Featherstone Introduction Polycheria osborni is an amphipod found quite abundantly in a variety of compound ascidian colonies in the lower intertidal and subtidal areas from Puget Sound, Washington, to Southern California. It was found in Aplidium (Amaroucium) by Colman (1898), Skogsberg and Vansell (1928), and in Clavelina sp. by Alderman (1936). Abbott and Newberry (1980) found Polycheria osborni in a variety of other ascidians. Skogsberg and Vansell (1928) described its structure and some of its behavior. In the present study the dis¬ tribution of Polycheria osborni in ascidians of the lower intertidal area of Mussel Point, Pacific Grove, California was observed. including the arrangement of the amphipods in the colony, the sizes of the amphipods in the various ascidians, and a sex ratio based on a subset of the population. Behavior of the Polycheria was also observed; their behavior in the burrow and the movement of them between burrows. Featherstone Distribution and location of Polycheria osborni Ascidians were collected at low tide in 3 areas at Mussel Point. The location termed West Point was exposed to considerable wave action at all but the lowest tides. In the second and third locations, the Bird Rock area and the Seal Island area, the force of the waves was broken by offshore rocks. Ascidians were also collected at Pescadero Point, on the open coast at the north tip of Carmel Bay. Ascidian samples were taken from the rocks with a putty knife. These were then brought into the lab where the ascidian samples were measured, the Polycheria osborni burrow positions were noted, counted and measured, and additional general observations were made. The ascidians with Polycheria osborni still in their burrows were maintained in a shallow trough provided with a continuous flow of fresh seawater at 10 to 15 7C. To find the distribution of Polycheria osborni in ascidians, many samples of the ascidians common on Mussel Point were collected haphazardly at various places and intertidal elevations, and studied under a dissecting microscope in the laboratory. Burrows appear as either zigzag seams or as oval openings on the free exposed surface of the colony in which Polycheria osborni could be seen. Surface area of each ascidian colony was calculated roughly from the length and width of the surfaces bearing zooid apertures. The number of Polycheria osborni burrows was counted, and the anterior- posterior length of each occupied burrow was measured to the nearest O.5 mm. The position of the Polycheria osborni in relation to the zooids of the colony were also recorded. Featherstone Eleven species of colonial ascidians from the Bird Rock and Seal Rock areas were examined for the presence of Polycheria osborni. Four of these (Archidistoma psammion, Synoicum parfustus, Aplidium solidum and Perophora annectens) contained no amphipods. Two more (Distaplia occidentalis and Clavelina huntsmani) contained only occasional amphipods. The five species in which Polycheria osborni were most frequently found were: Aplidium californicum, Polyclinum planum, Archidistoma diaphones, Archidistoma molle and Archidistoma ritteri. The average number of Polycheria osborni for these five ascidians ranged from O to 21.9/cm for most samples. There wasn't any trend of Polycheria osborni occurence between the West Point area and Bird Rock area. In each ascidian species there is great variance in the average number of Polycheria osborni/cm“. The population densities of amphipods found in particularly heavily infected regions of individual colonies ranged as high as 45/cm" for Archidistoma diaphanes, and 67/cm" for Archidistoma ritteri. The highest densities on Archidistoma ritteri occured on small colony lobes where the area of the lobe was less than 1 cm"; therefore, the actual number of Polycheria osborni counted was less than that recorded/cm. Extremely high populations, such as that of 45/cm" for Archi¬ distoma diaphanes, consisted very largely of juvenile Polycheria osborni in burrows about 0.5 mm long. The density of Polycheria osborni did vary with size. Fig. 2 shows diagrammatically the difference in numbers of Polycheria osborni/cm" by size in colonies of Archidistoma molle and Polyclinum planum. Although it is seen Featherstone here that the smaller amphipods occur in greater numbers/cm’. the large Polycheria osborni burrows may be as close together as the smaller. It has been observed that one large burrow may ac¬ tually run into another forming a 'T' or they may be end to end with a very narrow band of tunic between the two animals, Polyclinum planum was especially abundant at Mussel Point, so counts were made of numerous whole colonies of different sizes to see if a relationship exists between size of the ascidian