black dot
  black dot

Ink & opaline secretions


Defenses of nudibranchs and relatives include ink & opaline secretions, considered in this section, and
, and
, considered in other sections. 

Most of the 37 or so world species of sea hares Aplysia release a purplish-coloured ink when disturbed.  Additionally, some species release a white “opaline” secretion, also thought to be used for defense. Species eating red seaweeds also have a variety of secondary metabolites in their skins and digestive glands that may be defensive.  This section starts with a review article that provides a comprehensive overview of defenses in sea hares, and should be a “must-read” for anyone interested in the subject. After this entry the Research Studies are presented chronologically.

  black dot
Research study 1

drawing of a sea hare showing parts relating to ink and opaline-gland secretionsA review article from researchers at the University of Washington and Friday Harbor Laboratories, Washington provides clarification of several generalisations and misconceptions relating to ink and ink release in sea hares (Anaspidea), and a brief review of these may aid in understanding the results of Research Studies presented below.  Points of clarification about the ink include: 1) all sea hares have ink glands, most releasing a purple secretion, but some releasing white ink, 2) the ink gland is located in the mantle directly over the gill (ctenidium), with the ink being released through pores on the ventral surface of the gland, 3) the ink is composed primarily of pigments (phycoerythins) derived from red-algal foods, but does not contain secondary metabolites of the algae, 4) possible functions of the ink include anti-predator and predator-warning, and 5) release of ink is not necessarily high threshold or all-or-none, as has been described by other researchers. 

Points of clarification about opaline secretion: 1) all sea hares have opaline glands, located beneath the floor of the mantle cavity, 2) opaline secretion is released less readily and in smaller quantities than ink, 3) the secretion is highly proteinaceous and viscous, 4) its production is not dependent upon red algae being eaten, and 5) its function is not known. The authors comment that this part of sea-hare biology desperately needs to be researched.  Johnson & Willows 1999 Mar Freshw Behav Physiol 32: 147.

NOTE chemicals in the body that seem to have no role in metabolism; thought to have defensive functions


  black dot
Research study 2

Of the 2 west-coast aplysiid species, only Aplysia californica releases a purple ink that is derived from a diet of red seaweeds, while the sympatric A. vaccaria does not.  Early experiments at the Scripps Institution of Oceanography, California on feeding of A. californica on various red algal and biliprotein diets show that the main component of the, termed aplysioviolin, is derived from phycoerythrin of red algae, such as Laurencia pacifica.  On a diet of brown algae Egregia laevigata, A. californic becomes facultatively de-inked.  When returned to a diet of red algae the ability to produce ink is restored.  Within 72h some of the consumed phycoerythrin is transferred to the egg mass, where it imparts mauve-purplish colour.  The authors do not consider the aplysioviolin component of the ink to be defensive; rather, they suggest it may be a waste product.  They justify this statement by the fact that while the ink has an unpleasant “odious” smell, aplysioviolin itself is odourless.  They suggest that a possible source of the odour, and also a candidate for the defensive agent, is the brominated aromatic compound, aplysinol.  Chapman & Fox 1969 J Exp Mar Biol Ecol 4: 71.

NOTE  an earlier author, cited here, also working on ink composition in A. californica, uses the term aplysioviolin for the main blueish-coloured component of the ink, but has an additional term aplysiorhodin for a reddish component, and adds a third term aplysioazurin for another blue component.  What the present status of all these components is now, will require a biochemist's determination.  Winkler 1959 Pac Sci 13: 357

  black dot
Research study 3

graph showing all-or-none inking response in sea hares Aplysia californica to electrical shockInk-release in Aplysia californica is a high-threshold, all-or-none, behaviour controlled by just a few neurons located in the abdominal ganglion.  Note in the graph that a mild electrical shock must reach a threshold level before an animal inks. The ink is released into the mantle cavity and actively ejected from between the parapodia and the siphon by pumping movements of the mantle and parapodia (see photo series below). The authors note that a healthy Aplysia never releases ink spontaneously.  Carew & Kandel 1976 J Neurophysiol 40: 692; see also Carew & Kandel 1973 Fed Proc 32: 368.

