Ciliophorans are some of the most intriguing and diverse protists on the planet, having evolved many ways in which to exploit their surrounding marine environment. Aspects about their morphology, behaviour and evolution show surprising convergences with the Metazoans, despite their phylogenetic distance (Hausmann & Bradbury, 1996). The genus being described in this webpage is a colonial sessile ciliate known as Zoothamnium, which displays primitive cellular specialisation, settlement selection and an advanced reaction to stimulus in comparison to many other protists and even basal metazoans. Zoothamnium sp. was frequently found on the collected sediment plates within Moreton Bay, however due to their small size, they are often unnoticed or overlooked. This webpage aims to, possibly for the first time, compile all available knowledge about this genus and provide images as well as footage of a live specimen.             

External morphology

Ciliophoran protists are easily differentiated from other protists due to the presence of cilia which is used for locomotion and/or feeding (Brusca et al., 2016). Although most ciliates occur as mobile single cells, the genus being explored in this webpage is a sessile branching colony. Zoothamnium sp. can be easily identified into its subclass as a Peritrich due to its external morphology. Peritrichs have a central stalk connected to branching colonies often with ten to hundreds of individual zooids, allowing for an oral field covering the entire surface of the body (Hausmann & Bradbury, 1996)(Fig. 1). Despite being ciliates, Zoothamnium have greatly reduced stomatic ciliature as they use cilia for feeding rather than locomotion. Their central stalk is used for settlement as well as avoidance, contracting in response to a hazard in a zig-zag fashion (as can be seen in Fig 3).
Zoothamnium, like other ciliates, are polymorphic, meaning they have multiple physical forms of the same cell, which may be an early step towards cell specialisation seen in Metazoans. This is discussed further in Evolution and Systematics. Both macro and micro-zooids can be observed in Figure 2, with macro-zooids being located on the central stalk and micro-zooids located on the branching stalks (Herron et al., 2013). Micro-zooids are primarily specialised for feeding using cilia, with the terminal zooids (on the ends of the branches) being specialised for colony expansion via sexual reproduction (see Anatomy and Physiology). The macro-zooids can turn into swarmer cells (telotrochs) which are used in asexual reproduction (see Life History and Behaviour).

Size and colouration

Most protists are microscopic, so the ciliate protist Zoothamnium sp. is often overlooked by those without a keen eye. Despite ranging from 1.5cm to less than 1mm, in the scientific community the Zoothamnium sp. are known as some of the largest protists, often being referred to as ‘giants’ (Bright et al., 2014). In this case these protists can be larger than some metazoans (e.g., Loriciferans and others) (Ruppert et al., 2004).
Ciliates lack pigmentation, with the exception of symbiotic algae in some species which provide a brown/green tinge. Zoothamnium sp. from low magnification looks entirely clear (Fig. 1) however under higher magnification a symbiotic colouration was observed in the collected individual (Fig. 2). The symbiotic relationships of Zoothamnium are further explored in Anatomy and Physiology.

Feeding and locomotion

Being a sessile ciliate, Zoothamnium uses filter/suspension feeding to gain their nutrition for growth and reproduction. Each micro-zooid contains complex oral cilia which they use to feed on bacteria, diatoms and detritus (Ruppert et al., 2004) (Fig. 4). Studies on Zoothamnium intermedium have found that they have a predominantly algal or bacterium-based diet and can maintain the same growth rate regardless of which diet they use (Utz, 2008).

Zoothamnium sp. have a generally sedentary lifestyle, excluding the dispersal reproductive stage. Zoothamnium pelagicum is the only free-floating species of Peritrich ciliates and completes its whole life cycle in the open ocean (Gomez, 2017). When the reproductive stage of sessile ciliates occurs, the asexual cells known as telotrochs develop external cilia to aid in controlled movement which allows for settlement selection (Utz & Coats, 2008). Like many marine invertebrate larvae, settlement selection is of high importance for sessile Peritrichs as they need to settle in a prime area to survive without mobility.