colony, which is possibly proportionate to age, and the number of Polycheria osborni. The results, shown in Fig. 3, indicate that there is no correlation between the size of the colony and the number of amphipods/cm present. The Polyclinum planum varied in firmness and diatom cover within each size class, One feature of special interest from the standpoint of host/ commensal relations was the placement of Polycheria osborni burrows in relation to the zooids of the ascidian colony, The apertures of zooids, like the burrows of amphipods, generally occur on the distal free surface of the colony or its lobes, To determine the extent to which amphipods might be interfering with the activities of the host zooids, numerous colonies of 6 species of ascidians were examined and the positions of amphipods recorded. There were several possible locations for the burrows (Fig. 4). Three of the ascidian species, Aplidium californicum, Polyclinum planum, and Distaplia occidentalis have their zooids arranged in systems, with a central common cloacal aperture for the atrial outflow of several zoids, though each zooid has its own separate oral aperture. In these colonies there are three Featherstone possible burrow locations: in the center of the system, on or in the common cloacal aperture; amongst or on the zooids of the system, or in the tunic between the systems. The only Polycheria osborni found within the common cloacal aperture was a newly hatched one, less than 0.5 mm long which filled little of the aperture. The great majority of the amphipods were situated in the clear areas of tunic between adjacent systems, where interference with zooids was mini¬ mal. The ascidians Archidistoma molle, Archidistoma diaphanes and Archidistoma ritteri have both their atrial and oral apertures opening independently to the exterior of the colony, and they are not ordered in systems within the colony. The Polycheria osborni burrows can be either on the zooids or between the zooids in these ascidians, and most were found between zoids (Fig. 4). In either position Polycheria osborni often appear to have pushed the zooid aside to some extent at the top, as the zooid body descends into the tunic at an abnormal slant under the body of the amphipod. Seeing that Polycheria osborni occurs largely in areas betwen zooids it could be assumed that in such a position it is not hurting the zooids, whereas, if the Polycheria osborni is on top of the zooid there might be some damage to them. In addition, if the Polycheria osborni is competing for tunic with the zooids it might not be a comfortable burrow. This was observed in Distaplia occidentalis. In this ascidian the zooids and systems are close together. As a Polycheria osborni burrowed in, pulling on the tunic, the zooids were seen to contract, pulling the tunic out of the grasp of the Polycheria osborni. The closeness of the zooids in Distaplia occidentalis, a very soft ascidian, is possibly the Featherstone reason the incidence is so low. Size class structure and sexuality of the Polycheria osborni population. The body length of Polycheria osborni cannot be measured accurately without removing the animals from their burrows, How¬ ever, burrow length is easily measured without disturbing the am¬ phipods, and provides a good index to the distribution of size classes in the population. A total of 1866 burrows were measured to the nearest 0.5 mm in 5 species of colonial ascidians in the course of examining the distribution of Polycheria osborni in ascidians. Results show at the top of Fig. 5 that 64% of the population is in size classes 0-0.75 and 0.76-1.0 mm. The larger size class populations decrease sharply to 1.5% in the largest size class, 3.0 mm. When separate size class distinctions were calculated for different ascidian species, some interesting differences appeared. (Fig. 5). The Archidistoma diaphanes histogram is similar to that of the whole population. Aplidium californicum and Archidistoma molle show smaller percentages of the smallest animals, 0.5 mm, and still fewer appear in Archidistoma ritteri while Polyclinum planum shows a relatively even distribution of sizes. These differences are statistically significant indicating some relationship between amphipod size and the ascidian they burrow into (R X C contingency test P .005). Since there are often many small burrows around a large burrow it is assumed that those are newly hatched. The peak in the popu¬ lation of 0.5 mm Polycheria osborni in Archidistoma diaphanes icates Featherstone indicates that there may have been several newly hatched broods in the sample. Aplidium californicum and Archidistoma molle may have broods that were hatched several weeks early. Another possible determining factor is the toughness of the