Ophiodromus behavior Page 2 M. Serences INTRODUCTION Experimental research on commensal relationships in polychaetes has been largely limited to studies on host specificity and recognition in the families Hesionidae and Polynoidae (Davenport, 1950, 1953; Davenport, et. al., 1960; Davenport and Hickok, 1951, 1957;Bartel and Davenport, 1956; Hickok and Davenport, 1957). Much of this work has been devoted to the facultative commensal Ophiodromus pugettensis (Johnson) [-Podarke pugettensis]. Members of this species can be found free-living on wharf pilings and on mud flats (Bartel and Davenport, 1956;Reish, 1961) and as commensals on several Asteroid species including Luidia foliata Grube (Hickok and Davenport, 1957) and Patiria miniata Brandt (Bartel and Davenport, 1956). A single field study by Lande and Reish (1968) in southern California demonstrated the seasonal occurrence of O. pugettensis on P. miniata, with highs occurring in the winter and lows in the summer months. Commensal pop- ulations on Patiria were also shown to fluctuate and vary inversely with water temperature. Laboratory studies concerning the behavior of the commensal on the host, however, represent merely casual observations and the nature of this relationship has only received speculation. The purpose of this study, therefore, was to examine more thoroughly 0. pugettensis on P. miniata, both in the field and in the laboratory, in an effort to elucidate the commensal¬ host behavioral interactions. Observational notes on the commensal itself are also included. Ophiodromus behavior Page 3 M. Serences FIELD STUDIES MATERIALS and METHODS Field studies on Ophiodromus pugettensis were made over a six week period in April and May 1976 at Hopkins Marine Station, Pacific Grove, California. Forty-eight seastars were tagged, measured and the number of commensals per seastar recorded at a depth of approximately 30 feet using SCUBA on April 20, 1976. Three subsequent dives were made on April 21, May 7 and May 26, and the number of commensals per seastar counted on each occasion. The seastars were marked using numbered plastic laundry tags which were shot approximately 1 cm deep into the aboral surface of the seastar. No permanent injuries were sustained by the seastars using this method. Measurements to the nearest millimeter were made of the seastars from the tip of the longest ray to the opposite web. In addition, fifteen seastars with a total of 75 worms were collected on May 12 on SCUBA at a depth of 25 feet. Thirty-one seastars with a total of 111 worms were collected one week later skin diving at a depth of 20 feet approximately 25 feet towards shore from the previous site. Two weeks later seastars were collected on SCUBA from both areas; fourteen with 38 worms from the first area and thirteen with 41 worms from the second. The seastars were collected and later placed in an aquarium for from one to four hours. The commensals were Ophiodromus behavior Page 4 M. Serences then removed and placed in a glass dish filled with salt water. Measurements, to the nearest millimeter, were made after approximately ten minutes when the initial contraction response had subsided. RESULT Results for the field studies are summarized in Figures 1 and 2 and Tables 1 and 2. Figure 1 plots the number of commensals per host versus average size of P. miniata indicating an increase in commensal pop- ulations with increasing host size. Figure 2 summarizes the data on number of commensals per host versus frequency of occurrence. The index of dispersion for sample size 35 is 54.386 (PK.025) indicating clumping. A significant difference (P£.05) (X2 test, N-2 degrees of freedom) from this and a Poisson distribution is apparent. And, comparing expected versus observed values, a substantially higher occurrence of starfish without worms than expected is noted. Table 1 records changes in commensal populations over time for dive dates. The number fluctuations indicate that the commensals commonly move on and off of Patiria. It should be noted here that large 0. pugettensis were rarely noted in the field aborally on P. miniata. Table 2 lists the size range of the commensalscollected, area, date, sample size, mean, standard deviation and level of