Abstract: Diopatra ornata is a tube-dwelling polychaete commonly found in sandy subtidal regions from Mexico to California. Despite their abundance, little information has been published about D. ornata. This study examined tube construction, locomotion, feeding habits, and activity patterns of D. ornata, and its ability to regenerate lost or damaged regions of its body. D. ornata construct elaborate sand and mucus tube caps that are anchored in the sand and arch over with the aperture -1 cm above the sand and facing downward. Worms attach pieces of shell and drift kelp to the tube cap and can add on as much as 6 cm of new tube in one day. The portion of the tube that extends down into the substratum is more fragile and runs - horizontally at a depth of-10 cm below the surface of the sand. The worms move up and down inside their tubes using parapodia and can turn around completely inside it. They can extend their bodies up to approximately 8 cm from the aperture of the their tube to retrieve food or tube building material, but can quickly withdraw by sudden muscle contraction. They were never observed to leave their tubes completely. When foraging, D. ornata sit at the aperture of their tube cap and undulate their 5 cephalic cirri rapidly. They appear to sense the presence of food nearby using chemoreception or touch. Time lapse video showed that the worms have distinct day-night patterns of activity, with feeding or working on the buried portion of their tubes as the primary diurnal activity, and repair or addition to the tube cap at night. Regeneration of a head appears to require anterior segments with gills; more posterior sections of the body died and deteriorated within 2 days. Sections maintained in natural or artificial tubes (plastic straws) appeared to survive longer, but did not show any signs of regeneration in 22 days. Further study is needed to more fully understand some of these behaviors and examine other behaviors, such as their reproduction. Introduction: Diopatra ornata is member of the family Onuphidae (class Polychaeta, phylum Annelida). The majority of onuphids are tube-dwelling worms. Their body sizes range from a few millimeters to a few meters long and tend to be cylindrical and widest at the anterior (head) end (Rouse and Pleijel 1999). Onuphids are found throughout the world, in habitats ranging from the intertidal to the abyssal depths. D. ornata is commonly found in subtidal regions of California and Mexico. Polychaetes ("many setae") are characterized by distinct segments bearing a lateral pair of paddle-like parapodia, each with many bristles called setae (Levinton 2001). The setae of Diopatra ornata are hooked and have eight teeth. The well-developed sensory appendages located on the head end of D. ornata consist of three anterior antennae and two palps. The jaws, on the ventral side, have smaller toothed plates and a pair of curved tongs, and the pharynx is a ventral muscular proboscis (Rouse and Pleijel 1999). Several of the anterior segments have elaborate feathery gills. Lining the epidermal epithelium are mucus-secreting glands, and below the epithelium lie the circular muscle fibers and the longitudinal muscle fibers. D. ornata are a dull gray color with a iridescent cuticle (Moore 1911). D. ornata build tubes constructed of chitin, sand, pieces of kelp, and shells, held together by layers of mucus. These tubes are planted into the sand with thin segments under the sand, extending as far as a meter long, as well as elaborate hard, membraneous tube caps above the substrate (Fig. 1). D. ornata tubes form dense mats and cover the sand bordering kelp beds. Little information has been published about Diopatra ornata. This paper examines aspects of their behavior, in particular the way in which they construct their elaborate tubes. Materials and Methods: Diopatra ornata used in this study were collected from the sand bordering the kelp beds outside of Hopkins Marine Station in Monterey, CA. Animals were collected with intact tube caps but only 10-15 cm of the subsurface portions of their tubes. They were transported back to Hopkins Marine Station, where they were housed in five separate 40-liter aquariums. Four of these aquariums were half-filled with beach sand, and the fifth aquarium was half-filled with fine grade glass beads. All aquariums had à supply of running seawater. Density counts were performed in the field by counting the number of tube caps in a 0.0625 m’ plot. Samples were taken from 0.25 m’ plots and the different species present in that area were noted. Observations of locomotion were done by watching the worms' behaviors in their tubes. In addition, locomotion observations were made for 2 worms placed in to clear plastic straws. A high percentage