ascidians. It was seen that the young burrowed very quickly into Archidistoma diaphanes, a soft ascidian, agreeing with the peak in Fig. 5 for that ascidian, However, other soft ascidians, Archidistoma molle and Archidistoma ritteri, do not support the idea of soft ascidians being good hosts for the O.5 mm animals. Amphipods are slightly longer than the burrows they occupy, In order to be able to interpret burrow length in terms of amphipod length, 15 burrows were measured, then the amphipods were removed and measured. Results shown in Appendix A indicate a conversion factor of 1.5:2 for burrow length to amphipod length. Not only size classes but also the relation of size to sex and sexual maturity was investigated. Of special interest was the fact that in previous studies only female Polycheria osborni had been found among the larger sized animals (Skogsberg and Vansell, 1928; Colman, 1898). In order to look further for male Polycheria osborni, 107 amphipods of various sizes were removed from ascidians of different species and from different colonies, and preserved in 70% ethanol. The samples were haphazard with regard to ascidian source, but while the larger animals were all taken, the smaller juveniles were not collected in proportion to the numbers present in the colonies. The animals were all measured with an occular micrometer to the nearest 0.2 mm. On close examination, the popula¬ tion could be separated into juveniles, males and females. The Featherstone males had two distinct copulatory appendages ventrally on the pos¬ terior edge of the eighth thoracic segment (Fig. 6). These appen¬ dages were clear to white and varied somewhat in size with the size of the Polycheria osborni. Females could be identified by brood plates (oostegites) in the thoracic region. Juveniles were those Polycheria osborni which lacked both copulatory appendages and oostegites. Fig. 7 shows the number of juveniles, males and females at sizes from 0.5-3.6 mm. Juveniles were seen from the smallest sizes through 2.2 mm. Males and females began to appear at 1.8 and 1.6 mmrespectively. Interestingly, in this haphazard sample there were twice as many females as males. Several of the females observed were gravid. The eggs in 12 broods were counted; egg number ranged from 37 to 129 with a mean of 85.3 There was a clear correlation between the size of females and the number of eggs being carried (Fig. 8) but not between the age of the eggs and the number of eggs. The eggs in a single brood were at different stages of development but there were not distinct separations in development as would appear if 2 batches were brooding. Behavior of Polycheria osborni Burrowing Polycheria osborni removed from their burrows and placed on or near ascidians, are capable of forming new burrows; the process was observed in some detail by Skogsberg and Vansell (1928). In the present work, experiments were performed to compare the burrowing activities of Polycheria osborni of different sizes and of Polycheria osborni on different ascidian substrates. In these experiments, Polycheria osborni was removed from its burrow and placed on the same colony or a new ascidian colony or species where it was Featherstone observed at short intervals for up to 280 minutes. For the first 30 minutes the transplanted Polycheria osborni was watched con- tinuously so as not to miss the critical times for Polycheria osborni that burrowed quickly. The time for burrowing was divided up into the time that Polycheria osborni walked around on the ascidian or swam off, the time from beginning the burrow to being below the surface of the ascidian host, and the time until the burrow could be closed when Polycheria osborni was disturbed. Some amphipods when placed on any of the ascidians tested for burrowing behavior quickly swam away; others were immediately quiet, sitting on their backs on the ascidian whether they began to burrow or not. It was observed that if, when Polycheria osborni was placed on the ascidian, the antennae did not touch the host, the amphipods often swam away. In contrast, whenever the antennae contacted the ascidian the amphipods sat quietly on the ascidians or would climb on the colony if not yet already on it. The process of burrowing was described by Skogsberg and Vansell (1928), but their studies involved only one species of host ascidian. Present work shows that speed of burrowing may vary, not only with the individual and with its size but also with the host species (Fig. 9). Four Polycheria osborni placed on Archidistoma psammion did not burrow, but sat or walked on the ascidian, occassionally swimming off. Polycheria osborni burrowed most quickly into Archidistoma molle, Archidistoma diaphanes, and Aplidium californicum. Interestingly, all the