Michiya Kamio

l-Kynurenine, an amino acid identified as a sex pheromone in the urine of ovulated female masu salmon

Many animals employ sex pheromones to find mating partners during their reproductive seasons. However, most sex pheromones of vertebrates remain to be identified. Over the past 20 years, steroids and prostaglandins have been identified as sex pheromones in several fishes. These pheromones are broadly termed “hormonal pheromones” because they or their precursors act as hormones in these fishes. Hitherto, no other type of sex pheromone has been unambiguously identified in teleost fish. Here we report the identification of a “nonhormonal pheromone” in teleost fish. The urine of the reproductively mature female masu salmon (Oncorhynchus masou) contains a male-attracting pheromone. Bioassay-guided fractionation yielded an active compound that was identical to l-kynurenine in spectral and chromatographic properties. l-Kynurenine is a major metabolite of l-tryptophan in vertebrates. This pheromone elicits a male-specific behavior at even picomolar concentrations; its electrophysiological threshold is 10−14 M. l-Kynurenine is a reasonable substance for female masu salmon to advertise their readiness for mating.

Packaging of chemicals in the defensive secretory glands of the sea hare Aplysia californica

SUMMARY Sea hares protect themselves from predatory attacks with several modes of chemical defenses. One of these is inking, which is an active release of a protective fluid upon predatory attack. In many sea hares including Aplysia californica and A. dactylomela, this fluid is a mixture of two secretions from two separate glands, usually co-released: ink, a purple fluid from the ink gland; and opaline, a white viscous secretion from the opaline gland. These two secretions are mixed in the mantle cavity and directed toward the attacking predator. Some of the chemicals in these secretions and their mechanism of action have been identified. In our study, we used western blots, immunocytochemistry, amino acid analysis, and bioassays to examine the distribution of these components: (1) an l-amino acid oxidase called escapin for A. californica and dactylomelin-P for A. dactylomela, which has antimicrobial activity but we believe its main function is in defending sea hares against predators that evoke its release; and (2) escapins major amino acid substrates - l-lysine and l-arginine. Escapin is exclusively produced in the ink gland and is not present in any other tissues or secretions. Furthermore, escapin is only sequestered in the amber vesicles of the ink glandand not in the red-purple vesicles, which contain algal-derived chromophores that give ink its distinctive purple color. The concentration of escapin and dactylomelin-P in ink, both in the gland and after its release, is as high as 2 mg ml-1, or 30 μmol ml-1, which is well above its antimicrobial threshold. Lysine and arginine (and other amino acids) are packaged into vesicles in the ink and opaline glands, but arginine is present in ink and opaline at <1 mmol l-1 and lysine is present in ink at <1 mmol l-1 but in opaline at 65 mmol l-1. Our previous results showed that both lysine and arginine mediate escapins bacteriostatic effects, but only lysine mediates its bactericidal effects. Given that escapins antimicrobial effects require concentrations of lysine and/or arginine >1 mmol l-1, our data lead us to conclude that lysine in opaline is the primary natural substrate for escapin in ink. Furthermore, packaging of the enzyme escapin and its substrate lysine into two separate glands and their co-release and mixing at the time of predatory attack allows for the generation of bioactive defensive compounds from innocuous precursors at the precise time they are needed. Whether lysine and/or arginine are substrates for escapins antipredatory functions remains to be determined.

