Vampire teeth: Radula morphology of the vampire squid, Vampyroteuthis infernalis, indicates a soft-bodied diet

Overview

At the conclusion of the Fall 2018 quarter, my results indicated that Dosidicus gigas (Humboldt squid) and Doryteuthis opalescens (California market squid) both have a heterodont radula, while Vampyroteuthis infernalis (vampire squid) has a homodont radula.

Dosidicus gigas is known to surface at night to feed on teleostei, lanternfish, mackerels, eels, shrimp, sardines, and other cephalopods and crustaceans (Pardo-Gandrarillas et. al 2014). D. gigas can be found between 200-700 meters in the East Pacific Ocean from California to Tierra del Fuego (Nigmatullin et al. 2001).

Doryteuthis opalescens live within 320 kilometers off-shore in the East Pacific Ocean from British Columbia to the tip of the Baja Peninsula. D. opalescens live in the water column ranging from the surface to depths of up to 790 meters where they feed on small crustaceans, krill, small fish, and other squid (NOAA Fisheries Service Contributors, 2017).

V. infernalis occupies bathypelagic depths of typically between 600 and 1200 m throughout the world’s tropical and temperate oceans in the oxygen minimum layer where little light is found (Robinson et. al 2003). Vampyroteuthis has been known to feed upon prawns, cnidarians, and copepods (Young, 1977; Nixon, 1987). Hoving and Robinson (2012) report that ingested items extracted from captured specimen included the remains of gelatinous zooplankton, discarded larvacean houses, crustacean remains, fecal pellets, copepods, marine snow, eggs and diatoms.

Introduction

V. infernalis has been extensively investigated by Pickford (1946,1949) where she states that the majority of specimen found in the water has a salinity between 34.7% and 34.9%, oxygen content between 1.00 ml/L and 3.99 ml/L, a density (sigma-t) between 27.4 and 27.8, at temperatures between 2.0 and 6.0 degrees C, being most abundant between 1500m and 2500m in the Pacific and 1000-2000 meters in the Atlantic Ocean. Photos of the radula of V. infernalis under the SEM from 1975 were found also showing a homodont radula. Reserachers found that the radula of V. infernalis had an anterior prolongation of the rachidian tooth that provides support against stress during feeding (Solem, A. & Roper, C.F.E. 1975).

I theorized that possibly the deeper a cephalopod habituates, the less complicated the feeding structure may be which could be a potential reason for why V. infernalis has a homodont radula. Perhaps V. infernalis has a homodont radula indicating a soft-bodied diet, rather than the robust cutting heterodont radula of shallow predators. The radula of V. invernalis has long gracile teeth that appear to be well suited to tearing apart very soft tissue and perhaps picking apart marine detritus. Despite living in extremely low oxygen concentrations with very low metabolic rates, adult vampire squid can be fairly active swimmers and likely feed on a variety of soft-bodied organisms.

Materials and Methods

During the Spring 2018 quarter, I received Taonius borealis and Galiteuthis phyllura from (research cruise). Unfortunately while preparing samples, G. phyllura was damaged and is unusable. I also received several Euprymna scolopes (Hawaiian bobtail squid) specimen from Eric Koch at the Kewalo Marine Lab at the University of Hawai’i where his lab looks at beneficial host-microbe interactions with E. scolopes and its bioluminescent symbiont, Vibrio fischeri. I thank these researchers for donating specimen to my research.

The radulae of T. borealis and E. scolopes 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 

With both of these species, as with the previous 3 species from Fall 2017, their radula consisted of 7 longitudinal rows of teeth. T. borealis was caught on a research vessel trip in (NEED LOCATION). E. scolopes was caught in Hawaii and fixed in 5% formalin.

The following are SEM photographs of both squid species.