experimental amphipods burrowed into Clavelina huntsmani although they are found in that host infrequently in the field. The actual mechanism of burrowing was closely observed on Archidistoma molle supporting the behavior described by Skogsberg and Vansell (1928). Featherstone Polycheria osborni has been found to occur in a much greater variety of ascidian hosts than previously recorded. Observations on distribution of Polycheria osborni in different ascidian species, in relation to the zooids, and the length of time for burrowing in different ascidians suggests some reasons for the distribution to be as it is. No Polycheria osborni burrowed into Archidistoma psammion and none were found around China Point burrowed into this ascidian. However, two were found burrowed into Archidistoma psammion at Pescadero Point. This ascidian is very tough when pressed on. It is possible that this hardness creates a major barrier for this animal which is going to simply pull with its pereopods and push with its back to make a burrow. Several of the other ascidians looked at but found lacking in Polycheria osborni are encrusted with sand which would make it virtually impossible for Polycheria osborni to catch hold of the surface membrane to pull itself into a burrow. Polyclinum planum which had a medium density of Polycheria osborni in the field and into which only half the experimental animals burrowed into may also be firm enough to present some barrier to Polycheria osborni. Behavior of Polycheria osborni in its burrow. Studies were made to determine how much of the time the Poly- cheria osborni spend holding their burrows closed, holding their burrows open, or holding their burrows open while kicking their pleopods. Ten minute observations were carried out on 13 Polycheria osborni in 3 different ascidian species. An event recorder coupled with a strip chart recorder was used to keep records of when the animals closed their burrows, when they opened them, and when they kicked their pleopods. From these records, total duration for each activity in the 10 minute period could be obtained, 11 Featherstone In addition, further observations of the animal in its burrow were made to elucidate the direction of current flow, caused by the kicking pleopods, the part this current plays in feeding, and a little on how the animal feeds. The addition of a mixture of graphite particles and diatoms added to the water in which the ascidian sample was sitting allowed for easy observation of the current. Behavior in the burrow of the 13 amphipods studied for 10 minutes each showed variation; each individual Polycheria is represented by a separate vertical bar in Fig. 10. The Polycheria osborni in the 3 different ascidians showed no significant differences in behavior, therefore observations made in all 3 hosts have been combined. The amount of time the 0.5 mm long Polycheria osborni were closed, 0 minutes, is statistically significant in relation to the other sizes of Polycheria osborni (RX C contingency test P.005). Larger amphipods don't show behavior differences correlated with size. Polycheria osborni may kick its pleopods for 15 seconds without stopping or it may kick once, stop, and kick again. The number of starts in Fig. 10 refers to this and not to the total time spent kicking. Most of the Polycheria held their burrows open for at least 2/3 of the time observed, and were kicking for less than 1/3 of the time open. As Skogsberg and Vansell (1928) described, the current that the pleopods make brings in water predominantly from the sides of the burrow although water is also brought in from both ends. The water then shoots almost straight up from the central part of the body. This current brings fresh water by the gills, and also by any eggs that may be in the brood pouchs of females. In addition, some water does pass through the antennae bringing with it food particles that get caught in the antennae. One or two antennae 12 Featherstone are then bent quickly onto the burrow where the gnathopods scrape the food off from the base to the tip of the antennae. Graphite particles were observed in the antennae spread out and extended perpendicular to the surface of the colony. After having been scraped off by the gnathopods, the antennae again extended with no graphite on them. Another 'in burrow' behavior sometimes noted is that of Polycheria turning around in its burrow. An amphipod does this by rocking on its dorsal side up onto its head. It then rotates. head down in the burrow while using its pereopods to pull it around. Turning around takes about 3 seconds, and may be accom¬ panied by several false starts in which the amphipod rocks but not all the way to its head. Movement of Polycheria from one burrow to another. Published