note that only individuals that were added as immature specimens reproduced. It has been observed, in the benthic foraminifer Ammonia tepida, that any mature adults surviving after a reproductive event do not usually reproduce again [12]. It is possible that the large adult specimens of C. balkwilli collected from the field had already reproduced in their natural habitat, and would therefore not do so again, making it preferable to choose less mature specimens for culture experiments.

These experiments demonstrate that it is possible to successfully culture specimens of the benthic foraminfer Cornuloculina balkwilli from a single individual via asexual reproduction, and also show that only basic laboratory equipment is required, negating the need for sophisticated systems such as circulating and re-circulating marine aquaria and other flow-through systems or expensive commercially available chemostats. A continuously reproducing culture of C. balkwilli was obtained and will be maintained and ultimately used as a source of RNA and DNA for molecular marker discovery using next generation sequencing technologies. In addition, we propose that the simple culturing method used here, together with the species C. balkwilli would be ideally suited for use in environmental micropaleontological studies, for which laboratory cultures of other benthic foraminiferal species have already proven invaluable [11, 2934].

As a technical point, it should be noted that the DNA of the food organisms added to the culture system could become a source of contamination during subsequent molecular procedures. The genomic DNA of the food organisms would therefore need to be sequenced alongside that of the foraminifera, for comparison and elimination from further analysis. The same would apply if any symbionts or commensals were present, though this is not thought to be the case in this particular species. To minimize contamination from prey particles, all foraminifers should be kept without food for a period of time prior to the extraction of their DNA/RNA.


  1. Darling KF, Wade CM: The genetic diversity of planktic foraminifera and the global distribution of ribosomal RNA genotypes. Mar Micropaleontol. 2008, 67: 216-238. 10.1016/j.marmicro.2008.01.009.

    Article  Google Scholar 

  2. Anderson OR, Lee JJ, Faber WW: Collection, maintenance and culture methods for the study of living foraminifera. Biology of Foraminifera. Edited by: Lee JJ, Anderson OR. 1991, San Diego: Academic press, 335-357.

    Google Scholar 

  3. Arnold ZM: Culture methods in the study of living Foraminifera. Paleontol. 1954, 28: 404-416.

    Google Scholar 

  4. Arnold ZM: Biological observations of the foraminifer Spiroloculina hyaline Schulze. Univ Calif Publ Zool. 1964, 72: 1-92.

    Google Scholar 

  5. Arnold ZM: Field and laboratory techniques for the study of living foraminifera. Foraminifera. Edited by: Hedley RH, Adams CG. 1974, London: Academic Press, 153-206.

    Google Scholar 

  6. Grell KG: Der Generationswechsel der Polyalamen Foraminifera Rotaliella heterocaryotica. Archiv der Protistk. 1954, 100: 211-235.

    Google Scholar 

  7. Lee JJ, Pierce S: Growth and physiology of foraminifera in the laboratory: Part 4: Monoalgal culture of an allogromiid with notes on its morphology. J Protozool. 1963, 10: 404-411. 10.1111/j.1550-7408.1963.tb01697.x.

    Article  Google Scholar 

  8. Lee JJ, Sang K, ter Kuile B, Strauss E, Lee PJ, Faber WW: Nutritional and related experiments on laboratory maintenance of 3 species of symbiont-bearing, large foraminifera. Mar Biol. 1991, 103: 417-425.

    Article  Google Scholar 

  9. Lee JJ, Faber WW, Anderson OR, Pawlowski J: Life cycles of foraminifera. Biology of Foraminifera. Edited by: Lee JJ, Anderson OR. 1991, New York: Academic Press, 285-334.

    Google Scholar 

  10. Lutze GF, Wefer G: Habitat and asexual reproduction of Cyclorbiculina compressa (Orbigny), Soritidae. J Foram Res. 1980, 10: 251-260. 10.2113/gsjfr.10.4.251.

    Article  Google Scholar 

  11. Morvan J, Le Cadre V, Jorissen F, Debenay JP: Foraminifera as potential bio-indicators of the “Erika” oil spill in the Bay of Bourgneuf: Field and experimental studies. Aquat Living Resour. 2004, 17: 317-322. 10.1051/alr:2004034.

    Article  CAS  Google Scholar 

  12. Stouff V, Geslin E, Debenay JP, Lesourd M: Origin of morphological abnormalities in Ammonia (Foraminifera): Studies in laboratory and natural environments. J Foram Res. 1999, 29: 152-170.

    Article  Google Scholar 

  13. Stouff V, Lesourd M, Debenay JP: Laboratory observations on asexual reproduction (schizogony) and ontogeny of Ammonia tepida with comments on the life cycle. J Foram Res. 1999, 29: 75-84.

    Article  Google Scholar 

  14. Takata H, Dettman DL, Seto K, Kurata K, Hiratsuka J, Khim B: Novel habitat preference of Ammonia “Beccarii” forma 1 in a macrobenthos community on hard substrates in the Ohashi River, Southwest Japan. J Foram Res. 2009, 39: 87-96. 10.2113/gsjfr.39.2.87.

    Article  Google Scholar 

  15. Murray JW: British Nearshore Foraminiferids. Synopses of the British Fauna. Edited by: Kermack DM, Barnes RSK. 1979, New York, London: Academic press, 1-68. Volume 16

    Google Scholar 

  16. Sturrock S, Murray JW: Comparison of low energy and high energy marine middle shelf foraminiferal faunas. Microfossils from Recent and fossil shelf seas: Celtic Sea and western English Channel. Edited by: Neale JW, Brasier MD. 1981, Chichester: Ellis Horwood, 251-260.