of tubes in the field are occupied. This suggests that D. orndtd do move into tubes previously occupied by a different worm. To test the worm's ability to recognize its own tube and the tendency of a worm to settle in a different worm's tube, and plant that tube upright, Idisplaced 10 worms from their original tubes and placed 3 of them in unoccupied tubes made by another worm and 5 back into their original tubes. The worms were gently displaced from their tubes with tweezers, and they crawled into the new tubes when their heads were pointed inside the tube openings. After 2 days, the worms were observed to see if they had planted the tube into the sand or constructed a new tube. To measure the rate of tube building, 5 tube caps with worms were laid out horizontally on the sand. After a period of 24 hours, each tube was excavated and the length of new tube was recorded. This experiment was repeated twice. Shell fragments were placed 1 cm, 2 cm, and 3 cm away from occupied tubes to test how far the worms would extend from their tubes to collect shell material for their tube caps. The tendency to build curved tube cap openings was also tested by cutting back 26 occupied tube caps, leaving the tube caps sticking straight up with no curvature, and then allowing the worms to rebuild their tube cap. The distance from the tube cap to the substrate was measured both before the tube caps were cut back and after the worm had reconstructed the tube cap. The average of these distances was calculated and assumed to be D. ornata 's preferred distance from the substrate. Tube deterioration was examined by leaving unoccupied tubes in a tank supplied with running seawater. The quality of the tube structure was compared to those tubes occupied with worms (in the aquariums) by testing qualitatively for stiffness. The interiors of the tubes were also compared by cutting the tubes open and examining them visually. The day/ night activity patterns were recorded using time-lapse video of the activities of 3 worms over a course of 3 days. The recording speed was such that 4 minutes of real time was recorded on to 1 second of film (240 times faster than real time). The time-lapse video was also crucial in making many of the behavioral observations. Diopatra ornata have fragile bodies that often fragment during collection. Like many other polychaetes, this fragmentation is due to the worm's tendency to spontaneously autotomize. Many of the segments obtained during collection were kept in order to test the worm’s ability to regenerate lost segments. These regeneration experiments were performed in a tank with sand and running seawater. The worm segments were kept either in a petri dish, in a clear plastic straw, or on the sand. Survival and evidence for regeneration were monitored for 22 days. Results: Density and Neighbors: Tube beds of Diopatra ornata form dense patches in subtidal kelp beds. Densities range from 4 to 17 tube caps per 0.0625 m’, with an average of 10.69 tube caps (Table 1). When excavated individuals were allowed to replant their tubes in an aquarium, D. ornata planted their tubes at least one centimeter from their neighbors, and never with the tube cap openings facing each other. The densest patch of D. ornata tubes observed in the aquarium contained 8 tube caps in a 100 cm plot. Other polychaete species found in the D. ornata beds include Nothria elegans, Nothria iridescens, a terebellid, Halosydna brevisetosa, a maldanid, and a phyllodocid. Other species found in these beds include several species of snails, crabs, red algae, and unattached drift kelp (Macrocystis). The purpose of this sampling was to collect some of the species that co-exist with D. ornata, but this listis by no means inclusive of all the species living in the D. ornata beds. Locomotion: Diopatra ornata do not leave their tubes unles forced to do so. They are able to quickly move up and dovn their tubes and extend from the tube cap seither to catch prey or to grab building material for the tube cap). To move up and down in the tube, the worm relies on parapodia to crawl along the sides of the tube. The worm can extend out of the tube cap and then quickly withdraw by contracting the tail segments suddenly and forcefully. The secretions from the ventral surface of the worm allow the worm to slichtly side within the tube and aid in the worm's ability to hang from the tube cap. imn adition, small setae grasp the inner wall ofthe tube, making it easier for the wormn to stay inside the tube. It is difficult to dislodge a D. ornata from its tube, even when the tube is held vertically upside down. When Diopatra ornata is manually dislodged from its tube, the worm thrashes back and forth untilit has landed on the sand. Once on the sand, the