title for learn-about sections for chitons in A SNAIL'S ODYSSEY
  Feeding, growth, & shell repair

diagram of feeding structures of a chitonChitons are primarily herbivorous and feed on seaweeds using a radula. The radula is produced in a radula sac arising from the buccal cavity/pharynx, and is attached firmly to a pair of odontophore cartilages at the front (see diagram on Left). During feeding, large radula-retractor muscles rotate these cartilages downwards and outwards, and in so doing roll the radula out of the mouth to contact the substratum, and then rotate back againto create an upward scraping motion of the radula.  As the radula extends outwards, the cusps or teeth are splayed out against the substratum with their concave posterior surfaces facing in the direction of the scrape. On withdrawal, the cusps draw abraded material into drawing showing radula of a chiton in the buccal cavitythe buccal cavity. The abraded food material is then moved into the esophagus in ciliated tracts and thence to the stomach. As the cusps are worn away or break off, they are replaced at the posterior end of the radula by special secretory cells within the radular sac known as superior epithelial cells.  During this process the entire ribbon moves along like a conveyor belt.  Diagram upper left courtesy Nesson 1969 PhD thesis, Calif Inst Techn, Pasadena 250pp (see Research Study 3.1 below).

NOTE although attached to the odontophore cartilages, the radula is able to slide over them.  The mechanism is similar to the manner in which a human fingernail is fimly fixed to the soft supporting tissues of the finger, yet is able to slide over them during growth

NOTE  lit. “tooth-bearing” G.

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Radula & feeding

  Topics on chiton feeding, growth, & shell repair include radula & feeding, considered here, and DIETS, GROWTH, and SHELL REPAIR considered in other sections.

photograph of a chiton radula A chiton radula differs from that found in most other molluscs in 2 ways.  First, it consists of 2 major parallel rows of cusps with smaller, subsidiary cusps on the sides and, second, the major cusps are hardened by incorporation of the mineral magnetite, a naturally magnetic substance.  Magnetite has a hardness of about 6 on the Mohs scale, where 10 is the hardness of diamond, just slightly less than quartz, which is 7 on the Mohs scale.
NOTE magnetite has the chemical composition Fe3O4. Lost on the shore and can’t find the north star?  Simply dry a chiton radula, induce a magnetic moment with a handy magnet, and float the radula on water.  The radula will orient with one side facing the earth’s magnetic north pole. Although a natural magnetic moment is present, it is too weak to overcome the inertia of the radula’s mass; hence, the need for an initial magnetisation.  Sometimes even a dried whole chiton will orientate in this way if smal,l and if it can be floated in a “low-friction" manner.  The proviso is that you have to know the polarity of the magnet beforehand so that you can determine whether your “compass” is pointing north or south

NOTE  limpet radulae also have 2 major rows of cusps, similar in hardness to those of chitons (5-6 Mohs for limpets vs. about 6 for chitons) also by incorporation of iron oxide, but in this instance a mineral known as goethite (alpha Fe2O3 . H2O) that is not magnetic

Research study 1

Although the magnetic properties of chiton radula are known from early studies in California, discovery of magnetite in chitons is credited to Heinz Lowenstam in 1962.  Apparently, while sitting on a Bermudan shore, he noticed chevron-shaped markings being produced on a rock during the passage of a chiton.  Obviously, something harder than the rock was making the markings.  Through X-ray fluorescence analysis of cusps of west-coast chitons Cryptochiton stelleri and Katharina tunicata, and also of the Caribbean species Chiton tuberculatus, he later identified the substance in the cusps as magnetite, in concentrations of up to 65% dry-mass magnetite.  In that this was the first discovery of magnetite in a living system, he is also thought of as the originator of the scientific field of biomineralisation.  He also considered the possible role of magnetite in homing of chitons, and in so doing may have initiated research interest in the huge and interesting field of magnetite involvement in animal migration and orientation (fishes, newts, birds, and bees). Lowenstam 1962 Geol Soc Amer Bull 73: 435; for early study see Tomlinson 1959 Veliger 2: 36.