significance between the two areas sampled. Also included are size increases (mm/week) and corresponding levels of Ophiodromus behavior Page 5 M. Serences significance for similar area populations. Significant size and growth rate increases among 0. pugettensis as one moves from subtidal to intertidal areas is indicated. LABORATORY EXPERIMENT! T MATERIALS and METHODS P. miniata with commensals were collected both intertidally and subtidally off Hopkins Marine Station, Studies were conducted over a six week period during April and May 1976. Specimens were maintained in 40 liter salt water aquaria with circulating unfiltered sea water averaging 1° C above local water temperatures. To prevent loss of worms drainage holes were covered with fine mesh netting. Movement of the seastars and commensals was unrestricted except when isolation was necessary. Plastic containers 28x22x13 cm in which sea water could be cir¬ culated were then used. The following experiments were conducted altering the system in each case as described. Experiment 1--To determine whether commensals will colonize hosts in the laboratory, a Patiria with ten O. pugettensis was placed in a plastic tank with a con- specific from which commensals had been removed. Eight Ophiodromus were then introduced into the center of the tank. The number of worms per seastar were counted at irregular intervals over a period of 49 hours. Counts were made directly through the glass when possible but Ophiodromus behavior Page 6 M. Serences when commensals were obscured from view, seastars were removed and the number of worms counted outside the tank. Replacement in the tank was made as close as possible to the original position. The results are diagramed in Figure 3. Two worms were without hosts at the conclusion of the experiment. Initially, six out of eight worms migrated to the host lacking commensals implying preferential migration as well as dispersal of O. pugettensis. Experiment 2—-To discern whether field distributions of O. pugettensis on P. miniata could be replicated in the laboratory over a short time period, seventeen Patiria lacking commensals were added to an aquarium containing two conspecifics each with eighteen Ophiodromus. Seastars were removed after five days and the number of commensals per host recorded. Results are summarized in Figure 4. Two Patiria were without commensals following the experimental period. The index of dispersion for sample size 19 is 23.18 (P2.4) indicating randomness. Experiment 3--Hickok and Davenport (1957) reported com¬ mensals with Patiria selectively responding (PK.001) to Pisaster giganteus (Stimpson) in a choice apparatus. To determine whether Ophiodromus commensal with Patiria would accept alternate hosts in the laboratory, 24 commensals were equally divided among two P. gigantea and two Ophiodromus behavior Page 7 M. Serences Dermasterias imbricata (Grube). Each seastar species was maintained in a seperate aquaria and examined daily for Ophiodromus. Following day 1 only one commensal remained on each of the Pisaster and by day 2 no commensals were evident eitheronthe seastars or in the tank itself. No commensals were observed on the Dermasterias after the fifth day. Both observations suggest the commensals had been ingested by their prospective hosts. Experiment 4--To observe commensal behavior on an injured host, two slashes approximately one centimeter long were made through the aboral surface of a Patiria with eight Ophiodromus. No commensals were observed abandoning their host during eighteen hours of periodic observations. During the first hour, however, the commensals remained within or close to the ambulacral grooves and later move¬ ment was restricted to the oral surface. Experiment 5—-To determine if commensals would abandon their host after prolonged air exposure, a Patiria with ten Ophiodromus was placed in a plastic container filled with approximately fifteen millimeters of dry sand and observed periodically for 69 hours. Commensals remained on their host for at least 25 hours. After 48 hours, however, seven Ophiodromus had crawled off the host and after 69 hours only one worm Ophiodromus behavior Page 8 M. Serences remained on the seastar. The distances traveled from the host