worm quickly digs a hole by pushing the sand aside with its head. The worm enters the growing hole head first and then pulls istailend into the hole in spurts. Fist,it pulsome of its tail into the hole and then digs farther before pulling more of its tail into the hole.The worm completely buries itself before constructing a new tube cap. The entire burowing proces only takes a few minutes and is started as soon as the worm finds a suitable substrate. Tube recognition: Diopatra ornata does not immediately plant its tube cap into the substrate when inserted into a new tube or back into its original tube. After two days, however, two of the seven worms put into new tubes had planted the tubes and two of the worms had constructed new tubes. Two of the seven worms put back into their original tubes also planted their tubes after two days and again two worms constructed new tubes (Fig. 2 and 3). In one unusual case, a worm placed in a new tube created a hole into the side of the old tube (lying horizontally on the sand) and built the buried portion of its tube perpendicularly beneath the old tube. Tube building and tube structure: Tube caps of Diopatra ornata are always bent towards the substrate, with the aperture approximately one centimeter above the sand (Table 1). If the curved portion of the tube cap is cut, the worm adds to the existing tube cap, using surrounding sand and shells, until the tube opening is once again approximately a centimeter above the sand. D. ornata decorate and fortify their tubes caps with extra layers of sand, shells, and pieces of kelp. They pick up the shell (or piece of kelp) with their jaws, and then the shell sticks to the ventral surface of their body, just below the jaws. They then bring the shell back to the tube cap opening and hold the shell in the desired location. The secreted mucus allows the shell to stick to the tube cap, and the worm quickly pulls away from the shell, leaving it attached to the tube cap. The distance a worm reaches for a piece of kelp or a shell depends on the length of the worm. They do reach for shells farther than immediately under their tube, but the worms never fully leave the tube. D. ornata attach extra layers of sand to their tube caps in a similar fashion, by gathering sand on to their ventral surface and then attaching it to the tube cap. If the tube is excavated and/or damaged, Diopatra ornata repair them quickly, adding on approximately 4 centimeters underground over the course of one day as well as adding about 3 centimeters to the tube cap (Table 1). D. ornata always orient their tubes up with a curved cap and quickly plant the tubes in this manner if their tubes are excavated from the sand. To plant an already constructed tube cap, the worm first buries the back end of the tube and builds some of the tube under the sand. The worm then pulls the tube cap partly into the sand and continues to attempt to stand the tube up by emerging from the aperture and pushing against the substrate, levering the curved portion of the tube cap into an upright position. The worm continues this pulling and pushing until the tube is fully upright and stable. If the previously buried segment of the tube is still attached to the tube cap, the worm chews off that segment and starts building a new tube segment under the sand (attached to the tube cap). D. ornata tubes kept without worms become more flimsy and flexible over à period of one week. This alteration suggests that the worms do maintain the rigidness of their tubes, perhaps by continually secreting layers of chitin to the inside of the tubes. D. ornata also keeps the inside of its tube clean of sand and excrement, by dragging sand and fecal pellets to the top of its tube and then pushing it out of the tube cap with its anterior parapodia. Feeding: Diopatra ornata does not fully leave its tube but reaches several centimeters from its tube cap to capture passing food. When food was put into the aquarium, the worms showed increased tentacle movement and extended their heads out of the tube. However, when a plastic ruler was put into the aquarium, a worm only came out of its tube if it was directly disturbed. This difference in behavior suggests that D. ornata can sense when food is nearby, possibly using chemoreceptors. The worm reaches out to the food, captures the food with its jaws and anterior parapodia and then retreats quickly with the food back into its tube cap. If the food needs to be freed from something it is attached to, for example, the tube of another worm, the worm chews on the food until it can pull the food back into its tube cap. Neighboring D. ornata worms may work together on the same piece of food until they have each freed a segment to take back into their tube. Activity