photograph showing a sea hare Aplysia californica being given an electric shock
An electric shock is applied just posterior to the head, causing the sea hare to turn in the opposite way.
photograph showing a sea hare Aplysia californica releasing ink after being given an electric shock
The sea hare pulls in its head & expels ink from between its parapodia & from siphon.
photograph showing a sea hare Aplysia californica releasing ink after being given an electric shock
The ink is discharged in a series of pumping movements of the mantle and parapodia.
  black dot
Research study 4

diagram comparing mantle cavity features in a sea hare Aplysia californica when ejecting ink backwards and forwardsInk release by sea hares Aplysia californica following stimulation is usually in the direction of the source of the stimulus. A white opaline secretion may often be released in company with the ink.  Ink originates from the ink gland located on the mantle shelf just above the gills, while opaline secretion comes from glands located on the floor of the mantle cavity (see drawings). The secretions are directed to the front or back by contractions of the siphon, mantle, and parapodia as shown. Note the difference in the siphon in the 2 scenarios. In a "tail-shocked" individual the siphon stays level and ink is pumped posteriorad by mantle contractions. In a "head-shocked" individual the siphon constricts and the mantle contractions this time pump the ink secretions anteriorad through the flared margins of the parapodia. Note in the "Tail-shock series" below that the ink is released posteriorly as the individual crawls away. In the "Head-shock series" the individual pulls in its head and ejects its ink towards the front. Durations of the series are 15 and 25sec, respectively, after application of the shocks. Walters & Erickson 1986 J Comp Physiol A 159: 339.

NOTE  includes pinching with forceps, electrical shock, and application of crystals of NaCl

photo 1 in a series of 4 showing how ink release in a sea hare Aplysia californica tends to be directed towards the source of the irritating stimulus photo 2 in a series of 4 showing how ink release in a sea hare Aplysia californica tends to be directed towards the source of the irritating stimulus photo 3 in a series of 4 showing how ink release in a sea hare Aplysia californica tends to be directed towards the source of the irritating stimulus photo 4 in a series of 4 showing how ink release in a sea hare Aplysia californica tends to be directed towards the source of the irritating stimulus
photo 1 in a series of 4 showing how ink release in a sea hare Aplysia californica tends to be directed towards the source of the irritating stimulus photo 2 in a series of 4 showing how ink release in a sea hare Aplysia californica tends to be directed towards the source of the irritating stimulus photo 3 in a series of 4 showing how ink release in a sea hare Aplysia californica tends to be directed towards the source of the irritating stimulus photo 4 in a series of 4 showing how ink release in a sea hare Aplysia californica tends to be directed towards the source of the irritating stimulus
  black dot
Research study 5

In laboratory confrontations the predatory Navanax inermis will readily attack and eat juvenile sea hares Aplysia californica. The sequence photographed by the authors shows that shortly after contact by the predator, the prey is ingested by suction and swallowed whole.  The entire sequence lasts less than 30sec.  No ink is released during the attack, which may be relevant to the question posed in Research Study 6 below about the role of ink in defense.  Leonard & Lukowiak 1986 Behaviour 98: 320. 

NOTE  Navanax lives much deeper than the shallow-dwelling A. californica, so it is not clear how much natural predation there would be  

photograph 1 in a series of 3 showing predation by Navanax inermis on a juvenile sea hare Aplysia californica
On contact, the predator turns to face the prey.
photograph 2 in a series of 3 showing predation by Navanax inermis on a juvenile sea hare Aplysia californica
The sea hare is sucked in, with just its tail sticking out
photograph 3 in a series of 3 showing predation by Navanax inermis on a juvenile sea hare Aplysia californica
Within 30sec the sea hare is ingested
  black dot
Research study 6

histogram showing effect of ink on survival of sea hares Aplysia californica in face of anemone predationDoes the ink function in defense against predators?  Research on species of Aplysia other than A. californica suggests that the ink is, at the very least, an irritant to other animals, but results are mixed from experiments to test whether it is actually deters feeding by potential predators.  Experiments with A. californica and a potential sea-anemone predator Anthopleura sp. show that contact of a sea hare with the tentacles elicits copious ink discharge by the sea hare.  Because of its component of sticky mucus, the ink tends to coat the sea hare and continues to be released even as the sea hare is ingested by the anemone (see photo series below). 

In other experiments the ink triggers gastrovascular eversions in the anemones and causes them to reject whitefish, used to feed the anemones in the laboratory. If inkless sea hares are fed to sea anemones, many are eaten.  However, if at the same time freshly collected ink from other sea hares is squirted onto the anemone, a significant proportion of the prey is rejected (see histogram). Sea hares without ink, but with normal skin/digestive-gland “chemistry”, are eaten more than sea hares with ink but no skin “chemistry”, suggesting that the ink is the more important defense. The authors note that sea hares tend to avoid ink, thus possibly removing themselves from areas of ongoing predation.  Nolen et al. 1995 J Comp Phys A 176: 239.