accounts of Polycheria leave the impression that the amphipods, once burrowed in, remain permanently in place. Preliminary observations showed that this is not the case, and that Polycheria osborni does change burrows. To study amphipod movements, a colony of Archidistoma diaphanes (4.8 X 2.8 cm) with 110 Polycheria osborni burrowed into it (28 large and 82 newborn) was placed in a fingerbowl with a very gentle flow of fresh sea water. At numerous times during 9 days the colony was observed and the location of the Polycheria osborni burrows plotted, the orientation of the larger burrows noted and the general condition of the colony recorded, The plottings of consecutive observations were compared to see where old burrows and Polycheria osborni disappeared, where new burrows appeared and where large burrow orientations changed, 13 Featherstone The results of these observations show that the Polycheria popula¬ tion is quite dynamic. In Fig. 11, in all but 2 of the time inter¬ vals between successive observations, both large and small Poly- cheria moved, changing burrows or disappearing completely. By the end of 9 days all of the large amphipods had either changed burrow or orientation within their burrow. All but 9 of the small Polycheria had changed burrow location. During the time the colony was kept in lab the Hopkins Marine Station seawater system ex- perienced "bubble disease" and the Archidistoma diaphanes colony got many air bubbles just below the surface tunic. Two small Polycheria osborni were seen to be on top of these air bubbles but were not in the same locations at the next observation, Con¬ siderable debris also settled on the ascidian during the 9 days and no Polycheria osborni were observed in areas where the debris was especially heavy. To see if laboratory results reflected events actually occuring in the field, 2 colonies of Archidistoma diaphanes were observed on three successive days without disturbing them in any way. The first colony observed consisted of several lobes in a 3.5 cm X 4 cm area. In this sample the number of large (2 mm) and small ( 1 mm) Polycheria osborni/lobe were counted, then counted again after 23 hours and compared. In the second sample a colony of Archidistoma diaphanes (1.5 cm X 2 cm) was carefully diagrammed and the location and orientations of the burrows recorded, then observed again after 24 and 48 hours. The results show that Polycheria do indeed move about. Observations on the numbers of amphipods/lobe in the Archidistoma diaphanes colony (Fig. 12) indicate that there was only one lobe Featherstone which had the original number of Polycheria at the end of the second observations. In the colony where amphipod positions were mapped, (Fig. 13) the number of Polycheria osborni/lobe did not change much, but movement was still clearly evident. Studies of Polycheria osborni movement indicate that when amphipods leave burrows, these soon disappear, and few vacated burrows are normally seen on infested colonies. To investigate how quickly the ascidians were able to reshape the tunic after the burrow was vacated, Polycheria osborni (1.5 mm) were carefully removed from colonies of Polyclinum planum and Archidistoma diaphanes and the burrows observed intermittently for 3 days. Vacated burrows on Polyclinum planum took a mean of 20 hours to disappear, one shallow burrow took only 5 hours to disappear, while another deeper burrow took 30 hours. Burrows in Archidistoma diaphanes took on the average 24 hours to disappear. On Polycheria burrow extended all the way across a small lobe, and when it was removed the ascidian did not fill that burrow but instead remained as 2 smaller lobes. Featherstone Summary 1. In the period April-June, 1980, ascidians were collected with Polycheria osborni burrowed into them. 2. Polycheria osborni was abundant in Aplidium californicum, Archi¬ distoma diaphanes, A. molle, A. ritteri and Polyclinum planum; scarce in Distaplia occidentalis and Clavelina huntsmani; and absent in Archidistoma psammion, Synoicum parfustus, Aplidium solidum and Perophora annectens. 3. Found predominantly between zooids of the ascidians, the Polycheria don't appear to injure their hosts. 4. Two-thirds of the population of Polycheria observed were juveniles less than 1.25 mm long. 5. Different species of ascidian had different size amphipods predominating. 6. Male Polycheria osborni were reported for the first time and can be identified by 2 copulatory appendages on the eighth thoracic segment. 7. Polycheria sit in their burrows kicking their pleopods to make a current, feeding and occasionally closing the burrow over them. 8. The population of Polycheria is dynamic, the amphipods change burrow location and orientation. 