Cephalopod Radula under the SEM
by: Sam Mejia, The Evergreen State College

Introduction

Most molluscs have a radula, which is a specifically unique feeding organ that serves as a grinding mechanism to tear apart food into smaller pieces. In cephalopods, the radula consists of symmetrical rows of 7-9 teeth. The radula is most often studied using a scanning electron microscope (SEM) to observe the three-dimensional relationships between the rows of teeth.

Towards the front end of the radular ribbon, the teeth become worn in feeding and are replaced by lower teeth continuously forming in the radular sac. The whole ribbon moves forward while modified odontoblasts dissolve and absorb older teeth and membranes. This movement was compared by Huxley (1853) to a chainsaw, with backward movement of the ribbon thrusting food into the pharynx.

There are two basic forms of cephalopod radulas, the homodont radula with a single cusp on all teeth which are similar in form across a row and the heterodont radula that has more than one cusp on the rhachidian tooth and/or several teeth. Form and shape of molluscan radular teeth are commonly exclusive to a species or genus. Each row of radula teeth consists of one rhachidian tooth followed by one or more lateral teeth on each side, and then one or more marginal teeth.

(taken from the Tree of Life Web Project – Cephalopod Radula page)

SEM photographs were taken to further study the radula in detail. Several methods are available in regards to preparing and mounting the radula of several mollusc species for SEM observations, but only one in specific detail about cephalopods. Zheng (2002) was the primary resource of this paper.

Through emailing researchers and finding what is local and available, three species of squids were collected: Humboldt squid, vampire squid, and the California market squid. Scientific classifications and photos of each species are shown below.


(A female diver with a Humboldt Squid, Dosidicus gigas. Sea of Cortez, Gulf of California, Mexico. Photo by Carrie Vonderhaar at Ocean Futures Society with Getty Images)

(screenshot from a National Geographic & Monterey Bay Aquarium Research Institute youtube video)


(taken from the WDFW California market squid species info page)

Materials and Methods

Radulae or buccal cavities were retrieved from three cephalopods. Two radulae from Dosidicus gigas were given by the Gilly Lab from Hopkins Marine Station of Stanford University. Vampyroteuthis infernalis was given by Dr. Erik Theusen at The Evergreen State College. Doryteuthis opalescens was self-collected in San Francisco, CA.

The radulae were observed under the scanning electron microscope using the following protocol, Zheng (2002): radula was washed with diluted water three times in order to wash away food residue and other adhesive materials. The tissues were immersed into a 1% KOH solution for 24 hours, where specimen were then prefixed for 12 hours in 3% glutaraldehyde at 4°C.

After being washed with diluted water three times, the tissues were dehydrated rapidly through a graded ethanol series (30, 50, 70, 80, 90, 95%) for 10 minutes each, and placed through a grade isoamyl acetate series. The tissues were CO² critical point dried, and coated with gold. They were then examined with the JEOL JSM-6480LV scanning electron microscope. Some specimen were examined using the Z-stacking stereo microscope Leica MZ16.

Results and Discussion

In all three species, their radula consists of 7 longitudinal rows of teeth. D. gigas was caught off the coast in Peru and then frozen for several months. Radula from this specimen measured at 6.3 cm and 7.1 cm.  V. infernalis was preserved in formalin and the buccal cavity was extracted using forceps. The radula from this specimen measured at 4.5 mm. D. opalescens was caught off Pier 39 in San Francisco, CA, frozen for a week, where the buccal cavity was extracted using forceps. Radula from this specimen varied between 2.1 and 4.1 mm in length.

The following are SEM photographs of all three squid species.


Images from D. gigas display a heterodont radula with three cusps on the rhachidian tooth where the side cusps are almost the same length as the central cusp. The lateral tooth has two cusps, with the outside lateral tooth and marginal tooth having one cusp. This species does not appear to have any marginal plates.

Radula of D. gigas taken by Sam Mejia
Close up of the outside marginal tooth and two lateral teeth taken of D. gigas taken by Sam MejiaClose up of the rhachidian tooth and lateral tooth of D. gigas taken by Sam Mejia
Close up of lateral tooth and marginal tooth of D. gigas by Sam Mejia
Close up of marginal tooth of D. gigas by Sam Mejia


Images from V. infernalis show that this species has a homodont radula with a single cusp on all seven teeth, along with marginal plates along both sides of the rows.
Radula of V. infernalis taken by Sam Mejia
V. infernalis taken by Sam Mejia
further down the radula of V. infernalis taken by Sam Mejia

end of the radular ribbon of V. infernalis taken by Sam Mejia

Rhachidian tooth (middle) with lateral teeth on both sides of V. infernalis taken by Sam Mejia


Images from D. opalescens detail a heterodont radula with three cusps on the rhachidian tooth where the side cusps are distinctly shorter than the central cusp. The lateral tooth has two cusps with a larger cusp closest to the rhachidian tooth. The outside lateral and marginal teeth both have one cusp but are shaped remarkably different from each other. There are also marginal plates along both sides of the row.