Spiny lobsters use urine-borne olfactory signaling and physical aggressive behaviors to influence social status of conspecifics

SUMMARY Decapod crustaceans, like many other animals, engage in agonistic behaviors that enhance their ability to compete for resources with conspecifics. These agonistic behaviors include the release of chemical signals as well as physical aggressive and submissive behaviors. In this study, we report that Caribbean spiny lobsters, Panulirus argus, use both urine-borne chemical signaling and physical aggressive behaviors during interactions with conspecifics, and that these agonistic behaviors can influence the behavior and eventual social status of the interactants. Spiny lobsters that engaged primarily in physical aggressive behaviors became dominant, whereas spiny lobsters that received these physical aggressive behaviors responded with avoidance behaviors and became subordinates. Dominant animals frequently released urine during social interactions, more than when they were not in contact with subordinates and more than when they were not paired with another animal. Subordinates released urine significantly less often than dominants, and no more than when not paired. Preventing release of urine by catheterizing the animals resulted in an increase in the number and duration of physical interactions, and this increase was primarily driven by dominants initiating interactions through physical aggressive behaviors. Introducing urine from one of the catheterized animals into an aquarium reduced physical aggressive behavior by dominant animals to normal levels. Urine-borne signals alone were capable of inducing avoidance behaviors from solitary spiny lobsters in both laboratory and field conditions. We conclude that urine serves as a chemical signal that communicates social status to the interactants. Ablation experiments showed that that these urine signals are detected primarily by aesthetasc sensilla of the olfactory pathway.

Molecular identification of alarm cues in the defensive secretions of the sea hare Aplysia californica

Prey species possess numerous strategies to reduce predation. One tactic is to respond with antipredator behaviours when conspecific alarm cues are detected. The sea hare Aplysia californica defends itself from predators in many ways, one of which is releasing ink and opaline upon attack. Previous work showed that a mixture of ink and opaline from A. californica causes conspecifics to respond with antipredator behaviours such as moving away and/or ‘galloping’. We examined the specificity of the alarm response, including identifying the molecules mediating it. Either ink or opaline alone evokes the full alarm response, but conspecific mucus, conspecific haemolymph, odour from predatory spiny lobsters, or odour from algal food do not. Thus, the defensive secretions, ink and opaline, specifically act as alarm cues to nearby conspecifics. We isolated and identified the alarm cues in ink as the base uracil and the nucleosides uridine and cytidine. Each of these molecules individually elicits frequencies of alarm behaviours as great as ink. Ink without its alarm cue molecules does not elicit a significant frequency of alarm behaviours. Thus, these three molecules together are necessary and sufficient to produce alarm responses. Aplysia californica antipredator behaviours are also elicited by ink from the congener Aplysia juliana or Aplysia dactylomela. Furthermore, ink from the squid Lolliguncula brevis or the octopus Octopus bimaculoides also elicits antipredator behaviours by A. californica, owing to the presence of uracil and uridine. Thus, these alarm cues may be common among inking molluscs.

The purple pigment aplysioviolin in sea hare ink deters predatory blue crabs through their chemical senses

Sea hares release an ink secretion composed of purple ink and white opaline as a potential chemical defence against predators. The aim of our study was to identify deterrent molecules in the ink of Aplysia californica against an allopatric generalist crustacean predator, the blue crab Callinectes sapidus, and to define the mechanisms of action of the deterrents against crabs. We used two behavioural assays, a squirting assay and an ingestion assay, to show that ink is highly effective and that opaline is moderately effective in suppressing feeding of crabs. Results with reversibly blinded crabs demonstrate that the deterrence is mediated through the crabs’ chemical senses. We used bioassay-guided fractionation to identify the purple molecules aplysioviolin and phycoerythrobilin as a major and minor deterrent, respectively, in ink against crabs. These molecules derive from a light-harvesting protein in the photosynthetic system of dietary algae. This is the first demonstration of an animal converting a photosynthetic pigment into a chemical deterrent. Mixing opaline and ink enzymatically produces hydrogen peroxide, which also functions as a chemical deterrent against crabs. Our results and those of other studies show that sea hares use a diversity of molecules in their skin, mucus and ink secretion to chemically defend themselves against their potential predators. Aplysioviolin, phycoerythrobilin and hydrogen peroxide also exist in ink secretion of Aplysia dactylomela, a sea hare sympatric to blue crabs, and thus we posit that these molecules are potentially effective in ecologically relevant predator–prey interactions and need to be scrutinized in more ecologically relevant experiments.