9. Ascidian hosts usually reshape within 24 hours after a burrow has been vacated. 16 Featherstone Acknowledgements I would like to thank all the HMS spring students for making this an enjoyable project, Dr. Robin Burnett for his statistical help, and especially Dr. Donald P. Abbott for his assistance in identifying ascidians, and his great encouragement and advice throughout the project. Literature cited Abbott, D.P. and Newberry, A.T. 1980. Urochordata: The tunicates, Chapter 12, in R.E. Morris, D.P. Abbott, and E.C. Haderlie, Intertidal Invertebrates of California, Stanford University Press, Stanford, California. 928 pp. Alderman, A.L. 1936. Some new and little known amphipods of California, Univ. Calif. Publ. Zööl. 41 (7): 53-74. Colman, W.T. 1898. On a collection of crustacea from Puget Sound, Ann. N.Y. Acad. Sci. XI: 259 Skogsberg, Tage and Vansell, G.H. 1928. Structure and behavior of the amphipod, Polycheria osborni. Prod. Calif. Acad. Sci. (4) XVII: 267-295. 17 8 o — D-OO — DD to— D-OO—DO- 0 o P uoeuu eke s Fg 0— 600— o D——1 1 — uo/aequnu uuxeu 8 9 S O S O 5 — 1 2 2 15 121 6 + 10 19 20 29 surface area of colony cm 18 11 13 0 230 712 5 ) 3 5. 0.S 5 O O— % 8 „ spiooz u spiooz O uo ojjoui seuegdeip Hen Puoisipuoiyeuloisipiuoiy euoisipioit noe5 unuejd sijejuspiooo unipijdy nuijokjod eijdeisio siojsnjo O 8 uneg spiooz uO 1OUII 0 19jueo O uo O— S o 0 ° O oO o00 o m 0 131 30 20 104 40 20 60 40 20 40 201 60 40 204 40 20 0.5 — 10 135 Total, all species Archidistoma diaphanes 980 — Aplidium californicum 248 Archidistoma molle 169 Archidistoma ritteri 136 Polyclinum planum 333 530 2.0 1.5 length of burrow mm X — Fa 6 I 4 32 12 21 05 10 L L L 2.( 25 amphipod length mm 30 3.5 F7 150 125 100 50 undeveloped X early differentiation • differentiated 36 2.8 32 2.0 2.4 amphipod length mm 4 X 0 40 a 8 60 50 40 2 30 20 10 10 20 30 40 S 50 60 28 170 85 1 1 1 11 1 280 180 280 280 Fa 10 8 56 10 Nu21 0.5 10 burrow length mm 16 2.0 Hg 10 8 2 8 28 3 8 EE 2 2 S 8 8 88 peroui ou peroui jourou 2 sijnpe peroui ou porou jou ou BunoA F2 1 00 510 Archidistoma diaphanes colony 10 D +1 2 3 6 7 8 9 Lobe of colony 40 30 20 20 30 40 F6 12 A 0 Archidistoma diaphanes colony OG 5 6 o 9 55 E Fa 13 Figure Captions Fig. 1: Average and maximum distributions of Polycheria osborni/cm on different species of ascidians at West Point (left) and Bird Rock (right). rectangles: 1 mm-1 Polycheria osborni. Fig. 2: Number of Polycheria osborni/cm in relation to burrow lengths: 0.5, 1.0, 1.5 and 2.0. Burrows not to scale. Fig. 3: Average and maximum numbers of Polycheria osborni/cm' on Polyclinum planum colonies with surface areas from 1-66 cm". Each column shows mean, range, standard deviation and 95% confidence limits of the mean, Fig. 4: Position of Polycheria osborni in relation to the ascidian zooids. Fig. 5: Percent Polycheria osborni found in burrows to the nearest 0.5 mm burrow length in 5 ascidian hosts. Fig. 6: Morphology of the ventral side of the 8th thoracic and Ist abdominal segments of Polycheria osborni. Fig. 7: Total numbers of juvenile, male and female Polycheria osborni at body lengths from 0.6 to 3.6 mm measured to the nearest 0.2 mm. Fig. 8: Number of eggs in the female Polycheria osborni brood pouch for amphipod lengths 2.4-3.8 mm. Fig. 9: Length of time for Polycheria osborni to make a new burrow. Non-burrowing time is the time spent walking on the ascidian or swimming in the dish. Time for burrowing, solid line shows the time until the Polycheria osborni is below the surface of the ascidian, dashed line is the time until the burrow can be closed. Fig. 10: Behavior in the burrows of Polycheria osborni, 0.5-2.0 mm in length, for 10 minute trials. Showing the amount of time the burrow was closed, the amount of time was open and the pleopods were kicking (cross hatching). The number in the bar indicates the number of times the kicking starts. Fig. 11: Movement of Polycheria osborni in an Archidistoma diaphanes colony in the laboratory. The lines indicate the number of adults and young Polycheria osborni which have moved from an old or to a new burrow since the last observation. Nequals the number of individuals present at each obser- vation. Days 1-9 were 5/9-5/17. Fig. 12: Change in numbers of Polycheria osborni/lobe in an Archi¬ distoma diaphanes colony in the field. First bar of each pair is the ist day, the 2nd bar is the 2nd day. Fig. 13: Movement of Polycheria osborni within an Archidistoma diaphanes colony in the field. Graphical data, except for the total number of Polycheria osborni present, is since the previous observation. amphipod length 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Appendix A — 8084 — 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 burrow length 2 Archidistoma diaphanes Archidistoma ritteri o Polyclinum planum