Settlement selection

There have been several studies into habitat selection of telotrochs in Zoothamnium sp. One study on Zoothamnium niveum demonstrated that settlement success greatly depended on sulphide in the area, with more colonies settling in higher sulphide environments (Rinke et al., 2007). This may have been largely due to the symbiotic chemoautotrophic bacteria that is commonly observed on this particular species, which was not observed on the collected specimen.
Another study found that Zoothamnium intermedium was potentially an obligate epibiont as they settled on host organisms (for no obvious benefit) rather than on glass or plastic containers (Utz & Coats, 2008). Zoothamnium sp. have been commonly observed as epibionts on arthropods and even some fish (Hausmann & Bradbury, 1996; Utz& Coats, 2008). However there seems to be no direct benefit to the epibiont in these situations, suggesting that these organisms may emit a chemical cue which enhances telotroch settlement and growth. Chemical signalling in relation to conspecifics has also been studied and is further discussed in Behaviour.
Habitat requirements may also influence settlement location of sessile Peritrichs as it has been found that reduced light and limited wave action enhanced growth and proliferation of a sister genus (Vorticelids) (Langlois, 1975). Ciliates have also been known to have primitive visual spots to detect light and dark which can be used for phototaxis in telotrochs (Hausmann & Bradbury, 1996).

Behaviour
Despite being a sessile protist, there are several aspects of Zoothamnium behaviour that we can explore. The response to its surrounding environment through the contraction of its stalk can occur in the time scale of milliseconds, which can be argued to be much faster than many basal metazoans (Hausmann & Bradbury, 1996) (refer to the below footage). The mechanics behind this rapid contraction is further discussed in Anatomy and Physiology. This movement/avoidance behaviour of ciliates has been studied in terms of both desensitisation and habituation, both of which provide a primitive indication of cellular learning. It was found that the decreasing of a motor response (avoidance) in relation to a non-harmful negative stimulus has been observed consistently in contractile ciliates such as Zoothamnium, indicating habituation and therefore potential cellular learning (Hausmann & Bradbury, 1996).

Video depicting the contractile movements of Zoothamnium taken from a hand held camera in the lab. The specimen was living in a small amount of seawater with a coverslip to protect it. 


During the motile phase of the Zoothamnium life cycle, the telotroch (swarmer cell) exhibits primitive search and settlement behaviour. Studies have suggested the potential for chemical cues to be involved with settlement, as Peritrich telotrochs have been observed to settle near conspecifics (similar to metazoan larvae) (Langlois, 1975).
Settlement was not physically observed in the lab, however Zoothamnium individuals were observed within close proximity to one another and not commonly in their own space. This potentially supports conspecific chemical cues, however current evidence for this is inconclusive (Hausmann & Bradbury, 1996). Alternatively, this observed pattern may be due to settlement in a prime location based on other cues. For example, a study by Langlois in 1975, suggests that a bacterial film was a requirement for settlement of Zoothamnium and indicated a possible chemical attraction and/or food discrimination. This would show a potential behavioural aspect to settlement including sensory cues commonly seen in metazoan larvae.

Natural History
Zoothamnium sp. are predominantly asexual reproducers, with the capabilities of sexual reproduction through conjugation. Conjugation involves genetic recombination between the ‘conjugants’ of two individuals through a cytoplasmic exchange of genetic material via one haploid micronucleus to the other (Brusca et al., 2016). This produces a diploid nucleus in each conjugant which can be interpreted as analogous to cross-fertilisation in some metazoan invertebrates (Brusca et al., 2016).There have been few studies on sexual reproduction of Zoothamnium, mainly due to the lack of this process being observed (both in the lab and natural environment). A study by Summers in 1938 indicated that conjugation may occur during ‘epidemics’ during which individuals would synchronise and their cells would specialise further for sexual reproduction (see Anatomy and Physiology), but the extent or time in which these periods occur is largely unknown.

Asexual reproduction is far more commonly observed in Zoothamnium sp. and has distinct life stage phases (Fig. 7) A fully developed colony has three main cell types (discussed further in Anatomy and Physiology). The basic cell types are macro-zooids, micro-zooids and terminal zooids (Fig. 6). Macro-zooids are used in asexual reproduction and are a product of unequal binary fission common in sessile ciliates known as budding (Brusca et al., 2016). In Zoothamnium, it is common for multiple macro-zooids to be formed and released simultaneously as they separate from the main colony as swarmer (telotroch) cells (Fig. 5)(Summers,1938; Herron et al 2013). These swarmer cells then commence a ‘migratory’ pelagic phase in which they are mobile swimmers for a few hours before settling and attaching to the substrate by growing a stalk (Summers, 1938; Brusca et al., 2016). This apical cell then makes its first divide approximately 8 hours after settling, giving rise to its maximum size from 5 to 8 days after the first divide (Summers, 1938).
Despite its potential for exponential growth, the development of various parasites has been found to limit the growth of colonies beyond 10 days in both lab and natural conditions (Faure-Fremiet, 1930). Once the colony stops growing a period of senescence (growth arrest) occurs, with cell death gradually occurring from this point, evident from the stems towards the bottom of the organism deteriorating first due to their age (Bright et al., 2014)(Fig. 7).

