Bloom, Bowers, Cullenward -2- Biology of Phyllochaetopterus INTRODUCTION Phyllochaetopterus prolifica (Chaetopteridae) is a sedentary polychaete occurring in dense colonies of highly branched tubes (Potts, 1914). Like other Chaetopteridae, its body is divided into three distinct and highly differ¬ entiated regions (Figure 1). The external morphology of the species was investi¬ gated and described by Potts (1914), and also by Berkeley and Berkeley (1952). Work done on closely related species includes that done by Barnes (1964, 1965), who investigated tube building and feeding in Chaetopteridae. Extensive regeneration stud¬ ies on Chaetopterus variopedatus were carried out by Berrill (1928) and Faulkner (1932). The possibility of regeneration as a means of asexual reproduction for Phyllochaetopterus was discussed by Potts (1914), who suggested autotomy as a cause for the multiple occupancy of their tubes. However, Potts did not document his statements. This paper investigates various aspects of the biology of Phyllochaetopterus prolifica: its methods of tube irriga¬ tion and feeding, the functional morphology of the palps, its methods of asexual reproduction and regeneration, and aspects of its sexual forms. MATERIALS AND METHODS Colonies of Phyllochaetopterus prolifica were collected during April and May from three areas: the floats at the Monterey marina, the pilings at Monterey Wharf #2, and the kelp beds off Bloom, Bowers, Cullenward Biology of Phyllochaetopterus Hovden Cannery. Several of these colonies were wired to sty¬ rofoam and were floated in their natural orientation in running sea water aquaria. Parts of these colonies were periodically removed for experimentation; otherwise they remained undisturbed, Other colonies were dissected immediately after collection. Removal of individuals from natural tubes and their placement into plastic or glass tubes were carried out as described by Barnes (1965). The artificial tubes were kept in petri dishes or finger bowls in running sea water, and were observed with dissecting and compound microscopes. Plastic tubes were Intra¬ medic Polyethylene Tubing with internal diameters of .045 inches and variable lengths. Glass tubes were 0.9-1.1 mm in diameter and 100 mm in length. Observations of currents and feeding were made using carmine particles, the diatom Nitzschia longissima, and the green alga punalieila tertiolecta to trace water movement. These were suspended in sea water filtered through a Millipore filter, RESULTS AND DISCUSSION A. General Description The body of Phyllochaetopterus prolifica is divided into three distinct regions (Figure 1). The anterior region is cream¬ colored or light tan and contains ten to twelve setigerous segments. The rounded prostomium, which has two lateral eye¬ spots, is surrounded by the peristomial collar and lips. Paired palps and antennae arise at the base of the prostomium. The palps are approximately equal to the anterior region of the body in length. Their coloration varies from tan to Bloom, Bowers, Cullenward -4- Biology of Phyllochaetopterus reddish-brown. They are attached dorsally, anterior to the antennae. Directly anterior to the point of attachment, the dorsal and ventral surfaces are rotated 30-45° relative to the rest of the body; the ventral surfaces are thus tilted toward the midline of the body. The dorsal surfaces are rounded and crenulated; the ventral surfaces have two grooves which run the length of the palps: a shallow, ciliated furrow which car¬ ries particles outward, and a deep, ciliated trough which travel particles toward the mouth (Figure 2). The end of each palp is slightly knobbed. They can be held in three different posi¬ tions: extended in front of the body, tucked back along the dorsal surface, or tightly coiled adjacent to the peristomial collar. A ciliated, dorsal groove originates at the base of the palps and runs the length of the body. Food and fecal pellets move along it to the mouth and palps from more posterior regions. The neuropodia of the anterior region have simple setae and are used for locomotion. The fourth setigerous segment contains a large,dentate spine used for cutting. Whereas most of the dorsal surface of this region is covered with cilia, the ventral surface is unciliated and secretes the tube material (Barnes, 1965). The median region is greenish-gray and includes three to seventeen segments. The neuropodia are short ridges with uncini which are used to brace the individual against the tube wall. The noto- and neuropodia are covered with cilia, as is much of the dorsal surface of the median region. Between the lobes of the trilobed, foliaceous