Patterns: Diopatra ornata is consistent in its day/ night activity patterns. During the day, the worms wait at the aperture of their tube caps in their typical feeding posture with their tentacles waving just slightly outside of the tube. At night is when the worms make necessary additions to their tubes and tube caps (Table 2). Regeneration: Both anterior segments and posterior segments have been known to regenerate, though no new heads were regenerated in the time of this experiment. Tail segments left exposed to the outside sea water (not in a tube of any kind) quickly died and decomposed (in 2 days). Tail segments put in natural or plastic tubes lasted longer without decomposing, suggesting that the necessity of the tube for regeneration. However, after 22 days, the worms segments in plastic straws showed no signs of regeneration and had begun to decompose. One head segment found without any tail segments did not rebuild a tube and quickly decomposed. Heads with at least 10 tail segments were easily able to rebuild tubes and tube caps, though the tube caps were always substantially smaller than those built by intact worms. Worms with tail segments loose in their tubes often discarded those segments by pushing them out the tube cap opening. Discussion: Density: Mangum et al. (1968) found that the density of Diopatra cupred was correlated with current velocity: at higher current velocities, there was a higher density of D. cuprea. A possible explanation for this correlation is that the higher current speed allows the worm to come into contact with more water from which it can extract oxygen. However, D. cuprea is able to create its own small current by beating its antennae and most likely comes into contact with just enough oxygen-rich water from this"tube irrigation." The study suggested that the high current velocity was important because it meant that more water would flow by the tube cap, and therefore more food was likely to get caught on the hook of the tube cap (Mangum et al. 1968). When the tube cap densities of D. ornata were recorded, no measurement of current velocity was noted, but the same correlation may exist. Settling in dense patches may have other advantages for the worms. For example, the dense patches may offer them more protection from some predators. Other predators, however, may take advantage of the dense Diopatra beds. In addition, sometimes the worms feed from the kelp attached to the backs of other worms' tubes, and therefore the dense patches would create an opportunity for more food. Locomotion: The method in which Spiochaetopterus oculatus moves up and down inside its tube has been studied in great detail (Barnes 1964). This worm crawls headfirst by pushing its notopodia against the tube wall, one side at a time. To back down the tube, the worm slowly releases contact with the tube wall and slides down. Like D. ornata, S. oculatus can turn around inside its tube. The worm turns around by moving its head end down the tube, between the wall and the tail end (which is moving up the tube at the same time) (Barnes 1964). D. ornata was observed exhibiting the same behavior inside a clear plastic straw. Many tube-dwelling polychaetes are able to suddenly withdraw into their tubes. The nereids accomplish this " defense response" by quick contraction of the longitudinal muscles (Clark 1959). D. ornata also exhibit this response, though it is most commonly observed when the worm is bringing food back into its tube, and it is likely that the longitudinal muscles of D. ornata also allow for the quick withdrawal. Tube Recognition: It is still unclear whether D. ornata will inhabit vacant tubes constructed by other worms. All the worms displaced from their tubes and left on the sand quickly built a new tube and did not try to find an empty tube. In addition, 4 of the worms transplanted during the recognition experiment built new tubes. Tube building and tube structure: There are many possible explanations for why D. ornata and several other species of tube-dwelling worms make such elaborate additions to their tube caps (shells, kelp, etc), G.A. Brenchley (1976) suggested that the tube cap decorations might allow the worm to easily sense water motion caused by approaching predators. Worms in more heavily ornamented tubes were able to easily distinguish between small water disturbances, as might be caused by drift algae, and larger disturbances, as might be caused by potential predators. The worm can therefore detect whether the water disturbance is due to a predator or a food source and then behave accordingly. Worms in less heavily ornamented tubes did not show a significant difference in their responses to the different levels of water disturbance (Brenchley 1976). Another possible