Body plan and cellular specialisation
The body plan of Zoothamnium, as with all sessile Peritrichs, is optimised for a sedentary lifestyle. The division plane is parallel with the main axis of their body (usually vertical) with the daughter cells (macro-zooids) adhering to the central axis of the body until developed (Hausmann & Bradbury, 1996)(Fig. 8).
A cellular aspect that allows for both sexual and asexual reproduction in these organisms is nuclear dimorphism, which is present in all ciliates. In Zoothamnium each zooid or cell includes two types of nuclei. A large macronucleus controls the physiological/general homeostasis of the cell and a small micronucleus is used to maintain genetic diversity and is therefore only used in sexual reproduction (Huasmann & Bradbury, 1996; Brusca et al., 2016).

As briefly discussed above Zoothamnium is polymorphic, and throughout their life cycle six types of cells (zooids) may be formed, although not always at the same time (Summers, 1938).The nutritive micro-zooids are the most common cells and make up most of the colony. They are specialised for feeding and contain oral cilia which spin in a counter-clockwise direction for food capture (Summers, 1938; Hausmann & Bradbury, 1996). Terminal micro-zooids are present on the end of every branch, halting the progression of growth. These terminal micro-zooids are relatively indistinguishable from other micro-zooids. During sexual reproduction, the terminal micro-zooids can develop into micro-gamonts which become larger and develop locomotive cilia to break away from the colony and proceed with conjugation (Summers, 1938; Herron et al., 2013)(Fig.6). 
Macro-zooids are also present on almost all specimens observed of Zoothamnium and develop into swarmers (telotrochs) once they leave the parent cell for asexual reproduction as described above. A terminal macro-zooid is usually present at the top of the organism, however diminishes with growth, and beyond twenty branches it becomes indistinguishable. This was the case with the collected specimen. These terminal macro-zooids can develop into macro-gamonts which are possibly used in sexual reproduction, however it has never been witnessed (Summers, 1938).
 

Nutrition and Symbiosis In Zoothamnium each micro-zooid (excluding the terminal micro-zooids) has its own nutrition and energy sequestration system. Every feeding specialised micro-zooid has a buccal cavity which food is brought to via the oral ciliary organelles that can be seen in Figure 9 (Ruppert et al., 2004). From the buccal cavity the food particles are driven into the cytostome, which is a dedicated endocytic area of the cell which is free of cilia (Ruppert et al., 2004). From the cytostome the food particles pass through the cytopharynx and collected in a vacuole to be digested (Ruppert et al., 2004; Brusca et al., 2016). There is observed similarity in the physical morphology of feeding structures of the micro-zooids within the genus of Zoothamnium, despite the varying body morphology types (Wu et al., 2020)(Fig. 10). This shows that the conical feeding zooid with oral cilia is highly successful at food capture for these sessile ciliates. 

Ciliates are known to have symbiotic relationships as both the host and the epibiont. This is especially the case for Zoothamnium as they have been recorded to host chemoautotrophic bacteria as well as being non-invasive epibionts on arthropods (Hausmann & Bradbury, 1996; Bright et al., 2014). Zoothamnium niveum in particular has been recorded to frequently develop a relationship with a sulphide oxidising bacterium, allowing the protist to thrive in sulphide rich environments (Rinke et al., 2007). The specimen in this study did not have any visually evident chemoautotrophic bacteria. Algal symbiosis has also been recorded in some species of Peritrich, with each endosymbiotic algal cell located in a separate vacuole (Graham & Graham, 1978). This may explain the pigmentation observed in the collected specimen, however this was not investigated further.

Colony Cell Communication
When observed, Zoothamnium individuals appear to respond to negative stimulus in a similar way to metazoans, with a nerve-stimulated reaction. However, these organisms have no nerves or even a primitive neural network. Despite this, they are able to react to stimulus within a millisecond by using changes in membrane potential to trigger a mechanoreceptor response. This is explained by calcium action potential, which is known as the most remarkable electrical signal in all the ciliates,  responsible for the fastest reaction times related to calcium influx (Hausmann & Bradbury, 1996).
A spasmoneme, which has has protein filaments which shorten rapidly upon exposure to calcium, is commonly used in ciliates to detect these calcium changes (Fig. 8)(Ruppert et al., 2004). Zoothamnium sp. can quickly react to negative stimulus as it uniquely has a spasmoneme that runs uninterrupted through the entire colony, allowing it to contract and expand as a singular entity (Schuster & Bright, 2016). 