Behavioral and Electrophysiological Experiments Suggest That the Antennular Outer Flagellum Is the Site of Pheromone Reception in the Male Helmet Crab Telmessus cheiragonus

Sexually competent females of Telmessus cheiragonus (helmet crab) release two pheromones that elicit grasping and copulation behaviors in males (Kamio et al., 2000, 2002, 2003). Our study aimed to use behavioral and electrophysiological techniques to identify the site of reception of these sex pheromones. In behavioral experiments, either the inner or the outer flagella of the antennules were ablated bilaterally from male crabs, and responses of male crabs to female odor were examined. When the inner flagella were surgically ablated, the sexual response (i.e., grasping and copulation behavior) of male crabs was not significantly changed relative to control animals that had their second antennae ablated. In contrast, the sexual response was significantly reduced when the outer flagella of the antennules were ablated, suggesting that the outer flagellum is the receptor organ that detects the sex pheromones. In electrophysiological experiments, urine, which in females contains the pheromone that elicits grasping behavior by males but does not contain the pheromone eliciting copulation, whose release site is not known, was tested. Female and male urine as well as shrimp extract evoked phasic responses of chemosensory afferents innervating aesthetasc sensilla on the outer flagellum of male crabs. The response of the afferents had significantly higher magnitude and lower threshold when female urine was applied. Thus, behavioral and electrophysiological observations suggest that in male helmet crabs, the outer flagellum of the antennule is the chemosensory organ that detects female sex pheromone.

The Chemistry of Escapin: Identification and Quantification of the Components in the Complex Mixture Generated by an L‐Amino Acid Oxidase in the Defensive Secretion of the Sea Snail Aplysia californica

Escapin is an L-amino acid oxidase in the ink of a marine snail, the sea hare Aplysia californica, which oxidizes L-lysine (1) to produce a mixture of chemicals which is antipredatory and antimicrobial. The goal of our study was to determine the identity and relative abundance of the constituents of this mixture, using molecules generated enzymatically with escapin and also using products of organic syntheses. We examined this mixture under the natural range of pH values for ink-from approximately 5 at full strength to approximately 8 when fully diluted in sea water. The enzymatic reaction likely forms an equilibrium mixture containing the linear form alpha-keto-epsilon-aminocaproic acid (2), the cyclic imine Delta(1)-piperidine-2-carboxylic acid (3), the cyclic enamine Delta(2)-piperidine-2-carboxylic acid (4), possibly the linear enol 6-amino-2-hydroxy-hex-2-enoic acid (7), the alpha-dihydroxy acid 6-amino-2,2-dihydroxy-hexanoic acid (8), and the cyclic aminol 2-hydroxy-piperidine-2-carboxylic acid (9). Using NMR and mass spectroscopy, we show that 3 is the major component of this enzymatic product at any pH, but at more basic conditions, the equilibrium shifts to produce relatively more 4, and at acidic conditions, the equilibrium shifts to produce relatively more 2, 7, and/or 9. Studies of escapins enzyme kinetics demonstrate that because of the high concentrations of escapin and L-lysine in the ink secretion, millimolar concentrations of 3, H(2)O(2), and ammonia are produced, and also lower concentrations of 2, 4, 7, and 9 as a result. We also show that reactions of this mixture with H(2)O(2) produce delta-aminovaleric acid (5) and delta-valerolactam (6), with 6 being the dominant component under the naturally acidic conditions of ink. Thus, the product of escapins action on L-lysine contains an equilibrium mixture that is more complex than previously known for any L-amino acid oxidase.

Spiny lobsters detect conspecific blood-borne alarm cues exclusively through olfactory sensilla.