explanation is that the shells and kelp may provide a habitat for small invertebrates that the D. ornata çan later catch as food. In addition, the tube caps may serve as protection for the worm, though there is no evidence to support this hypothesis. Diopatra ornata tube caps are always curved, and this orientation allows the worms to wait in the front of their tubes, with their tentacles waving out the opening of 14 the tube cap, yet remain hidden from passing predators. The curved tube may also assist in catching food particles, such as algae, that float by the tube. Diopatra ornata are extremely adept at quickly constructing new tubes or repairing damaged tubes. This ability suggests that they might have to abandon or repair their tubes frequently in nature. Their tube beds appear to be stable, but this stability may be more the result of the worm’s quick building skills than a lack of environmental disturbances. Feeding: There are several species of tube-dwelling polychaetes that live off the central California coast. Many of these polychaetes, including the serpulids, the sabellids, the sabellariids, and the chaetopterids, are suspension feeders. These worms show little activity compared to Diopatra. The chaetopterids, for example, obtain food by filtering the surrounding water through a mucus bag (Barnes 1965). Öther less active polychaetes include the terrebellids, which are deposit feeders. However, Diopatra ornata are much more active, raptorial feeders with powerful jaws, despite living in permanent tubes. Their major food source is the giant kelp, Macrocystis pyrifera (Kim 1992). However, worms from the family Onuphidae (includes D. ornata) often catch small invertebrates for food. Some worms periodically move large amounts of water through their tubes, in an attempt to obtain more oxygen from the water. Diopatra cuprea, however, does not withdraw much oxygen during respiration, and therefore may be using this "tube irrigation" primarily as a way of checking the surrounding water for chemicals that may indicate nearby food particles (Mangum and Cox 1971). It is likely that D. ornata senses the presence of food in the water in a similar fashion. The commonly observed D. ornata behavior of waiting at the opening of the tube cap with tentacles waving is probably the way in which the worm moves the water through their tubes. A study of Harmothoe imbricata revealed that either the palps or the cirri are able determine the location of vibrations in the water. In addition, the palps contain chemoreceptors that transmit chemical information, allowing the worm to determine if the vibration was caused by food or a predator (Daly 1973). The sensory appendages of D, ornata may have similar abilities and could be at least partially responsible for the ability of D. ornata to determine sense the presence of food nearby in the water. Other species of polychaetes are similar to D. ornata in that they will not leave their tubes completely to feed. For example, nereid worms reach from their tubes to grab food, but have not been observed to extend more than one-third of their body length to feed (Clark 1959). Nereis virens burrows into the sand and moves to a position closer to the food, if it could not reach the food from the tube (Clark 1959). This behavior has not been observed in D. ornata. Activity Patterns: D. ornata's day and night activity patterns (limited activity during the day and tube building or remodeling at night) suggest that the worm may be able to sense light and exhibits a photonegative response, as seen with other polychaetes. Studies on Nephtys show that the worms preferred the dark half of the water dish. In addition, these worms swam more frequently in lighter conditions, presumably looking for a place to bury into the sand, and did bury themselves faster, if given the opportunity (Clark 1956). D. ornata may similarly stay in their tubes during the day and primarily make tube additions at night in an attempt to avoid the light. The worms may also be building at night in order to avoid day-time predators, such as the benthic sand-dab fish. Further directions: There are still several questions surrounding the behavior and biology of Diopatra ornata. For example, it is known that some tube-dwelling polychaetes leave their tubes and go to the surface of the water to reproduce, while others breed in their tubes. However, the reproductive behavior of D. ornata is not completely understood. Conclusions: Diopatra ornata, a little studied species of polychaete, is able to construct elaborate tubes, made of sand, mucus, and pieces of kelp and shells. They build the underground segments of their tubes, averaging 4 cm a day, and prefer to keep the apertures of their tube caps approximately 