Local habitat

The specimen of Zoothamnium used for this page was collected from Moreton Bay, QLD from a sediment plate suspended in the water column (see Fig.11). From observations in the lab, Zoothamnium was most common on the edges of the submerged plates and hardly observed on the top or bottom of the plates. This distribution may be influenced by the need for sunlight, as some species of sessile Peritrchs contain endosymbiotic algae which provide energy to the host (Graham & Graham, 1978). It is however unclear if this relationship occurs in Zoothamnium.
Another explanation for this distribution is access to food sources, as it was observed that more algae was growing on the sides of the plate compared to the top and bottom, which may be their primary food source. Zoothamnium niveum has been found to proliferate areas with high algal/bacterial content such as hard substrate close to seagrass, directly on the seagrass, sunken wood and mangrove peat where degrading vegetal debris is present (Bright et al., 2014).

Global distribution

Zoothamnium sp. has been found in areas of varying salinity, pH and water temperatures in the marine environment. In a study of ciliates in the South China Sea, almost all the Zoothamnium species were found in mangrove environments (Hu et al., 2019). They have also been observed in the Indian Ocean and distribution was influenced by suitable substrate availability rather than water quality or salinity (Munir & Sun, 2018). Studies into the distribution of Zoothamnium niveum have shown occurrences along most shallow areas of the tropical/sub-tropical regions of the globe (Fig. 12) (Bright et al., 2014).

Phylogeny case study: convergent traits between Zoothamnium sp. and Metazoans

The Ciliophorans, which sit in the clade of the Alveolates, are phylogenetically very distant to the Metazoans (the Holozoan clade) (Fig. 13). Despite this distance, ciliophorans appear to share some similarities with the Metazoans, suggesting the evolution of multiple convergent traits. One of the most obvious convergences, particularly pronounced in Zoothamnium, is the convergent development of multicellularity. Studies have shown that multicellularity has evolved separate from the Metazoans several times (Sebe-Pedros et al., 2017). Zoothamnium not only has multicellularity but primitive cell specialisation and organisation, suggested to equal or even surpass the complexity seen in specialised tissue cells (Hausmann & Bradbury, 1996).
However, one of the most surprising convergences between the Metazoans and Zoothamnium is their sensory perception and motility. Primitive eye spots, calcium action potentials and chemical receptors can generate behaviour at an organisational level in the ciliates which is comparable to small metazoans (Hausmann & Bradbury, 1996). In the case of the calcium action potentials which provide mobility, they can react to stimulus within milliseconds, a much shorter time frame than most basal Metazoans. These calcium action potentials are also present in Metazoan tissue (e.g. soma of neurons, skeletal muscle and smooth muscle), indicating that this primitive sensory system evolved within the animal kingdom more than once.

Classification and systematics

Major Clade: Alveolate (Cavalier-Smith, 1991)
Phylum: Ciliophora (Doflein, 1901)
Subphylum: Intramacronucleata (Lynn, 1996)
Class: Oligohymenophorea (de Puytorac et al, 1974)
Subclass: Peritrichia (Stein, 1859)
Order: Sessilida (Stein, 1933)
Family: Zoothaminiidae (Sommer, 1951)
Genus: Zoothamnium (Bory de St. Vincent, 1824)

The genus Zoothamnium was originally within the family of Vorticellae, classified by Ehrenberg in 1838, however due to the zig-zag contractile nature of the stalk it was separated into its own family by Sommer in 1951 (Clamp & Williams, 2006). The family Vorticellae included species which contracted in a cylindrical fashion and did not have colonies of zooids like the Zoothamnium (Hausmann & Bradbury, 1996).

Conservation actions are quite limited within the general area of protozoa, mainly due to the lack of understanding of their importance in the wider ecosystem. Ciliates have high significance in marine ecosystems, with observations of their feeding habits indicating that they play an important role in reducing the number of bacteria and protophytes in marine sediments (Langlois, 1975). In natural environments where nutrients are limiting, protozoa may also play an important role in concentrating bacteria which increases organic compounds (Lackey, 1936). Zoothamnium in particular also plays an important role in the marine food chain, as it has been found that young snails and copepods preyed heavily on both experimental and natural populations of species of Zoothamnium (Spoon, 1967). It is therefore important to continue with comprehensive studies into population changes and threats on marine ciliates such as Zoothamnium. Due to a wide understanding of their life history, their sessile behaviour and proliferation on sediment plates, Zoothamnium could be used as a representative for conservation in the world of the Protozoa. 