NOTE homing in chitons is considered elsewhere in this learn-about section: ECOLOGY: ORIENTATION & HOMING

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Research study 2

photograph of back end of radula of chiton Katharina tunicataphotograph of front end of radula of chiton Katharina tunicataThe cusps at the back of the sac are at first colourless, consisting mainly of a proteinaceous framework to which the various hardening minerals will be added (see photo on Left).  In front of these first 8-or so rows are 4-8 rows of reddish-brown cusps, followed by 25-70 rows of black-capped cusps (see photo on Right). The number of rows of cusps varies with species.  Chemical analysis of the cusps of the chiton Mopalia hindsii show that cusp-rows 0-8 contain essentially no iron (<1% dry mass Fe), the reddish-brown cusp-rows 8-11 have 11% iron, cusp-rows 11-23 have 41% and, at the terminal region of the radula, cusp-rows 24-35 have 47%.  These values correspond to about 16, 57, and 64% dry mass magnetite, respectively.  The black-coloured cusps are strongly reactive to a magnet, and even the reddish-brown cusps are slightly reactive.photograph of a cross-section of a radula of chiton Cryptochiton stelleri

The secretory cells in the radular sac that surround the developing cusps contain iron in soluble form.  Iron is first demonstrated histochemically in Mopalia hindsii radulae in the posteriormost reddish-brown cusps, indicated as "start of Fe deposition" in the photo above Left).  In the cusp rows posterior to this, iron is present only in the distal regions of the closely adhering superior epithelial cells, appearing as a narrow blue-staining band around the cusps about 20µ in thickness.  This is shown in the cross-sectional view on the Left, including deposition in the cusps themselves. The origin of the iron is unknown, but could come from the chitons' iron-rich algal foods.  Chitons have myoglobin-rich radular muscles characterised by being dark red in colour; hence, pathways for obtaining and metabolising iron are present.  Carefoot 1965 Proc Malac Soc Lond 36: 203.

NOTE 0.2-0.9% dry mass iron measured in several encrusting and filamentous species

Research study 3

photograph of mouth of chiton Cryptochiton stellerischematic showing mineralisation in the cusps of a chiton radulaThe mineralisation process can be followed in electron-microscopic views of the radula of the chiton Cryptochiton stelleri (see photos below).  At the level of the reddish-brown cusps, crystalline iron is beginning to be precipitated within the proteinaceous framework of the cusps.  At the level of the black cusps, magnetite solidly fills the meshwork spaces.  Average magnetite content within the cusps of Cryptochiton is 52%.  Towe & Lowenstam 1967 J Ultrastr Res 17: 1.

Note the authors have drawn the cusps on the radula facing the wrong direction

Mouth of chiton Cryptochiton stelleri 1X

The photographs below show various stages in mineralisation of the radular cusps:

electron microscopical view of unmineralised protein matrix 42,000X
Electron microscopical view of the unmineralised protein matrix of a newly forming cusp 42,000X
electron microscopical view of early mineralisation in the cusp of a chiton radula
Magnetite deposition begins at the level of the reddish-brown cusps 40,000X
electron microscopical view of a mineralised cusp during radula development in a chiton
In the fully formed black cusps the magnetite solidly fills the meshwork spaces of the protein framework
Research study 3.1

