SHOWEN: DEFENSE RESPONSE OF ARCTONOE VITTATA INTRODUCTION: The keyhole limpet Diadora aspera (Eschscholtz, 1833) is one of the many hosts of the scaleworm Arconoe vittata. (Grube, 1955). Margolin (1964) found that Diadora raises its membranous, frilled mantle, virtually covering its shell in response to contact with a predatory seastar or upon exposure to seawater in which a seastar had been kept. Dimock and Dimock (1972) report that A. vittata will bite the tube feet of various seastars, including Pisaste: ochraceus (Brandt, 1835) following seastar-limpet con- tact and initiation of the mantle response. They also present evidence that no substance released from Diadora was involved in eliciting the response from A. vittata and that a worm isolated from its host would not bite a starfish. Their paper was a brief note based on small sample size. I here further elucidate the mechanism of this behavior. MATERIALS AND METHODS: Diadora aspera, two to five cm in length, with commensal ctonoe vittata were collected during April and May of 1976 from a submerged rocky outcropping known as the Pinnacles (36°33' North by 121°58' West) in 70 to 95 feet of water. Intertidal Pisaster ochraceus were used as the test animals. Animals were kept in aquaria with running sea water at Hopkins Marine Station, Pacific Grove, California. Due to the difficulty in collecting numbers of 4 vittata most worms were used in more than one experiment. Basic Experiment: D. aspera with commensal worms were placed on glass and observed from the underside, rather than in enameled trays from above as in Dimock and Dimocks work. An arm of a Pisaster was brought into SHOWEN: DEFENSE RESPONSE OF ARCTONOE VITTATA contact with the edge of the Diadora shell. Often the starfish would move itself upon the glass, cover¬ ing a large part of the shell. Muring the ten minute trial period notes were taken on the movements of the starfish, the host and the commensal. Starfish Water Experiment: To determine if either the host's mantle response or water which had contacted Pisaster could cause a worm to search without direct contact of the starfish to the limpet, two starfish were introduced to test aquaria but not permitted to contact the host. The animals were observed for ten minutes. "Diadora Juice" Experiments: Five limpets and an isolated worm were placed in a small plastic tray of sea water and a seastar was touched to the worm. Observation was for ten minutes in each of three trials. Additionally, mucus and seawater from the mantle cavity of a Diadora in which a mantle response had been previously elicited by a starfish was pipetted onto an isolated Arctonoe in contact with a seastar. The worms were observed for three minutes in each of five trials. MgCl. Experiment: In order to test for the necessity of the physical movement of the mantle response for biting to occur limpets with their worms removed were placed in isotonic Mgcly solution, an anaesthetic, for several hours. The relaxed Diadora were then placed upside down in aquaria and held in place with a lump of clay. The worms were then returned to the hosts and after the worm was settled a starfish was placed in contact with it. The observation period was ten minutes in each of three trials. SHOWEN: DEFENSE RESPONSE OF ARCTONOE VITTATA Gelatin Groove Experiment: To test for the necessity of the Diadora's presence for biting to occur an arm of a starfish was touched to the anterior portion of an Arctonoe placed in a shallow groove in fresh, firm g dry gelatin in 100 ml seawater). The groove was roughly "worm sized" in all dimensions, so as to allow the worm to fit in snuggly. Tests were scored only it the worm remained in the groove for the ten minte trial period. As a control isolated worms were tested against a starfish on a gelatin surface without a groove. The observation period for the control experiments was also ten minutes for each of five trials. Palps Excised Experiment: Worms without palps were tested using the "Basic Experiment" procedure. RESULT The Arctonoe vittata were variable in their markings. One type had the posterior edge of the first pair of elytra marked with a black or brown band, whereas the other type had no such marking. Except for this marking, the worms were white or cream colored. None of the worms had the dark band across segments seven and eight commonly associated with the species yet prostomial and parapodial characteristics are those of A. vittata. Eight of 17 marked worms were observed to bite whereas only one of 12 unmarked bit. The difference is signi¬ ficant at the .025 level according to the G-test. Prior to the introduction of the seastar the worm was generally motionless in the mantle cavity, with its dark tipped palps usually retracted but occasionally extended and waving. After introduction of the star- fish the Arctonoe would remain motionless until the onset of the mantle response. The first response of the worm noted was waving of its palps and the anterior portion of its body. It would then emerge from beneath SHOWEN: DEFENSE RESPONSE OF ARCTONOE VITTAT the shell and display the searching behavior described by Dimock and Dimock. Nine worms never showed any search activity at all. While a few worms moved directly towards the tube feet, as described by Dimock and Dimock, many seemed to be searching randomly. Twelve worms showed search behavior but never contacted a starfish and bit. Only if the starfish was within a centimeter or two was the worm able to pinpoint the point of tube foot contact. Nine of the worms, upon contact with a tube foot, ran their palps along the tube foot and everted their proboscis and bit, often holding on to the tube foot for several seconds. All data following applies to marked worms only. Starfish Water