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| Defenses | ||||||||
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| Ink & opaline secretions | ||||||||
Defenses of nudibranchs and relatives include ink & opaline secretions, considered in this section, and
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 the Research Studies are presented chronologically. |
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Research study 1 |
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Points of clarification about opaline secretion include: 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. NOTE chemicals in the body that seem to have no role in metabolism; thought to have defensive functions
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Research study 2 |
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Of the 2 west-coast aplysiid species, only A. 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 Aplysia 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, Aplysia 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. |
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Research study 3 |
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Research study 4 |
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NOTE includes pinching with forceps, electrical shock, and application of crystals of NaCl |
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Research study 5 |
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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. NOTE Navanax lives much deeper than the shallow-dwelling A. californica, so it is not clear how much natural predation there would be |
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Research study 6 |
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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. 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 |
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Research study 7 |
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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 |
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Research study 8 |
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The active ingredients are millimolar quantities of amino acids2 that stimulate chemoreceptor neurons in the lobster’s nervous system. The secretions appear to function in at least 3 ways: the first, by a novel and previously undescribed form of chemical defense termed phagomimicry3, 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 lobsters are not known to be major predators of sea hares worldwide, the study shows that the potential effects of ink-opaline secretions 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 NOTE1 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 these include large amounts of taurine, which is a known phagostimulant for many marine invertebrates, as well as lysine, and histidine, and including relatively large concentrations of ammonium NOTE3 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
Sea hare Aplysia californica releasing ink 0.6X |
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Research study 9 |
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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 ways. NOTE 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 finds that sea-hare inks act as sensory irritants to various invertebrates and fishes. Opaline secretion is phagostimulatory to sea anemones, but not to lobsters, and so on NOTE this is a major protein in the ink and has antimicrobial activity |
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Research study 10 |
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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 escapin1. This 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”. 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 |
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A 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 
Ink-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. 
Ink release by sea hares Aplysia californica following 
Does the ink function in defense against predators? Research on other species of Aplysia 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. 
A detailed study of the processing, storage, and secretion of ink by A. californica by scientists in 
Sea hares Aplysia californica 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 
A 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.