NOTE  these experiments are done at the University of Miami using sea hares raised from eggs at the University’s Aplysia Mariculture Facility and anemones shipped in from California.  The anemones are described as being A. xanthogrammica, but one of the researchers acknowledges in a later paper that the identification was likely mistaken, and that the anemones used were probably Anthopleura sola (see Research Study 9 below)

NOTE  inkless specimens are obtained in 2 ways, the first by feeding sea hares green alga Ulva that lack the necessary phycoerythrobilin pigments used to manufacture ink.  These inkless animals also lack any skin "chemistry" or digestive-gland “chemistry”.  The second way is to “de-ink” is by massaging the ink gland over successive days.  This eventually discharges all ink but leaves the sea hare with its complement of potentially toxic skin and digestive-gland secondary metabolites obtained from its normal diet of red algae

photograph 1 in a series of 5 showing a sea hare Aplysia californica escaping predation by a sea anemone Anthopleura sp.
A sea hare approaches a sea anemone in an aquarium tank...
photograph 2 in a series of 5 showing a sea hare Aplysia californica escaping predation by a sea anemone Anthopleura sp. caught up in the sea-anemone's tentacles...
photograph 3 in a series of 5 showing a sea hare Aplysia californica escaping predation by a sea anemone Anthopleura sp.
...and ingested. The sea hare releases copious amounts of ink...
photograph 4 in a series of 5 showing a sea hare Aplysia californica escaping predation by a sea anemone Anthopleura sp.
...that shortly is seen diffusing from the gastrovascular cavity...
photograph 5 in a series of 5 showing a sea hare Aplysia californica escaping predation by a sea anemone Anthopleura sp.
...the sea hare crawls out, seeming not much the worse for wear.
  black dot
Research study 7

diagram showing morphology of ink gland in a sea hare Aplysia californicaA detailed study of the processing, storage, and secretion of ink by A. californica by scientists in Florida leads to the following. The purple-pigment component of the ink is a phycoerythrobilin, and is stored, along with a protein of unknown function in muscular ink-release vesicles within the ink gland. The ink gland is located in the mantle shelf part of the mantle cavity and, when the vesicles discharge, the ink is carried out in the exhalent respiratory flow through the siphon and parapodial margins. After a red alga, in this case Gracilaria tikvahiae, is consumed, the ink pigment phycoerythrobilin is cleaved from its protein component in vacuoles in cells of the digestive gland and carried in the hemolymph to the ink gland.  The ink is transported to and stored in membrane-bound vacuoles in the ink gland for variable lengths of time, then later incorporated into ink-release vesicles. The protein component of the ink, representing 35% of the dry mass of the secreted ink, is added at this time.  Variable amounts of mucus are also released with the ink component.  The diagram shows the different kinds of vesicles present in the ink gland, the main ones being the so-called Red-Purple Vesicles (RPV) that contain the ink. Also present in the ink gland are Amber Vesicles and Clear Vesicles. The ducts and pores from the RPV are shown, along with valves that control release of ink from the RPV. Prince et al. 1998 J Exp Biol 201: 1595; see also Coelho et al. 1998 J Exp Biol 201: 425 for details of metabolism of phycoerythrins in the digestive gland of Aplysia californica.

NOTE  the sea hares used in the study come from a culture facility at the University of Miami, NIH National Resource for Aplysia, Miami, Florida

NOTE  in a later paper the senior author at the University of Miami provides a similarly detailed description of the opaline gland in A. californica, an organ whose function is much less well known than the ink gland. Prince 2007 J. Moll Stud 73: 199.