Images from T. borealis show a heterodont radula with three cusps on the rhachidian tooth where the side cusps are significantly shorter than the central cusp. The lateral tooth has two cusps with the outside cusp being significantly shorter than the inside cusp. With D. gigas, the lateral tooth also had two cusps but are remarkably different than T. borealis.
Radula of T. borealis taken by Sam Mejia
Radula of T. borealis taken by Sam Mejia

Radula of T. borealis taken by Sam Mejia
Radula of T. borealis taken by Sam Mejia

Radula of T. borealis taken by Sam Mejia Close up of rhachidian tooth and lateral tooth of T. borealis by Sam Mejia


Images from E. scolopes depict a homodont radula with a single cusp on all seven teeth with no marginal plates along both sides of the rows. The marginal teeth are noticeably longer than the other teeth.
Radula of E. scolopes taken by Sam Mejia

Radula of E. scolopes taken by Sam Mejia

Radula of E. scolopes taken by Sam Mejia

Radula of E. scolopes taken by Sam Mejia

Radula of E. scolopes taken by Sam Mejia

Radula of E. scolopes taken by Sam Mejia

Radula of E. scolopes taken by Sam Mejia

Radula of E. scolopes taken by Sam Mejia

Radula of E. scolopes taken by Sam Mejia
with measurements


MORE RECENT IMAGES OF V. INFERNALIS
Note the similar homodont structures with E. scolopes. Each tooth looks to be the same length with V. infernalis, where E. scolopes has significantly longer marginal teeth.
Radula of V. infernalis taken by Sam Mejia

Radula of V. infernalis taken by Sam Mejia

Radula of V. infernalis taken by Sam Mejia

Radula of V. infernalis taken by Sam Mejia

Radula of V. infernalis taken by Sam Mejia

Radula of V. infernalis taken by Sam Mejia
with measurements

Radula of V. infernalis taken by Sam Mejia

Radula of V. infernalis taken by Sam Mejia

Radula of V. infernalis taken by Sam Mejia

Close up of marginal plate, marginal tooth, and lateral teeth of V. infernalis by Sam Mejia

Discussion

T. borealis is a glass squid from the family Cranchiidae which comprises of approximately 60 species occuring in surface and midwater depths in oceans around the world (Norman, M. & Lu, C. 2000). An attacking behavior was filmed 948 meters deep off the Pacific coast of Northeast Honshu, Japan by a cranchiid squid (likely Taonius borealis) on a feather-star; however Cranchiid squids are thought to feed on small planktonic crustaceans or fishes between 200-400 meters (Lindsay, D.J. & Yamakita, T. 2015).

From Okutani et. al 2015

Eupyrmna scolopes, otherwise known as the Hawaiian bobtail squid, is from the family Sepiolidae which is native to the central Pacific Ocean, particularly the shallow coastal waters 2-4 cm deep of Midway Island and the Hawaiian Islands. Most sepiolid squids habituate very deep waters, however this specific squid is quite different. E. scolopes feeds on mysid shrimp, younger squids consuming Artemia crustaceans, and is known for being a “sit and wait” predator by burying itself in the sand with its tentacles and waiting for prey to pass by at night, aiming all their arms at the prey and striking it with two tentacles (Fleisher K. & Case, J. 1995).

My hypothesis of shallow predators having a heterodont radula compared to deeper water cephalopods having homodont radula was proven wrong with Eupyrmna scolopes; however, E. scolopes is an outlier in itself within the Sepiolidae family typically living in deeper waters.

More measurements need to be taken, specifically on teeth length. More research will be done regarding stable isotope analysis in cephalopods and other deep-sea invertebrates.

Further Research

Paul Bennetts from Alaska Pacific University is donating radula from Enteroctopus dolfleini and I expect to receive it in the next couple weeks.

Denise Whatley from Tonmo is donating the radula of a bandensis cuttlefish, Octopus briareus, Octopus hummelincki, as well as a nautilus.

Bret Grasse, Lisa Abbo, and Taylor Sakmar with Marine Biological Laboratory, Woods Hole “Cephalopod Breeding Initiative” are also donating several species of cephalopods for my research. I will be working on these specimen over the next couple months.

I submitted an abstract to the 2018 Cephalopod International Advisory Council Conference on behalf of myself and my thesis professor Erik V. Thuesen titled “Vampire teeth: Radula morphology of the vampire squid, Vampyroteuthis infernalis, indicates a soft-bodied diet” where it was accepted as a Poster presentation in the Physiology session in November 2018 in St. Petersburg, Florida.