1 cm from the substrate. They have been shown to be able to regenerate anterior or posterior parts, though they may need to be in their tubes in order to do so. They seem to be able to sense the presence of nearby food, and feed by grabbing the food and quickly bringing it back to the tube. They move within the tube using their small parapodia and setae, but also have the ability to suddenly withdraw into the tube with a quick contraction of their longitudinal muscles. D. ornata most actively add to their tubes at night, possibly in an attempt to avoid light. Acknowledgements: I would like to thank my project advisor, Jim Watanabe, for all his advice, attention, and enthusiasm. In addition, I would like to thank Lisa Walling and Freya Sommer for their expertise and for their assistance in collecting the worms. Thanks also to Chris Patton for sharing his technical wisdom and for allowing me to use the time- lapse recorder. Lastly, thanks to my fellow spring quarter students and our wonderful TA, Carrie Kappel, for their support and for always asking, "how are the worms? Literature Cited: Barnes, R. D. 1965. Tube-building and feeding in chaetopterid polychaetes. Biol. Bull. 129: 217-233. Barnes, R. D. 1964. Tube-building and feeding in the chaetopterid polychaete, Spiochaetopterus oculatus. Biol. Bull. 127: 397-412. Brenchley, G. A. 1976. Predator detection and avoidance: Ornamentation of the tube¬ caps of Diopatra spp. (Polychaeta: Onuphidae). Mar. Biol. 38: 179-188. Clark, R. B. 1956. The eyes and photonegative behavior of Nephtys (Annelida, Polychaeta). J. Exper. Biol. 33:461-477. Clark, R. B. 1959. The tubicolous habit and the fighting reactions of the polychaete Nereis pelagica. Anim. Behav. 7: 85-90. Daly, J.M. 1973. The ability to locate a source of vibrations as a prey-capture mechanism in Harmothoe imbricata (Annelida: Polychaeta). Mar. Behav. Physiol. 1: 305- 322. Kim, S.L. 1992. The role of drift kelp in the population ecology of a Diopatra ornata Moore (Polychaeta: Onuphidae) ecotone. J. Exp. Mar. Biol. Ecol. 156: 253-272. Levinton, J. S. 2001. Phylum annelida: segmented worms. pp. 245-247 in J. S. Levinton. Marine biology: function, biodiversity, ecology, Oxford University Press, New York, NY. Mangum, C. P., and C. D. Cox. 1971. Analysis of the feeding response in the onuphid polychaete Diopatra cuprea (Bosc). Biol. Bull. 140: 215-229. Mangum, C. P., S. L. Santos, and W. R. Rhodes, Jr. 1968. Distribution and feeding in the onuphid polychaete, Diopatra cuprea (Bosc). Mar. Biol. 2: 33-40. Moore, J. P. 1911. The polychaetous annelids. Proc. Acad. Nat. Sci. Philadelphia for 1911: 234-318. Rouse, G. W., and F. Pleijel. 1999. Onuphidae Kinberg, 1865. p. 169-171 in G. W. Rouse and F. Pleijel, ed. Polychaetes, Oxford University Press, Oxford, England. Table 1 - Summary of results obtained from the natural density measurements, the distances from the substrate, and the tube building rates. Diopatra ornata densities in the field Average number of tube caps per 0.0625 m 10.69 4.66 Standard deviation Tube Cap Curvature 1.087 cm Average distance from the substrate 0.592 Standard deviation Tube Building Rates 4.2 cm Average tube length added in 24 hours 1.308 Standard deviation Table 2 - The time of day, over a three day period, a group of three worms were observed performing certain activities. Activity Time antenna waving 11:45 AM 1:13 PM antenna waving antenna stopped waving 7:45 PM adding shell to tube 8:41- 10:21 PM adding sand to tube 12:05 AM 1:25 AM less activity 25-3:45 AM adding sand to tube sunrise 6:13 AM 3 AM - 8:05 PM antenna waving sunset 8:05 PM 25 - 10:45 PM adding to tube adding to tube 12:05 - 12:25 AM 25 - 3:29 AM adding to tube sunrise 6:05 AM antenna waving 6:17 AM - 7:57 PM sunset 7:57 PM adding to tube 9:25 - 11:21 PM adding to tube 2:25 - 12:45 AM sunrise 5:49 AM 6:33 AM - 8:37 PM antenna waving sunset 8:37 PM minor tube additions 8:45 PM adding to tube 10:49 PM adding to tube 3:25 AM adding to tube 4:05 AM sunrise 5:49 AM antenna waving. 5 AM Figure legends: Fig. 1. A characteristic Diopatra ornata tube. Fig. 2. The percentage of worms that chose to stay in the tube, plant the tube, change the structure of the tube, or build a new tube, when disturbed from their original tube and placed back into a different tube. Fig. 3. The percentage of worms that chose to stay in the tube, plant the tube, change the structure of the tube, or build a new tube, when disturbed from their original tube and placed back into that same tube. Fig.1 mnabe, with permission 35 30 25 15 10 28.6 % planted Fig. 2 28.6 28.6 14.2 % still in % changed % in new tube but not tube tubes planted structure 45 40 30 20 10 % planted Fig. 3 % still in % changed % in new tubes tube but not tube planted structure