A later study done by a PhD candidate at the California Institute of Technology, Pasadena adds considerable detail to our knowledge of formation and mineralisation of the cusps in chitons Mopalia muscosa and Lepidochitona hartwegi.  Iron is transported in the hemolymph in a protein-bound ferritin form.  At the proximal ends of the superior epithelial cells, which abut on a dorsal hemolymph sinus, are numerous vesicles filled with ferritin.  At the apical ends of the cells is a large concentration of iron-containing membrane-bound granules (see photograph).  Note in the magnified inset view of the same photograph that at the boundary of the granular region of each cell and the newly forming cusps are 500-1000 microvilli arising from a layer of mitochondria-rich cytoplasm.  The ferritin, probably in a soluble ferruginous form moves either by diffusion or active transport from the microvilli into the protein scaffolding of a newly secreted cusp and is deposited as ferric oxide (magnetite).  The author calculates that each mature lateral cusp contains about 40ug Fe, for a total of 80ug per transverse row.  However, as the minor cusps also contain some iron, the total per row may be closer to 100ug.   Through Fe59-labeling experiments, the author determines that replacement rate in M. muscosa is about 0.6 rows . d-1 at 15oC, so a total of about 60ug iron is required per day for the mineralisation process.  Based on estimated iron content of the seaweed foods of M. muscosa, and if absorption is 100%, this requirement could be met through consumption of just 100mg fresh mass of algae per day .  The author’s focus in the study is on the fine structure, both light-microscopical and electron-microscopical, of the secretory tissues, but the thesis also contributes to the identification of ferritin-protein being transported in the hemolymph.   Nesson 1969 PhD thesis, Calif Inst Techn, Pasadena 250pp.

Research study 4

photograph of a radula of a 25d-old chiton Lepidochitona fernaldiphotograph of an adult radula of a chiton Lepidochitona fernaldis showing all 17 cusp rowsStudies on early development of Lepidochitona fernaldi at Friday Harbor Laboratories, Washington show that there is no sign of a radula structure in the trochophore stage.  However, by about a week after metamorphosis , which is about 20d after fertilisation, the tiny juveniles are foraging. Their radulae at this stage consist of several longitudinal rows of cusps capped with black magnetite and a few associated lateral cusps (see photo on Left).  Specifically, only cusps 2, 5, and 8, are present, and a medial cusp is absent.  It seems that the postmetamorphic juveniles secrete a minimal number of cusps, but still have a functional radula. Only later are the other lateral cusps added until the adult complement is reached.

Most molluscan radulae have multiple rows of cusps but with much less differentiation in size and function in the cusps as present in chitons.  Chitons actually have 8 lateral cusps per side as shown in the photograph on the Right, plus a common medial Cusp M.  The median cusp M is flanked by a small Cusp 1, a large Cusp 2, which is the dominant one for scraping and is the only cusp in a transverse row that is hardened with magnetite.  Then come 2 small Cusps 3 and 4, a large Cusp 5 that is thought to protect the soft parts around the mouth from the hard and sharp Cusp 2 as it rolls back and forth during feeding and, lastly, there are 3 insignificant Cusps 6, 7, and 8.  This order is repeated on the other side making 17 cusps in total along a single transverse row.  Eernisse & Kerth 1988 Malacologia 28: 95. 

NOTE Lepidochitona fernaldi broods its larvae in the pallial grooves until the larvae reach the crawling stage, within 1-2d of metamorphosis.  The trochophore stage is passed through in the egg capsule

photograph of the habitat of a gumboot chiton Cryptochiton stelleri taken from a video

CLICK HERE to see a video of Cryptochiton stelleri in the field, motionless, but possibly feeding.

NOTE the video replays automatically

Research study 5

diagram showing composition of a cusp of a chiton radulaRecent studies on the composition of cusps of chiton radulae show that their structure is more complex than first thought.  The base is made up of the supporting material chitin and protein, while the anterior portion is a calcium-phosphate compound known as apatite.  The magnetite portion lines the entire posterior surface of the cusp, and is protected from oxidation by a thin layer of ferrihydrite, a hydrated form of magnetite: 5Fe2O3. 9H2O.  Despite much recent work on biomineralisation of magnetite, it is not known how it is synthesised in living tissue.  Brooker et al. 2003 Mar Biol 142: 447.