ranged from between .5 to 11 cm (mean of 4.1 cm). Experiment 6—-To observe what would happen when a large number of Ophiodromus were forced upon a Patiria, 25 Ophiodromus, of average size 9 mm, were placed on two Patiria of sizes 5.5 and 6.3 cm, respectively. The 5.5 cm Patiria (A) was not fed throughout the experiment while an unlimited supply of squid was fed the 6.5 cm host (B). No more than eighteen commensals would remain on a given seastar for longer than an hour despite repeated replacements and, of those, at least four were found aborally. After 24 hours eleven and six of the initial worms remained on the seastars A and B,respectively. Forty-eight hours later three were present on A and one commensal remained on B. Experiment 7—-To determine whether Ophiodromus are commensal with Patiria for feeding purposes, a small piece of mussel was placed in a crystallization dish filled with sea water and a Patiria with sixteen commensals, which had been starved for three days, was placed on top of the food. The worms'reaction was observed using mirrors positioned so the oral surface of the host could be seen through a dissecting microscope. During the course of an hour four of the commensals came down to the food and everted their proboscises as many Ophiodromus behavi Page 9 M. Serences as four times. The commensals would venture underneath and above the everted stomach of the seastar, but rarely went between the folds of the stomach or close to the mouth. Experiment 8—-To determine whether the commensals were in¬ deed eating the food of the seastar, fifteen feeding experiments were conducted. In each case, 25 ml of un¬ filtered salt water, unless specified, was used to prepare 17gms of unflavored gelatin. This was poured approximately 1/2 cm thick into petri dishes and additional substances were added as noted. Methylene blue was used to dye the first gelatin and carmine red was used in subsequent ex¬ periments. Patiria with Ophiodromus were starved for periods noted in Table 3. For each experiment, several small pieces of gelatin were placed in a crystallization dish of appropriate size filled with sea water. A seastar with six commensals was placed over the food. Once the Patiria had hold of the gelatin, it was placed in a plastic tank with circulating sea water. Commensals were examined under a dissecting scope after six, nine and 24 hours following feeding for any signs of dyed particles within the gut. Results and observations are listed in Table 3. The slashes indicate results from duplicate runs of the same experiment. Commensal feeding while the host was ingesting food was observed in six out of sixteen experiments. Ophiodromus behavior Page 10 M. Serences Experiment 9—-To quantify the startle response, a sudden contraction of commensals, the observation container was given a sudden shake and the reaction of the Ophiodromus when on top of, near the edge of, or not touching the host food was noted. Results are summarized in Table 4. A significantly fewer number of contractions occurred when the commensal was not touching the food than in either other case (PA.005, 2x2 Contingency Test). Experiment 10—-To determine whether diurnal patterns of commensal movement were obvious, freehand sketches to scale of commensals positions on a Patiria were made on graph paper and average distances from the nearest am- bulacral grooves determined from this. The results are a compilation of observations made irregularly over a nine day period and are graphed in Figure 5 as the average distance of commensals to the nearest ambulacral groove of the host as a function of the time of day. No periodicity in movement is apparent. Experiment 11--To see if free-living Ophiodromus could adapt to a commensal existence on P. miniata, three frée¬ living conspecifics, obtained among tunicates from wharf pilings, were placed on a Patiria and observed for five days. After this period, the seastar was fed a mixture of unflavored gelatin, carmine red and squid extract. Following initial placement on the Patiria, free-living Ophiodromus behavior Page 11 M. Serences Ophiodromus remained on the seastar throughout the five day period. Movement was predominately on the oral surface although worms were