  black dot
Research study 8

photograph of lobster attacking a sea hare courtesy Kicklighter et al. 2005Sea hares Aplysia californica appear to defend themselves from attack by spiny lobsters Panulirus interruptus by releasing ink and opaline secretions.  The authors use a novel experimental approach.  They remove ink and/or opaline glands from test individuals and present the treated1 sea hares to the lobsters.  Results show that sea hares with both glands intact, or only opaline gland intact, escape predation in about 64% of the encounters with spiny lobsters, while sea hares with neither gland or with just an ink gland escape predation in only about 18% of encounters (see histogram, which represents only part of a large data set). Encounters in which sea hares release secretions and survive indicate that the secretions have different effects on the lobsters, with ink inducing ingestive behaviour, opaline secretion inhibiting ingestive behaviour and also evoking escape responses, and both secretions stimulating grooming. The photograph on Left shows the end of an attack by a lobster on a sea hare. After much release of ink the sea hare is allowed to escape and the lobster displays feeding behaviour. The active ingredients in the ink are millimolar quantities of amino acids that stimulate chemoreceptor neurons in the lobster’s nervous system.  Included are large amounts of taurine, which is a known phagostimulant for many marine invertebrates, as well as lysine, and histidine. The secretions appear to function in at least 3 ways: the first, by a novel and previously undescribed form of chemical defense termed phagomimicry2, in which stimulation of feeding pathways deceive the lobsters into responding as though food stimuli were present (the behaviours include moving the 1st two pairs of legs to the mouth and “digging” movements); the 2nd, by sensory disruption; and the 3rd, by chemical deterrence.  The authors describe phagomimicry as a “sensory trap” because the lobster’s chemosensory system is “trapped” to respond in a certain “preprogrammed” way.  Although lobsters3 are not known to be major predators of sea hares worldwide, the study shows that the potential effects of ink-opaline secretions4 are far more complex than previously envisaged.  What is needed now, of course, is comparable investigations of other potential predators of Aplysia californica.  Kicklighter et al. 2005 Current Biol 15: 549; see also Derby & Aggio 2011 Integr Comp Biol 51: 771 for an up-to-date review of chemical defenses in marine molluscs and other animals. The latter paper also provides wonderful photograph of an inking Aplysia (lower Right) done by Genevieve Anderson, Santa Barbara Community College, California.

photograph of sea hare Aplysia californica releasing ink courtesy Genevieve Anderson, Santa Barbara Community CollegeNOTE1  individuals with one of the secretory glands missing are still able to release secretion from the other and, according to the authors, all surgically treated animals appear to be “in good health” on the day following their surgery when they are used in experiments

NOTE2 videos of ink and opaline gland release, and induction of phagomimicry responses in the lobsters can be found at SEA HARES USE NOVEL ANTIPREDATORY CHEMICAL DEFENSES. These videos are well worth watching, especially if you have never seen ink and opaline being secreted by a sea hare

NOTE3 a study by researchers at Chapman University, California shows that an attack by lobsters Panulirus interruptus will act as a sensitising stimulus to the head/siphon withdrawal response in A. californica equal to that of a strong electric shock.  In demonstrating that electrical shock does indeed mimic at least one natural stimulus, the authors provide an ecological context for its use in learning experiments with A. californica Watkins et al. 2010 J Neurosci 30 (33): 11028.

NOTE4  ink and opaline secretions are acidic, ranging from pH5-8.  Recent studies suggest that secretions of greater acidity lead to greater enhancement of the phagomimetic chemical defense.  Shabani et al. 2007 J Comp Physiol A 193: 1195

Sea hare Aplysia californica releasing ink 0.6X

  black dot
Research study 9

histogram showing anti-feedant effect of ink of sea hares Aplysia californica on tentacles of a potentially predatory sea anemone Anthopleura solaA follow-up investigation to Research Study 6 above investigates which components of the ink and opaline secretions of Aplysia californica are aversive to sea anemones Anthopleura sola, as demonstrated by tentacle shriveling and/or retraction. Tentacle shriveling and/or retraction would perhaps lead to anemones dropping ensnared sea hares.  Sea hares are first fed the red alga Gracilaria ferox and then their ink and opaline-gland secretions are applied to sea-anemone tentacles in 50µl doses with a syringe, with the same volume of seawater being used for a control. 

Results show that the ink is aversive to the sea anemones, but that the opaline secretion actually elicits feeding by the sea anemones. Tests with algal extracts elicit no response in the anemones, suggesting that the aversive components are actually produced by the sea hares.  One of the ink’s components, a protein called escapin, is tested alone and  together with its substrate L-lysine, but neither elicits tentacle shriveling and/or retraction different from the seawater controls.  The authors conclude that escapin plays little or no role in defense, at least not against A. sola.  By use of fractionation techniques the authors determine that several components, including both lipophilic and hydrophilic ones, may be involved in the aversive responses. The authors suggest that the multiple components in the ink of Aplysia spp. may explain why different potential predators are affected in different photograph of sea anemones Anthopleura solaways.  For example, opaline secretion is phagostimulatory to sea anemones, but not to lobsters, and so on. Kicklighter & Derby 2006 J Exp Mar Biol Ecol 334: 256.