NOTE the study uses Acanthopleura echinata, not a west-coast species

NOTE  a mineral Ca5(PO4)3 containing varying amounts of attached hydroxyl, fluorine, and chlorine.  Calcium phosphate is the major component of mammalian teeth and bones

Research study 6

photograph of a black leather-chiton Katharina tunicata in a feeding patchRadula cusps, as in a limpet, are thought to operate like a wood rasp scraping across the substratum. However, close scrutiny of radular action in black leather-chitons Katharina tunicata at Friday Harbor Laboratories, Washington shows that the cusps actually work by cutting against one another as they are drawn over an alga.  This action may be more effective at slicing through the succulent flesh of Katharina’s preferred food algae than, say, scraping a diatom-covered rock.  Padilla 2003 Am Malacological Bull  18: 163.




Black leather-chiton Katharina tunicata through its
browsing has created a cleared patch amongst
anemones Antopleura elegantissima 0.25X

Research study 7

histograms comparing elemental composition of exterior and core material in a chiton cusp Cryptochiton stelleriA stylish paper by a consortium of scientists largely from the University of California provides an up-to-date look at biomineralisation of chiton radular cusps, using samples from Cryptochiton stelleri.  Through modern microscopical and nanomechanical-characterisation techniques the authors confirm that chiton radula cusps are harder and stiffer than any other biominerals described to date.  Notably, they are 3-fold harder than the enamel of vertebrate teeth and the nacre of molluscs, and perform as well as hard ceramics.  Mature cusps in C. stelleri are tri-lobed and have 2 distinct mineral phases (see diagrams below).  The core consists of a softer iron phosphate while the exterior is hard ferromagnetic iron oxide (magnetite).  Additional elemental components of the cusps are C, Ca, K, Na, Mg, and Si (see histograms).  Abundant chitin fibres throughout provide structural support.  The leading edge of each cusp is about 15% harder than the trailing edge and, just like a beaver tooth, is self-sharpening.  Cracks propagated during wear of the magnetite veneer tend to travel parallel to the long axis of the cusp and, if they intercept the core material, tend to be deflected at the interface.  This strategy of deflection minimizes catastrophic failure of the cusp.  The magnetite is organized into crystalline bundles (250nm in width) oriented parallel to the long axis of the cusp.  Each bundle is surrounded by a thin organic layer.  The authors provide abundant additional information on ultrastructural and biomechanical properties of the cusps.  Weaver et al. 2010 Materials Today 13 (1-2): 42.

NOTE  determined through scanning-electron microscopy and energy-dispersive spectroscopy

  schematic showing radular features of the gumboot chiton Cryptochiton stelleri
Research study 8

illustration of radular cusps of chiton Cryptochiton stelleriAlthough the precise mechanism of mineralisation of chiton-radula cusps is not yet known, it seems likely that just as in formation of nacre, teeth, and bone, it is the initial protein/carbohydrate scaffolding that initiates nucleation, and guides the orientation and growth of the iron-oxide crystals.  Recent work by American and Japanese researchers on cusp formation in the gumboot chiton Cryptochiton stelleri has added greatly to our understanding of the process (see also Research Study 7 above).  The cross-section microscopical view of a radular cusp of chiton Cryptochiton stelleriresearchers first confirm through use of transmission-electron microscopy that mineral deposition occurs only on organic fibers in the cusp portions of the radula teeth and not in the base regions (see illustration).  Close inspection of the cusp in tooth-row 16 reveals a much greater density of organic fibers in the leading edge than in the trailing edge and, indeed, this leads to more mineral-binding sites being available in the leading edge than in the trailing edge.  Leading-edge deposits are not just more closely packed than trailing edge ones, but they are much smaller in diameter (means of 35 vs. 150nm, respectively).  Extraction and analysis of proteins from the teeth regions identify 6 proteins that are cusp-specific, of which myoglobin and an acid peptide are likely to be most involved in the mineralisation process.  As formation of an iron-oxide crystal requires precise control of oxygen concentration, the authors suggest that it is role of myoglobin to do this.  Other proteins identified and possibly involved in mineralisation are ubiquitin, ferritin, and several newly identified cusp-specific acid peptides.  Nemoto et al. 2012 Proteomics 12: 2890.