occasionally observed aborally. Although two eversions of the proboscis were noted during host feeding, no red particles were detected in the guts after 24 hours. DISCUSSION Experimental and field results suggest that commensal distributions on Patiria are at least partially regulated through behavioral controls. Field distributions showing a higher occurrence of seastars lacking commensals than expected from a Poisson distribution could be an indication of preferential host colonization. When Ophiodromus were given a choice between two Patiria in the laboratory, host selectivity was again indicated although a larger sample size would be required to support this hypothesis. A random distribution was obtained in the laboratory after extremely clumped initial conditions yet a longer dispersal time may be required to obtain experimental distributions approximating those in the field. Unnatural surroundings or restrictions of space and food in the laboratory may also account for this discrepency. Although commensal densities per seastar increased with increasing host size in the field, hosts artificially crowded in the laboratory were unable to maintain large worm densities. Inter- or intraspecific behavioral interactions may be important in Ophiodromus behavior Page 12 M. Serences controlling commensal densities on hosts. Size differences between commensals from the two subtidal sampling sites may indicate that growth rates for Ophiodromus are greater in nearshore and intertidal habitats than in subtidal regions. Additional ecological studies are needed, however, to determine the cause of these findings. Host abandonment was not observed in the laboratory except under extreme conditions such as prolonged exposure to air or overcrowding. Commensal intolerance of such conditions, rather than host survival, may determine whether Ophiodromus remains associated with the host. Ophiodromus may not associate with P. gigantea and D. imbricata due to host ingestion of the commensals. Evidence from feeding experiments suggest 0. pugettensis is commensal with P. miniata for feeding purposes as well as protection. Commensal movement on the host showed no periodicity and was generally restricted to the oral surface of the seastar where protection and food supply seem greatest. Yet, commensals seemed more selective in food preferences than host Patiria. This may be of adaptive significance, however, considering Patiria are scavengers and also of a cannibalistic nature. It also implies that something other than stomach eversion may be necessary before a feeding response is elicited in the commensals. Neither benefit or harm to P. miniata by its commensal was indicated. And, initial observations with free-living Ophiodromus behavior Page M. Serences Ophiodromus indicate that at least temporary existence on Patiria can be tolerated. Further studies are needed to conclude whether such results can be maintained over long periods of time. Larval settling studies, as begun by Davenport and Hickok (1957), are also necessary to elucidate the evolution and nature of these commensal associations. Ophiodromus behavior Page 14 M. Serences SERVATIONAL NOTE Morphological studies on the polychaete are extremely limited. Observational notes made in the laboratory are included here in hopes of aiding further research in this area. Seemingly well adapted to its commensal existence, the polychaete can wedge itself between the oral plates of the seastar or within the ambulacral grooves to the point of concealment. Although frequently oriented along the edge of the ambulacral grooves, movement among the tube feet is also common and increases when the seastar moves. Often the worm can be seen extending its body into the water before crawling from surface to surface of the host, and will move among the granular tufts when aboral. No aggressive behavior between commensals and host or between worms was observed, although the polychaetes rarely lie close to one another. If inverted, eversions of the proboscis can occur, apparently in an attempt to right the worm. Commensals re- moved from their host were not observed to eat small pieces of squid after a two hour period. Contained within the posterior segments of the poly- chaete, is