notopodia there is a loop Bloom, Bowers, Cullenward Biology of Phylloch lined with large, flagellum-like membranelles; their beat draws water posteriorly through the loops. The dorsal groove passes under the medial loop and widens posterior to each noto¬ podium, forming a cupule. A small (0.15-0.20 mm), clear ball made of mucus secreted by the medial loop rotates in the cupule. The balls catch and tightly pack particles which are driven past them by the beating membranelles. The medial loop period¬ ically spreads apart, and the balls move forward along the dorsal groove to the mouth where they are ingested (Barnes, 1965). The posterior region is dark green and has a highly variable number of segments, never exceeding forty-five. Each notopodium is elongate and has a ciliated tip from which protrudes one seta. The bilobed neuropodial flaps have uncinal plagues which are used to brace the animal against the tube. This region is used primarily for locomotion and stability. The asexual form of P. prolifica inhabits large, dense clumps of twisted, weakly annulated tubes. These tubes consist of a parchment-like substance which is deposited in layers (Barnes, 1965). Older sections of tubes are opaque and thickened, whereas sections of new growth are thin, translucent, and more regularly annulated. Areas of new growth can expand to accom¬ modate additional internal layering. The colonies have a matted base of tightly packed tubes which is attached to a substrate. The tubes radiate from the substrate and are frequently and irregularly branched, especially near the base. A high degree of interconnection exists between tubes, and it is possible that the entire colony has arisen from the extensive growth and branching of one or a few tubes. The Bloom, Bowers, Cullenward -6- Biology of Phyllochaetopterus clumps tend to have an even upper boundary with the majority of tubes opening at this surface. The tubes range in diameter from .5-1.2 mm, and are inhabited by as many as twelve individuals in a one-foot section. Individuals in the tubes often build transverse partitions between each other. These opaque white partitions contain two holes made by the modified fourth setiger. It is presumed that the fourth setiger is also used to make the small pores which appear irregularly along the sides of the tubes. B. Irrigation and Feeding For thirty-seven tubes, there was an average of one animal in every five centimeters of tube (r=0.84, p(.01,Figure 3). The number of pores in a tube was counted by constricting one end with cotton thread, gently forcing water into the other end with a syringe, and counting the number of leaks. These were unevenly spaced, but they averaged one to every 3.3 centi¬ meters of tube (r=0.73, p(.01,Figure 4). There was an average of 1.7 pores per animal (r=0.74, p«.01, Figure 5). All slopes. had 95% confidence limits which did not include zero as a pos¬ sible slope (Sokal and Rohlf, 1968). P. prolifica filters particles from the irrigation water using the mucus ball method described earlier. Animals were always seen producing mucus balls with all except the first foliaceous notopodia, unless the animals were doubled over or passing a tube-mate. The balls average 0.15-2.0mm in diameter when passed forward to the mouth. When three animals were placed in con¬ centrations of Nitzschia which ranged from 10'to 10 cells per Bloom, Bowers, Cullenward Biology of Phyllochaetopte ml, the time taken for ball formation was significantly less at higher concentrations (p(.001, Figure 6). Many of the particles which passed through a foliaceous parapodium were not caught in the ball immediately behind it. Water which passed by seven or eight balls still carried par¬ ticles. Some of these were caught in the dorsal groove in the posterior region and passed forward. Carmine particles moved through the transparent foregut at about 2 mm/min. Thereafter, they moved through the dark hindgut at an average rate of 0.43 mm/min. Hartman (1969) reports that P.prolifica ranges in length from 1-6 cm. A typical animal has a hindgut of about 2 cm; therefore, food would travel through it in about 1.5 hours. Barnes (1964, 1965) noted that P. socialis and related species create currents primarily with the beating membranelles of the foliaceous parapodia. P. prolifica also depends prim¬ arily on this method. In addition, some animals occasionally show slow posterior to anterior peristaltic waves which set up currents. The movements of animals through tubes also serve to circulate water. P. prolifica does not show an ability to pump a unidi¬ rectional flow of water through a tube. Carmine particles were