SUMMARY When attacked by predators, diverse animals actively or passively release molecules that evoke alarm and related anti-predatory behavior by nearby conspecifics. The actively released molecules are alarm pheromones, whereas the passively released molecules are alarm cues. For example, many insects have alarm-signaling systems that involve active release of alarm pheromones from specialized glands and detection of these signals using specific sensors. Many crustaceans passively release alarm cues, but the nature of the cues, sensors and responses is poorly characterized. Here we show in laboratory and field experiments that injured Caribbean spiny lobsters, Panulirus argus, passively release alarm cues via blood (hemolymph) that induce alarm responses in the form of avoidance and suppression of feeding. These cues are detected exclusively through specific olfactory chemosensors, the aesthetasc sensilla. The alarm cues for Caribbean spiny lobsters are not unique to the species but do show some phylogenetic specificity: P. argus responds primarily with alarm behavior to conspecific blood, but with mixed alarm and appetitive behaviors to blood from the congener Panulirus interruptus, or with appetitive behaviors to blood from the blue crab Callinectes sapidus. This study lays the foundation for future neuroethological studies of alarm cue systems in this and other decapod crustaceans.

Mycosporine-like amino acids are multifunctional molecules in sea hares and their marine community

Molecules of keystone significance are relatively rare, yet mediate a variety of interactions between organisms. They influence the distribution and abundance of species, the transfer of energy across multiple trophic levels, and thus they play significant roles in structuring ecosystems. Despite their potential importance in facilitating our understanding of ecological systems, only three molecules thus far have been proposed as molecules of keystone significance: saxitoxin and dimethyl sulfide in marine communities and tetrodotoxin in riparian communities. In the course of studying the neuroecology of chemical defenses, we identified three mycosporine-like amino acids (MAAs)—N-ethanol palythine (= asterina-330), N-isopropanol palythine (= aplysiapalythine A), and N-ethyl palythine (= aplysiapalythine B)—as intraspecific alarm cues for sea hares (Aplysia californica). These alarm cues are released in the ink secretion of sea hares and cause avoidance behaviors in neighboring conspecifics. Further, we show that these three bioactive MAAs, two [aplysiapalythine A (APA) and -B (APB)] being previously unknown molecules, are present in the algal diet of sea hares and are concentrated in their defensive secretion as well as in their skin. MAAs are known to be produced by algae, fungi, and cyanobacteria and are acquired by many aquatic animals through trophic interactions. MAAs are widely used as sunscreens, among other uses, but sea hares modify their function to serve a previously undocumented role, as intraspecific chemical cues. Our findings highlight the multifunctionality of MAAs and their role in ecological connectivity, suggesting that they may function as molecules of keystone significance in marine ecosystems.

Evaluation of the bioactivities of water-soluble extracts from twelve deep-sea jellyfish species

Many polypeptides isolated from shallow water cnidarian species have been utilized as valuable biochemical tools in both basic and applied biological sciences. Deepwater cnidarian species might be another potential resource for novel biochemical tools. However, because of limited access to cnidarian samples from deep-sea environments, bioactive polypeptides have never before been reported from this group. In this study, we collected twelve deep-sea jellyfish species (nine hydrozoans and three scyphozoans) using a plankton net that was specially designed for collecting deep-sea organisms, and prepared water-soluble extracts, presumably containing polypeptides, of these jellyfishes. The extracts were subjected to cytotoxicity, hemolytic activity, and crustacean lethal toxicity tests. In the cytotoxicity test, six out of the nine tested hydrozoan species showed activity. In the hemolytic activity test, only three hydrozoans showed activity and none of the scyphozoan jellyfishes showed activity. In the crustacean lethality test, two hydrozoan jellyfishes and all three of the tested scyphozoan jellyfishes showed lethal activity. These results revealed a high incidence of water-soluble bioactive substances occurring in these deep-sea jellyfishes. Furthermore, all the heat-treated and the methanol-treated crude jellyfish extracts lost their bioactivities. Thus, it is likely that the bioactive compounds in the water-soluble extracts were unstable polypeptides (proteins). This is the first published report on bioactivities in extracts from deep-sea jellyfishes.