a white substance of unknown constitution and function. Examination of 66 commensals showed 48 (72.7%) with white coloration in posterior segments and 18 (27.3%) without. Of those commensals reported without the white coloration, eight had emitted the substance after pro¬ vocation while in a dish of slightly warm stagnant sea Ophiodromus behavior Page 15 M. Serences water. The material was not always totally discharged, however, and anything remaining was concentrated in the anterior portion of the segments. If the worm is anteriorly provocated with a sharp instrument it will contract slightly and back up tail first rather than turning around on itself. Commensals without or with what appeared to be re- generated tail segments were also observed. Movement on and off the seastar did not seem to be hindered by such alterations. Tail cirri were dyed in methylene blue to see if any functional adaptations could be detected. Although generally appearing to drag passively behind the polychaete, they were frequently observed rigidly extended. Curved positions, inwardly and outwardly, around tube feet or the bases or oral plates were also noted. When a swimming commensal was observed the tail cirri appeared to move in a whip-like fashion. Ophiodromus b age 16 M. Serences SUMMARY 1. The host-directed behavior of Ophiodromus pugettensis, a commensal polychaete, was investigated in the field and laboratory on Patiria miniata over a six week period during April and May 1976 at Hopkins Marine Station, Pacific Grove, California. Field studies indicate: 2. A. An increase in commensal populations with increasing host size. B. Commensal populations of 0. pugettensis fluctuate with time. C. Clumped distribution of 0. pugettensis on P. miniata. D. An increase in size and growth rate of O. pugettensis moving intertidally. 3. Laboratory results suggest: A. Commensal distribution on P. miniata after small time periods is random. B. Feeding as well as protection may be the nature of the commensal existence of 0. pugettensis on P. miniata. C. Large number of commensals on individual P. miniata are uncommon for long periods of time. D. O. pugettensis populations on P. gigantea and D. imbricata may be uncommon in the field due to host ingestion of the polychaete. E. Commensal O. pugettensis abandonment of P. miniata is uncommon unless conditions are extreme. Ophiodromus behavior Page 17 M. Serences ACKNOWLEDGMENTS I would like to express my thanks to Chris Harrold, for helping me with my field studies, Larry Harding and the Hopkins Marine Station faculty and staff. My deepest thanks, however, go to Dr. Robin Burnett whose diving ability, patience, willing advice and profound enthusiasm made working on this project one of the most memorable experiences I have ever had. Ophiodromus behavior Page 18 M. Serences LITERATURE CITED Bartel, A. H. and D. Davenport 1956. A technique for the investigation of chemical responses in aquatic animals. J. Anim. Behav. 4:117-119. Davenport, D. 1950. Studies in the physiology of commensalism. I. The polynoid genus Arctonoe. Bid. Bull. 98:81-93. TT 1953. Studies in physiology of commensalism. III. The polynoid genera Acholoe, Gattyana and Lepidasthenia Studies in physiology of commensalism. I The polynoid generaP. noe, Lepidasthenia and Harmothoe. J. Mar. Bio. Assoc. U. K. 32:272-288. Davenport, D., G. Camougis, and J. F. Hickok 1960. Analysis of behavior of commensals in host-factor. I. A hesionid polychaete and pinnotherid crab. Anim. Behav. 8:209-218. Davenport, D. and J. F. Hickok 1951. Studies in physiology of commensalism. II" The polynoid genera Arctonoe and Halosydna. Biol. Bull. 00:71-83. 257. Notes on the early stages of the facultative commensal Podarke pugettensis Johnson (Polychaeta: Hesionidae). Ann. Mag. Nat. Hist., London, Ser. 12., 10:625-631. Hickok, J. R. and D. Davenport 1957. Further studies on the behavior of commensal polychaetes. 