observed flowing simultaneously into both ends of a section of natural tube. Most characteristic was an ebb and flow pattern at the mouth of the tube. Six 15 cm long plastic tubes containing three individuals each were covered at one end with phytoplankton netting. These were placed in a 10 cells/ mi solution of Dunaliella tertig Bloom, Bowers, Cullenward -8- Biology of Phyllochaetopter The individual furthest from the open end typically did not begin to catch the algae in its mucus bags until after 30 min¬ utes exposure. Most had caught algae after an hour. Three of these tubes had three pin holes each, located along the length of the tube. A t-test showed no significant difference in rates of Dunaliella pick-up between the group with holes and the group without (Table 1). Water flows in the tubes at about 1 mm/sec until it pas¬ ses the foliaceous parapodia, slowing to 0.2 mm/sec past the posterior region. The cilia covering much of the dorsal surface set up eddies, resulting in turbulent water flow. The arrows in Figure 7 show the directions of water flow around the body. P. prolifica was able to tolerate oxygen stress. Ten individuals were placed in each of five glass-stoppered reagent bottles. Nitrogen was bubbled through three of these for 15 minutes, while air was bubbled through the other two. Oxygen tension in the nitrogen bottles was determined with an oxygen electrode to be less than 10% saturation. The bottles were sealed and left in running sea water for 19 hours. After re¬ moval and examination, all animals were alive, and no differ¬ ences could be detected between the individuals in low oxygen tension and the controls. The movement of individuals in artificial tubes was periodically observed during a five hour period. Three indivi¬ duals were placed in each of four 15 cm long plastic tubes, one end of which was covered with phytoplankton netting. These were vertically suspended in a running sea water aquarium. Individuals were distinguished by the different number of fol¬ Bloom, Bowers, Cullenward -9- Biology of Phyllochaetopterus iaceous notopodia on each. Figure 8 summarizes the observations. Each individual was closest to the tube opening relative to the others in its tube for three or four of the ten observa¬ tion times. An analysis of variance showed no statistical difference in the average times spent in each position. The average interval between times spent in the closest position was 1.8 hours, and the average distance travelled was 7.5 cm/hr. * * * The asexual form of P. prolifica is a highly modified filter feeder which lives in a complex and densely populated community. It therefore must have evolved a method for ensuring that all tube-mates get adequate food and oxygen. Efficient tube irrigation would seem necessary for success. However, the long length, small diameter, branches, and closed ends of the tubes might make irrigation difficult. Instead, P. prolifica has evolved an energetically economical system which involves a minimum of pumping and the need for little coordination among tube-mates. The slow speed at which water passes the animals' posterior segments and the slow rate at which individuals further from the tube opening pick up particles suggest that water moves only slowly from the opening into the further reaches of the tube. There the water may be stagnant and depleted of food particles. However, animals continually change positions in the tube; each animal therefore spends time at the tube mouth. Food would travel through the gut of a typical animal in about 1.5 hours, and the average time spent away from the opening of a tube is 1.8 hours. Thus the animal may move to the opening Bloom, Bowers, Cullenwa -10- 3iology of Phyllochaetop when it needs to empty its gut and move away when its gut is full. MacGinitie (1945) found that Chaetopterus yariopedatus can filter particles as small as proteins with its mucus net. P. prolifica may have the same ability, and it can filter out 54 Dunaliella cells and also catch macroscopic particles with its palps. This ability to exploit a large range of particle sizes, coupled with the ability to change the speed of mucus ball formation with particle concentration, would allow the animal to feed quickly. However, the sigmoid nature of the curve in Figure 5 suggests that at high concentrations, P. prolifica is limited by the rate at which it can produce and consume mucus. At low concentrations the animals probably have a maximum residence time for mucus balls. The sections of tube near the opening are probably deficient in oxygen, since a variety of tubiculous polychaetes have been shown to remove 