NOTE  overall protein content of the cusp is 0.2% dry mass

Cross-sectional view of right-hand radular cusp of chiton Cryptochiton stelleri in the process
of being mineralised. Iron stains blue and note that it appears in the cusp and in the secretory
superior-epithelial cells above the cusp, but not in the cusp base or radula membrane 12X

Research study 9

photo-schematic of orientation of magnetite molecules in radula of gumboot chiton Cryptochiton stelleriAnother investigation by a consortium (12 in number) of largely California researchers also on gumboot chitons Cryptochiton stelleri, provides information specific to chemical and structural schematic showing transformation of ferrihydrite to magnetite in the radular cusps of a chiton Cryptochiton stelleritransformations that occur during cusp formation. The process occurs in 4 stages. The first is the formation of the organic scaffolding of the cusps consisting primarily of crystalline fibers of ⍺-chitin, with associated protein. Next comes the addition of precursor iron (ferrihydrite) crystals oriented within the organic framework. Third, a solid-state phase-transformation of ferrihydrite to magnetite occurs. This transformation begins at Cusp#1-2 (see schematic above Left), with high levels of ferrihydrite, and ends at Cusp#5 with all crystalline mineral now being in the form of magnetite. Finally, the magnetite crystals grow and expand to form continuous parallel rods within the maturing cusps. The size and curvature of the rods are dictated by the underlying organic matrix, which ultimately affects both shape and curvature of the cusps, and their self-sharpening ability. The rods actually change their orientation from front to back of the cusp, bending around the iron-phosphate core in the transitional zone between them (see orientation of dashed white lines in the photograph on Right). Wang et al. 2013 Advanced Functional Mater 23: 2908.

NOTE instrumentation used in the study includes synchroton X-ray diffraction, synchroton micro-X-ray fluorescence, transmission electron microscopy, and scanning electron microscopy

NOTE ferrihydrite (Fe3+)2O3. 0.5H2O is a common mineral in the earth’s crust

Research study 10

A related study published later in the same journal also on gumboot chitons Cryptochiton stelleri and done by some of the same researchers featured in RS9 above, provides further details on the unique molecular structure that imparts wear- and stress-resistant properties to the cusps. Each mature cusp comprises an array of organically encased crystalline and nano-diagram showing features of the radula of a gumboot chiton Cryptochiton stellerisized magnetite rods surrounding a soft core of organic-rich iron phosphate (see figure on Left). In the authors’ words the “hard shell/soft core design…contain(ing) rod-like architectures…result in resistance to fatigue loading and deformation”. The nanorods of magnetite are aligned parallel to the cusp’s surface along its contours (see diagram below). Other fine-scale features of construction include the cusp’s curvature that favours redistribution of stress to the base of the cusp, without which the tip would be more likely to fracture. Additionally, the microrods on the front edge of the cusp are about 20% smaller in diameter than those on the back edge, increasing overall hardness. The nanorods are themselves bound together by mineral bridges, adding to lateral toughness. Propagation of cracks is impeded by the layered microstructure, crystalline boundaries, and organic scaffolding, all acting to maximise energy dissipation. Finally, differential hardness from front to back of the cusp leads to self-sharpening.The researchers’ primary interest in the work is in developing abrasion-resistant materials for commercial tooling, machining, and coating applications, but both publications make fine and interesting reading for biologists. In fact, the picture now seems so complete that a reader may wonder (naively) what more can possibly be added. Grunenfelder et al. 2014 Adv Funct Mater 24: 6093.photographs of microstructure of radular cusps of a gumboot chiton Cryptochiton stelleri

Research Studies 9 and 10 above have 12 and 9 authors, respectively. This seems a crowd, but is partly explained by the sophisticated instrumentation involved in such technical work. More unusual is that despite 4 of the authors being involved in both studies and being at the same University (UC Riverside, California), there is only a bare mention of the earlier paper in the second. Given that there is more than a little duplicative material involved, one wonders about this; in any case, a reader would have appreciated being informed up front that the work involves 2 papers