3:397-406. Biol. Bull. 11 Lande, R. and D. J. Reish 1968. Seasonal occurrence of the commensal polychaetous annelid Ophiodromus pugettensis on the starfish Patiria miniata. Bull. So. Calif. Academy Sciences 67:104-111. Reish, D. J. 1961. The use of sediment bottle collector for monitoring polluted marine waters. Calif. Fish and Game 47:261-272. C Ophiodromus behavior M. Serence Figure 1 —-Plot of average size P. miniata versus number of 0. pugettensis per host. This indicates an increase in the number of commensals per host with increasing host size. indicates two data points. Page 19 2 4 0 Ophiodromus behavior M. Serences FIGURE 1 0 0 o oo 0 a ooo0 234 50 789011231 AVERAGE SIZE P. MINIATA (CM) Page 20 Ophiodromus behavior M. Serences Figure 2--Histogram of number of commensals per host. Clumping is indicated. Darkened bars indicate Poisson distributions; slashed bars observed distributions. Page 21 Ophiodromus behavior M. Serences FISURE 2 LAA NUMBER COMMENSALS /SEASTAR Page 22 Ophiodromus behavior Page 23 M. Serences Figure 3--Graph of number of commensals per host over time period of 48 hours. represents host initially lacking commensals. e represents host with ten commensals initially. Two worms were without hosts at the conclusion of the experiment. S 0 Ophiodromus behavior. 1. Serences 141 12 91 81 4 4 6 12 FIGURE 3 18 24 30 36 42 TIME (HRS.) 48 Page 24 phiodromus behavior M. Serences Figure 4--Graph of the number of commensals per P. miniata versus frequency of occurrence in the laboratory. A random distribution is indicated. Page 25 8 Ophiodromus behavior M. Serences FIGURE 4 4 A 14 1 O 3 4 2 5 6 NUMBER COMMENSALS /SEASTAR Page 26 hiodromus behavior M. Serences Figure 5--Graph of time of day versus average distance of commensals from ambulacral grooves of the host (cm). Averages were taken from a number of commensals on various hosts and com¬ piled over a nine day period. No obvious patterns were detected. Page 27 Ophiodromus behavior M. Serences 1O I9 L E L O 1 4 FIGURE 5 Page 28 —— 8 TIME O 12 DA 8 12 phiodrom Serences Table 1--Records number of commensals o seastars for dive dates: Indicates fluctuations in commensal populations per host. Ophiodromus behavior M. Serences TABLE 1 PATIRIA TAG NUMBER NUMBER 2220 4 O 4 3 2 14 O 4 16 18 2 2. 26 3 27 3 20 3. 3 3 38 49 40 Page 30 OF COMMENSALS 4721 577 52 4 5 5 4 6 10 2 2 2 3 3 O 2 3 2 4 6 6 4 Ophiodromus behavior M. Serences Table 2--Area and date of collection, size range (mm) of 0. puget collected, sample size, mean, standard deviation and level of significance. Area 1 was at a greater depth and dis- tance from shore. Significant mean size differences in commensals between areas is indicated. Area, dates of comparison and level of significance between similar pop- ulations sampled. Indicates a sig- ficant change in size of commensals. Page 31 Ophiodromus behavior M. Serences TABLE 2 51ZE AREADATE N )( RANGE 39 75530/1892 12 5/ 3-15 111 78472488 2 3-13 38 6.1322244 20 2508 5- 41 9244 2 AREA VS. 20 2 VS. 26 P VALUE O0 01 (O3 (002 Page 32 Ophiodromus behavior M. Serences lable 3--Number of commensals with dye in gut af er time periods of 6 9 and 12 hours after exposure to gel atin mixture, with indicated addition and after starvation of indicated tervals. Slashes indicate resu from duplicate runs of the same periment. Six worms were used each tria e Ophiodromus M.Serences MIXTURE LMETHVLENE BLUE CARMINE +RED SQU1D LJUICE LGIGARTINA CORYMBIFERA +EGREGIA CARMINE f RED FPHYLLOSPADIX GIGARTINA CORYMBIFERA + EGREGIA CARMINE RED SEASTAR + JUICE LPHYLLOSPADIX SEASTAR JUKE FPHYLLOSPADIX CARMINE RED SMITHORA + NAIADUM behavior TABLE STARVATION PERIOD 3 DAYS O DAYS O DAYS D DAYS 6 DAYS 7 DAYS 8 DAYS 6 DAYS 3 NUMBER MITH DYE Oh Ohr 124 OO O/ /0 10 1 24 4 0 O 4 4 O O O 4 3 6 O O 2 2 O O O O 10 16 1 O O O O O Page 34 OBSERVATIONS NO EVERSIONS EVERSIONS EVERSIONS SEASTAR DID NOT INJEST FOOD NO PROBOSCIS EVERSIONS PRIOR TO STOMACH EVERSIONS STARTLED' NEAR FOOD NO PROBOSCIS EVERSIONS PRIOR 0 STOMACH EVERSIONS PROBOSCIS EVERSIONS STARTLED NEAR FOOD STARTLED' NEAR FOOD SEASTAR DID NOT INJE FOOD SEASTAR DID NOT INJEST Foop todrones behavio --Reco ple sult onse Jumb rimen tin the contraction response was obsery rnumber of trials is lis ted while commensal was touch not touchir near the host foo Indication hat commensals: re more al hen edin age 4. 1 L 1 Ophiodromus behavior M. Serences TABLE NUMBER TIMES RESPONSE PER NUMBER TRIALS CONTRACTION WHILE TOUCHING TOP OF FOOD 2 CONTRACTION WHILE TOUCHING EDGE 2 OF FOOD CONTRACTION WHILE NOT TOUCHING 12 FOOD Page 36