Experiment: The three limpets displayed the mantle response when near but not touching the Pisasters. All the worms displayed search behavior. Four worms with palps removed also showed searching behavior using the same test procedure. Additionally, this searching behavior was observed at least ten times when water in which starfish had been kept was allowed to flow into aquaria containg Diadora with commensal worms. "Diadora Juice" Experiment: In five experiments in which fluid from the mantle cavity was applied to an isolated worm in contact with a Pisaster, no aggressive behavior occurred and no increase in activity was noted. The worm often remained among or next to the tube feet. In three trials in which an isolated Arctonoe vittata was placed in contact with a Pisaster in seawater with five disturbed Diadora, no searching or aggressive behavior by any worm occurred. MgCl, Experiment: Arctonoe returned to Diadora rendered limp from MgCly treatment behaved normally, although they were not as protected as in a normal limpet. TTAT SHOWEN:DEFENSE RESPONSE OF ARCTONOE When a starfish was touched to the limpet but not the worm, the Arctonoe always remained inactive. Only when the tube feet actually contacted the prostomial region did the worm become aroused and exhibit searching be- havior. This resulted in biting in three of the six worms tested. Gelatin Groove Experiment: The response of Arctonoe in a gelatin groove was similar to their behavior on a limpet treated with Mgolø solution. Again, in six trials the worms never turned in the di- rection of a tube foot, stretched out towards a tube foot, or bit until the prostomial region was touched with a tube foot. Six worms remained in the gelatin groove for ten minutes; three of them bit. Approximately two thirds of the worms left the groove before ten minutes. Worms did not remain in gelatin preparations more than several hours old, possibly due to the presence of increased numbers of microorganisms. Palps Excised Experiment: Worms with palps removed behaved like normal worms with one important exception. Two of seven ctonoe tested made attempts at biting; both of them missed the tube feet. This phenomenon was also observed informally on three other occasions. No worms missing palps was ever observed to successfully bite a tube foot. DISCUSSION: The fact that Arctone bearing a dark band bite signi- cantly more than unmarked worms indicates the two sympatric groups vary in more than one genetic trait. If this indicates gene isolation between the forms is not yet known. Dimock and Dimock indicated the presence of a Pisaster is insufficient to cause biting in an isolated worm. Some stimulus, associated with the presence of the host is required. That worms do not search on MgClz treated limpets indicates that mere presence of the host and starfish m SHOWEN:DEFENSE RESPONSE OF ARCTONOE VITTATA is not adequate to elicit searching. Some cue from the iadora must be missing, either a physical movement, a chemical released during the mantle response, or a chemical the effects of which are negated by Mgcl2. It was observed that Arctonoe on normal limpets either isaster in contact with or merely in the same tank with will search only after initiation of the mantle response. This confirms that mere presence of a seastar in the environment will not elicit searching, and indicates that some aspect of the mantle response must initiate search behavior. Dimock and Dimock did not observe that worms searched on limpets exposed to seawater in which starfish had been kept. However, Margolin (1964) reports that 100% of Diadora tested gave the mantle response in reaction to seastar water whereas only one of ten limpets did so in the Dimocks experiment. That worms do not become aroused either in the presence of disturbed Diadora or upon direct application of mucus from a disturbed limpet indicate that: 1. No water soluble substance released from Diadora during the mantle response or insoluble substance in the mucus is responsible for initiating searchinging. 2. There is no chemical whose effects are negated by Mgcl,. The physical movement of the mantle is necessarry and sufficient to elicit searching. That worms will bite on MgCl, treated limpet or in a gelatin groove once contacted by a tube foot indicates that the worm must merely be in a groove and directly contact a starfish for biting to occur. Experiments involving worms without palps dekmonstrate palps are not needed for detection of or reaction to a Pisaster but that they are required for proper coordination of the bite. Biting then results from a two part behavior; searching C ABCT FNC SHOWEN:DEFENSE RESPONSE OF ARCTONOE VITTATA in response to the physical movement of the mantle followed by biting in reponse to position in a groove and contact with a starfish. SUMMARY: 1. Commensal Arctonoe vittata marked with a dark band ter ochraceus significantly were shown to bite I more than unmarked worms on host Diadora as 2. Arctonoe were shown to search in response to the physical movement of the mantle reponse. 3. Arctonoe were found to bite, once aroused, if they were situated in a groove and contacted a tube foot. 4. Worms with palps removed were found to miss the tube foot when attempting tobite. ACKNOWLEDGEMENTS: Many thanks to Dr. Robin Burnett for much help in thinking about this project, to Jim Watanabe for assistance in collecting, and to the entire faculty and staff of Hopkins Marine Station for making this project possible. e SHOWEN: DEFENSE RESPONSE OF ARCTONOE VITTATA LITERATURE CITED: Dimock, Ronald V. and Joyce G. 1969 A possible "defense" response in a commensal polychaete. The Veliger 12:65-68 Margolin, Abraham Stanley 1964. The mantle response of Diadora aspera. Animal Behavior 12:187-194 0