Resources

Childress J. J. 1995. Are there physiological and biochemical adaptations of metabolism in deep-sea animals? Trends Ecol. Evol. 103036. doi:10.1016/S0169-5347(00)88957-0

Fleisher, K., Case, J. 1995. Cephalopod predation facilitated by dinoflagellate luminescence. Biology Bulletin, 189: 263-271.

Hoving, H. J. T., and Robison, B. H. 2012. Vampire squid: detritivores in the oxygen minimum zone. Proc. R. Soc. B Biol. Sci. 279, 4559–4567. doi: 10.1098/rspb.2012.1357

Nigmatullin, C., K. Nesis, A. Arkhipkin. 2001. A Review of the Biology of the jumbo squid Dosidicus gigas (Cephalopoda: Ommastrephidae). Fisheries Research, 54: 9-19.

NOAA Fisheries Service Contributors, 2017. “FishWatch: Doryteuthis opalescens” (On-line). NOAA National Marine Fisheries Service.

Mark Norman & C.C. Lu. 2000. “Preliminary checklist of the cephalopods of the South China Sea”. The Raffles Bulletin of Zoology (Supplement No. 8): 539–567.

Okutani, T., Fujiwara, Y., Tsuchida, S., Lindsay, D.J., Kawato, M., Yamakita, T. 2015. Attack by a cranchiid squid on a deep-sea feather star. Chiribotan Vol. 45, No. 4: 292-295.

Olsen, R., J. Young. 2007. The role of squid in open ocean ecosystems. Report of a GLOBEC-CLIOTOP/PFRP workshop, 16-17 November 2006, Honolulu, Hawaii, USA.. GLOBEC Report, 24: 1-94.

Pardo-Gandarillas, M.C., Lohrmann, K.B., George-Nascimento, M. & Ibáñez, C.M. 2014: Diet and parasites of the jumbo squid Dosidicus gigas in the Humboldt Current System, Molluscan Research, DOI: 10.1080/13235818.2013.860870.

Pickford, G. E. 1946. Vampyroteuthis infernalis Chun an archiac dibranchiate cephalopod I: Natural History and Distribution. Number 29 in Dana Report. Copenhagen: 1-40.

Pickford, G. E. 1949. Vampyroteuthis infernalis Chun an archaic dibranchiate cephalopod. II. External anatomy. Dana-Report No. 32: 1-132.

Pickford, G. E. 1949. The distribution of the eggs of Vampyroteuthis infernalis Chun. Journal of Marine Research, 8(1):73-83.

Roper, C. F. E. and R. E. Young. 1975. Vertical distribution of pelagic cephalopods. Smithsonian Contributions to Zoolog, 209: 1-51.

Seibel, B., E. Thuesen, J. Childress. 1997. Decline in pelagic cephalopod metabolism with habitat depth reflects differences in locomotory efficiency. The Biological Bulletin, 192: 262-278.

Seibel, B., E. Thuesen, J. Childress. 1998. Flight of the Vampire: ontogenetic gait-transition in Vampyroteuthis infernalis (Cephalopoda: Vampyromorpha). The Journal of Experimental Biology, 201 (16): 2314-2324.

Solem, A., Roper, C.F.E., 1975. Structures of Recent cephalopod radulae. The Veliger, 18: 127-133.

Villanueva R, Perricone V and Fiorito G. 2017. Cephalopods as Predators: AShort Journey among Behavioral Flexibilities, Adaptions, and Feeding Habits. Front. Physiol. 8:598. doi: 10.3389/fphys.2017.00598

Young, J. Z. 1977. Brain, behaviour and evolution of cephalopods. Symp. Zool. Soc. Lond. 38: 377–434.

Young R. E.Vecchione M.Donovan D. T. 1998. The evolution of coleoid cephalopods and their present biodiversity and ecologyS. Afr. J. Mar. Sci. 20393420. doi:10.2989/025776198784126287

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.

Thank you for your time.

 


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