NOTE  this is a major protein in the ink and has antimicrobial activity

NOTE  also, in Research Study 8 above, components of the ink of A. californica are found to act as phagomimics, that is, the lobsters exhibit feeding behaviours in the presence of the ink, but not directed to the sea hares. Other research on a related species Aplysia dactylomela in Jamaica finds that sea-hare inks act as sensory irritants in various ways to invertebrates and fishes.  Carefoot & Pennings 1999 J Exp Mar Biol Ecol 234: 185

Sea anemones Anthopleura sola in an aquarium tank (thus explaining their bleached
appearance). This new species represents the old "solitary" form of the aggregating
anemone A. elegantissima. It is much larger than the aggregating form, has different
colour patterns to manubrium and tentacles and is, of course, solitary 0.6X

  black dot
Research study 10

Researchers at Georgia State University reiterate the chemical makeup of ink and opaline secretions in Aplysia californica, and provide further detail on how the chemicals are packaged.  The main defensive chemical is an L-amino acid-oxidase called escapin1This compound is exclusively produced in the ink gland, has antimicrobial activity, and is thought by the authors to function chiefly in an antipredator role.  Within the ink gland, escapin is only present in the so-called amber vesicles and not in the red-purple vesicles2 (refer to figure in Research Study 7 above).  These latter contain the phycoerythrobilin pigments derived from Aplysia’s red-algal food that give the ink its characteristic purple colour.  Both of the chief amino-acid components of the ink, namely, lysine and arginine, are involved in escapin’s bacteriostatic effects, but only lysine is involved in its bacteriocidal effects. Whether these antibacterial properties play a significant role in the everyday biology of Aplysia, or whether they are just a side-effect of the ink's antipredatory chemistry is not known. Lysine is also present in opaline secretion but in much higher concentration than in the ink.  On the strength of this, the authors propose that the lysine in the opaline secretion acts as a substrate for the enzyme escapin in the ink, and that the simultaneous3 release of ink and opaline allows for the generation of antipredatory defensive compounds “from innocuous precursors at the precise time they are needed”.  Johnson et al. 2006 J Exper Biol 209: 78.

NOTE1  the major amino-acid components in escapin are L-lysine and L-arginine

NOTE2  a third type of vesicle, clear, is also present, but appears to be more common in sea hares eating green algae or other foods such as lettuce that lack the pigments necessary to produce purple ink

NOTE3  the authors’ assertion that ink and opaline are commonly released simultaneously by Aplysia may provoke comment from other researchers.  Although this may be usual in A. californica, in other Aplysia species the 2 secretions seem often to be released separately, ink more readily than opaline.  In fact, it is often difficult to stimulate a sea hare to release opaline as it appears to have a higher stimulus threshold. In one of the videos accessible in Research Study 8 above, however, A. californica can be clearly seen releasing both secretions simultaneously

  black dot
Research study 11

photo graph of catfish Ariopsis felisRelated studies by researchers at Georgia State University on defensive functions of ink and opaline secretions of sea hares Aplysia californica provide evidence that the aplysioviolin and phycoerythrin components are deterrents to feeding by different fishes. By recording impulses from nerves that innervate the chemosensory barbels1 of the marine catfish Ariopsis felis the authors determine that both ink/opaline components strongly reduce responses of the barbels to amino acids and bile salts, both known stimulatory compounds for teleost taste systems. Complementary studies on the tropical bluehead wrasse Thalassoma bifasciatum show that release of ink both interferes with the fish’s olfactory sensitivity and, when incorporated into food pellets2, with taste sensation within the mouth. When the wrasse’s olfaction is occluded by obstruction of the nares with petroleum jelly, the deterrent effect of the ink cloud is eliminated but not the ink’s effect on food acceptance. Thus, ink but not opaline secretion interferes with the olfactory-capture phase of feeding but not with the nonolfactory-acceptance phase. The studies3 add to our understanding of the functions of ink and opaline secretions in sea hares. Nusnbaum & Derby 2010 Anim Behav 79: 1067; Nusnbaum et al. 2012 J Comp Physiol A 198: 283.photograph of bluehead wrasses Thalassoma bifasciatum

NOTE1 the catfish have taste buds not only on the barbels, but also on the face, mouth, and general body surface

NOTE2 the ink components are incorporated into pellets made from shrimp powder and agar

NOTE3 the researchers’ choice of fishes to test is unusual, as neither species lives even in the same ocean as sea hares A. californica, let alone (in the case of bluehead wrasses) utilises sea hares of any species as food. These wrasses are primarily planktotrophic, subsisting mainly on copepods and various larvae, and sometimes small benthic crustaceans and snails. Also, a more logical choice for ink and opaline secretions to use on wrasses would be Aplysia dactylomela and, for catfish, A. brasiliana, because both pairs would then be cohabitants of the same oceans with potential for at least some history of co-evolution. The authors choose California sea hares for the vast body of knowledge available on their ink and opaline secretions


Top: Florida catfish Ariopsis felis

Bottom: bluehead wrasses; Left:
initial-phase; Right: terminal phase

  black dot