Radula of D. opalescens taken by Sam Mejia

Close up of the marginal plate, marginal tooth, and two lateral teeth of D. opalescens taken by Sam Mejia

Close up of the rhachidian tooth and lateral teeth of D. opalescens taken by Sam Mejia

Close up of marginal plate, marginal tooth, and two lateral teeth of D. opalescens taken by Sam Mejia


Conclusion

The diet of each of these species differ entirely. Being the largest of the three,  D. gigas feed on micronektonic prey such as other smaller squids, crabs, fish, and krill. Next in size, V. infernalis feed on organic debris that sink down from the ocean surface.  D. opalescens, the smallest of the three species, feed on polychaetes, crustaceans, and small fishes.

Both D. gigas and D. opalescens has a heterodont radulae, while V. infernalis feed has a homodont radula. This research helps support findings that V. infernalis does not feed on micronektonic prey, but “marine snow”.

Further research should compare more cephalopod species, especially in order to find any relationship between predator and prey. More research should go into comparing the size and function of each tooth, as well as the function or purpose of marginal plates. There is a lot more research to be done in regards to cephalopod radulae.


Acknowledgements and Thanks

I would like to thank Dr. Erik Thuesen for allowing me to do this research project to further my knowledge of SEM techniques and cephalopods, as well as for donating Vampyroteuthis infernalis to my project. I would like to thank Patrick Daniel from the Hopkins Marine Station of Stanford University for responding to my inquiries about specimen needed and donating the radulae of Dosidicus gigas from their Bay Area Science Festival’s Discovery Day display at AT&T Park in San Francisco, CA. I would finally like to thank my parents for helping me catch several specimen of Doryteuthis opalescens and cooking squid adobo.

References

  1. Hoving, H. J., & Robison, B. H. (2012). Vampire squid: detritivores in the oxygen minimum zone. Proceedings of the Royal Society of London B: Biological Sciences, rspb20121357.
  2. Messenger, J. B. (1999). The radular apparatus of cephalopods. Philosophical Transactions of the Royal Society B: Biological Sciences354(1380), 161–182.
  3. Pickford, G.E., (1946). Vampyroteuthis Infernalis Chun, An Archaic Dibranchiate Cephalopod. C.A. Reitzels Forlag.
  4. Samuel, D. V., & Patterson, J. (2003). A comparative study on the radula of three Coleoid Cephalopods. South Pacific Study24(1), 33-38.
  5. Stewart, J. S., Hazen, E. L., Bograd, S. J., Byrnes, J. E., Foley, D. G., Gilly, W. F., … & Field, J. C. (2014). Combined climate‐and prey‐mediated range expansion of Humboldt squid (Dosidicus gigas), a large marine predator in the California Current System. Global change biology20(6), 1832-1843.
  6. Uyeno, T. A., & Kier, W. M. (2005). Functional morphology of the cephalopod buccal mass: a novel joint type. Journal of morphology264(2), 211-222.
  7. Venkatesan, V., Ramesh Kumar, P., & Babu, A. (2016). Scanning electron microscope studies on the radula teeth of four species of marine gastropods from the Gulf of Mannar, India. Indian Journal of Fisheries63(1), 140-145.
  8. Young, R.E., Vecchione, M., Mangold, K.M. (2000) Cephalopod Radula. Tree of Life Web Project. 
  9. ZHENG, X. (2002). dong, Wang, Ru- cai., Ocean University of Qingdao, Qingdao266003, China); Morphological study on radula of nine cephalopods in the coastal waters of China [J]. Journal of Fisheries of China5.
  10. Scanning Electron MIcroscopic Studies on Radula

More photos


D. gigas under the Z-stacking stereo microscope taken by Sam Mejia


Close up of rhachidian tooth of D. gigas taken by Sam Mejia

Close up of lateral tooth of D. gigas taken by Sam Mejia

Extreme close-up of lateral tooth of D. gigas taken by Sam Mejia
Close up of marginal tooth of D. gigas taken by Sam Mejia

Extreme close up of edge of marginal tooth (where marginal plate would be) taken by Sam Mejia

Rhichadian and lateral teeth of D. gigas taken by Sam Mejia

Z-stack microscope: V. infernalis taken by Sam Mejia

 
Z-stack microscope: V. infernalis taken by Sam Mejia
Z-stack microscope: V. infernalis taken by Sam Mejia

Z-stack microscope: V. infernalis taken by Sam Mejia

Z-stack microscope: V. infernalis taken by Sam Mejia

V. infernalis radula taken by Sam Mejia

V. infernalis taken by Sam Mejia

Lateral teeth of V. infernalis taken by Sam Mejia

Close up of the marginal plate and marginal tooth of V. infernalis taken by Sam Mejia

Marginal plate and ripped marginal tooth of V. infernalis radula taken by Sam Mejia

Detailed measurements of V. infernalis taken by Sam Mejia

Close up of marginal tooth of D. opalescens taken by Sam Mejia

Extreme close-up of marginal tooth of D. opalescens taken by Sam Mejia

Detailed measurements for D. opalescens taken by Sam Mejia


The TEXT of this page is licensed under the Creative Commons Attribution-NonCommercial License – Version 3.0. Note that images and other media featured on this page are each governed by their own license, and they are available for reuse with proper citation.

SEM & Z-stack microscopy photos taken by Sam Mejia, please cite as:

Mejia, S. (2017) Cephalopod Radula under the SEM. The Evergreen State College.

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