    Google Scholar 

  17. Goldstein ST: Foraminifera: A biological Overview. Modern Foraminifera. Edited by: Gupta BKS. 2002, Boston: Kluwer Academic Publishers, 37-55.

    Google Scholar 

  18. Jepps MW: Studies on Polystomella Lamarck (Foraminifera). J Mar Biol Assoc UK. 1942, 25: 607-666. 10.1017/S0025315400055168.

    Article  Google Scholar 

  19. Lister JJ: Contributions to the life history of the Foraminifera. Phil Trans Roy Soc B. 1895, 186: 401-453. 10.1098/rstb.1895.0008.

    Article  Google Scholar 

  20. Schaudinn P: Uber den Dimorphisms bei Foraminiferen. SitzBer Gesell naturf Freunde Berlin. 1895, 5: 87-97.

    Google Scholar 

  21. Bradshaw JS: Laboratory studies of the rate of growth of the Foraminifera “Streblus beccarii (Linné) var. tepida (Cushman)”. J Paleontol. 1957, 31: 1138-1147.

    Google Scholar 

  22. Dettmering C, Rottger R, Hohenegger J, Schmalijohann R: The trimorphic life cycle in foraminifera: Observations from cultures allow new evaluation. Eur J Protistol. 1998, 34: 363-368. 10.1016/S0932-4739(98)80004-7.

    Article  Google Scholar 

  23. Goldstein ST: On the life cycle of Saccammina alba Hedley, 1962. J Foram Res. 1988, 18: 311-325. 10.2113/gsjfr.18.4.311.

    Article  Google Scholar 

  24. Goldstein ST, Moodley L: Gametogenesis and life cycle of the foraminifer Ammonia beccarii (Linné) forma tepida (Cushman). J Foram Res. 1993, 1999 (23): 213-220.

    Article  Google Scholar 

  25. Harney JN, Hallock P, Talge HK: Observations on a trimorphic life cycle in Amphistegina gibbosa populations from the Florida Keys. J Foram Res. 1998, 28: 141-147.

    Article  Google Scholar 

  26. Leutenegger S: Reproduction cycles of larger foraminifera and depth distribution of generations. Utre Micropal Bull. 1977, 15: 26-34.

    Google Scholar 

  27. Röttger R, Kruger R, De Ruk S: Trimorphism in foraminifera (Protazoa): Verification of an old hypothesis. Eur J Protistol. 1990, 25: 226-228. 10.1016/S0932-4739(11)80173-2.

    Article  PubMed  Google Scholar 

  28. de Nooijer LJ, Langer G, Nehrke G, Bijma J: Physiological controls on seawater uptake and calcification in the benthic foraminifer Ammonia tepida. Biogeosciences. 2009, 6: 7083-7102. 10.5194/bgd-6-7083-2009.

    Article  Google Scholar 

  29. Bernhard JM, Mollo-Christensen E, Eisenkolb N, Starczak VR: Tolerance of allogromiid Foraminifera to severely elevated carbon dioxide concentrations: Implications to future ecosystem functioning and paleoceanographic interpretations. Global Planet Change. 2009, 65: 107-114. 10.1016/j.gloplacha.2008.10.013.

    Article  Google Scholar 

  30. Fujita K, Hikami M, Suzuki A, Kuroyanagi A, Sakai K, Kawahata H, Nojiri Y: Effects of ocean acidification on calcification of symbiont-bearing reef foraminifers. Biogeosciences. 2011, 8: 2089-2098. 10.5194/bg-8-2089-2011.

    Article  Google Scholar 

  31. Munsel D, Kramar U, Dissard D, Nehrke G, Berner Z, Bijma J, Reichart GJ, Neumann T: Heavy metal incorporation in foraminiferal calcite: Results from multi-element enrichment culture experiments with Ammonia tepida. Biogeosciences. 2010, 7: 2339-2350. 10.5194/bg-7-2339-2010.

    Article  CAS  Google Scholar 

  32. Saraswat R, Kurtarkar SR, Mazumder A, Nigam R: Foraminifers as indicators of marine pollution: A culture experiment with Rosalina leei. Mar Pollut Bull. 2004, 48: 91-96. 10.1016/S0025-326X(03)00330-8.

    Article  PubMed  CAS  Google Scholar 

  33. Toyofuku T, Kitazato H: Micromapping of Mg/Ca values in cultured specimens of the high-magnesium benthic foraminifera. Geochem Geophys Geosyst. 2005, 6: Q11P05-doi:10.1029/2005GC000961

    Article  Google Scholar 

  34. Williams DF, Röttger R, Schmaljohann R, Keigwin L: Oxygen and carbon isotopic fractionation and algal symbiosis in the benthic foraminiferan heterostegina depressa. Palaeogeogr Palaeoclimatol Palaeoecol. 1981, 33: 231-251. 10.1016/0031-0182(81)90041-9.

    Article  Google Scholar 

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We would like to thank Dr Ian Boomer and Professor John Murray for their help in identifying foraminiferal species from the field. This work was supported by the BBSRC (PhD studentship BBS/S/K/6 2004/11230).

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Correspondence to Christopher M Wade.

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Authors’ contributions

HAS and CMW conceived and designed the study. HAS and CMW conducted the fieldwork. HAS conducted the laboratory work. HAS and CMW analyzed data. HAS and CMW wrote the manuscript. Both authors read and approved the final manuscript.

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Seears, H.A., Wade, C.M. A note on the laboratory culture of the benthic foraminifer Cornuloculina balkwilli (MacFadyen). BMC Res Notes 6, 369 (2013).

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