Bright, M., Espada-Hinojosa, S., Lagkovardos, I.& Volland, J. 2014. The giant ciliate Zoothamnium niveum and its thiotrophic epibiont Candidatus Thiobios zoothamnicoli: a model system to study interspecies cooperation. Frontiers in Microbiology 5(145): 1-13.

Brusca, R. C., Moore, W. & Shuster,S. M. 2016. Invertebrates. Third Ed. Sinauer Associates Inc USA.

Clamp, J. C. & Williams, D. 2006. A Molecular Phylogenetic Investigation of Zoothamnium (Ciliophora, Peritrichia, Sessilida). Journal of Eukaryotic Microbiology 53(6): 494-498.

Faure-Fremiet, E. 1930. Growth and Differentiation of the Colonies of Zoothamnium alternans (Clap. And Lachm.). Biological Bulletin 58(1): 28-51.

Gomez, F. 2017. Motile behaviour of the free-living planktonic ciliate Zoothamnium pelagicum (Ciliophora. Peritrichia). European Journal of Protistology 59:65-74.

Graham, L. E. & Graham, J. M. 1978. Ultrastructure of Endosymbiotic Chlorella in a Vorticella. Journal of Protozoology 25(2): 207-210.

Hausmann, K. & Bradbury, P. C.1996. Ciliates: Cells as Organisms. Gustav Fischer USA.

Herron, M.D., Arshidi, A., Shelton, D. E. & Driscoll, W. W. 2013. Cellular differentiation and individuality in the ‘minor’ multicellular taxa: Differentiation and individuality. Biological Reviews 88(4):844-861.

Hu, X., Lin, X. & Song W. 2019. Class Oligohymenophorea. In: Hu X., Lin X., Song W. (eds) Ciliate Atlas: Species Found in the South China Sea. Springer, Singapore.

Lackey, J. B. 1936. Occurrences and distribution of the marine protozoan species in the Woods Hole area. Biological Bulletin 70:264-278.

Langlois, G. A. 1975. Effect of Algal Exudats on Substratum Selection by Motile Telotrochs of the Marine Peritrich Ciliate Vorticella marina. The Journal of Protozoology 22(1):115-123.

Munir, S. & Sun, J. 2018. The first snapshot study on horizontal distribution and identification of five peritrich ciliates (Genus Vorticella Linnaeus and Zoothamnium Bory de St.Vincent) from the eastern Indian Ocean. Acta Oceanologica Sinica 37(10):79-85.

Rinke, C., Lee, R., Katz, S. & Bright, M. 2007. The effects of sulphide on growth and behavior of the thiotrophic Zoothamnium niveum symbiosis. Proceedings of the Royal Society B 274: 2259-2269.

Ruppert, E. E., Fox, R. S., &Barnes, R. D. 2004. Invertebrate Zoology: A Functional Evolutionary Approach. Seventh Ed. Brooks/Cole USA.

Schuster, L. & Bright, M. 2016. A Novel Colonial Ciliate Zoothamnium ignavum sp. nov. (Ciliophora, Oligohymenophorea) and Its Ecotsymbiont Candidatus Navis piranensis gen.nov., sp. nov. from Shallow-Water Wood Falls. PLoS ONE 11(9):e0162834.

Sebe-Pedros, A., Degnan, B. M. & Ruiz-Trillo, I. 2017. The origin of Metazoa: a unicellular perspective. Nature Reviews: Genetics, 18, pp 498-512.

Spoon, D. M. & Burbanck, W. D. 1967. A new method for collecting sessile ciliates in plastic petri dishes with tight-fitting lids. Journal of Protozoology. 14, 735-739.

Summers, F. M. 1938. Some Aspects of Normal Development in the Colonial Ciliate Zoothamnium alternans. Biological Bulletin 74(1): 117-129.

Utz, L. R. P.& Coats, D. W. 2008. Telotroch formation, survival, and attachment in theepibiotic peritrich Zoothamnium intermedium (Ciliophora, Oligohymenophorea). Invertebrate Biology 127(3): 237-248.

Utz, L. R. P. 2008. Growth of theperitrich epibiont Zoothamnium intermedium Precht, 1935 (Ciliophora, Peritrichia) estimated from laboratory experiments. Brazilian Journal of Biology 68(2): 441-446.

Wu, T., Li, Y., Lu. B., Shen, Z., Song, W. & Warren, A. 2020. Morphology, taxonomy and molecular phylogeny of three marine peritrich ciliates, including two new species: Zoothamnium apoarbuscula n. sp. and Z. apohentscheli n. sp. (Protozoa, Ciliophoa, Peritrichia) Marine Life Science & Technology 2: 1-15.