50-60% of the oxygen in the water which passes their bodies (Dales, 1963). P. prolifica can tolerate low oxygen tensions. The animals lie extended in tubes with a maximum surface area exposed. The stirring action of the cilia may serve to increase oxygen uptake by maximizing exposure to the surrounding water. Furthermore, the pores in natural tubes might aid in diffusion of water and food particles into the upper reaches of the tubes, although such an effect was not seen in the thicker-walled plastic tubes. Thus all tube-mates are able to endure sub-optimal conditions in between periods of heavy feeding. Bloom, Bowers, Cullenwa Biology of Phyllochaetopterus C. Function of Palps P. prolifica uses its palps to remove fecal pellets and other debris from the tubes. Fecal pellets are passed up the dorsal, ciliated groove and carried by ciliary action to the base of the palps. They are subsequently passed out to the ends of the palps where they are prevented from moving further by the knoblike processes located there. When the palps are extended outside of the tubes, they pull apart from each other and the particles are released. Two types of particles are regularly carried up the dorsal, ciliated groove: fecal pellets and food balls. However, only fecal pellets are ejected by the palps. In order to determine how this selectivity in rejection of particles is accomplished, particles in the vicinity of the mouth were observed for extended periods of time. Only particles less than 2504 in diameter were ever ingested. The diameter of fecal pellets was found to be 250-350 and that of the mucus balls was 150-2504. It was also observed that the arrival of particles at the mouth was usually followed by a short retraction of the body. During this retraction, fecal pellets were caught in the outgoing groove of the palps, whereas the mucus balls remained positioned at the mouth until swallowed. An experiment was performed to study the effects of palp removal on both the clearing and building of tubes. 25 individ¬ uals were confined to 10 mm sections of natural tubing. The palps were removed from 10 of these individuals; the first parapodia were removed from five others as controls. Tube¬ building was recorded daily, and other observations were made. The individuals with palps and the controls had similar rates Bloom, Bowers, Cullenwa Biology of Phyll -12- of tube-building. Those without palps had much slower rates (Figure 9). The individuals without palps made only small additions to the tubes and kept both ends closed with partitions. These worms cleared their tubes by pushing feces out through holes in the partitions with their peristomial collars. The feces aggregated about the ends of the tubes. After about seven days, the partitions were removed and the rate of tube-building increased. The palps were 1-1.4 mm in length, long enough to be extended out in front of the body. A study was undertaken to determine the percentage of time that individuals occupying the ends of tubes spend with their palps protruding. Ten individuals in natural tubes were placed in a glass bowl filled with sea water and watched continuously for five hours. Similarly, fifteen individuals were observed for two hours in their natural habitat at the Monterey marina. Although the values obtained varied consid¬ erably from individual to individual, the palps were protruded 50% of the time (Table 3). During the seven hours of obser¬ vation, the palps were frequently seen carrying small particles to the mouth; only five instances of ejection of debris from the tubes were recorded. In order to test the responsiveness of the palps to mechanical stimulation, an artificial colony of individuals in natural tubes was set up in a large glass bowl of sea water. One end of each tube was held in place by clay, and the other end was left free. In separate trials, l gm and .l gm weights (only the difference in magnitudes is important) were dropped from a height of 1 inch onto palps protruding from the ends of Bioom, Bowers, Culienwar Biology of Phyllochaetopterus -13 tubes. The retraction of the individuals into the tubes was measured. The process was repeated 30 seconds after the palps reappeared at the end of a tube, and time intervals between stimulations were recorded. The process was repeated 20 times with 10 individuals. Table 4 shows the average distance retracted in response to the different weights. The difference is significant to a level of .001. Thus the response is graded; stimulation with the heavier weight causes a greater withdrawal. No habituation to stimulation was found after 20 trials. Autotomy of the palps was tested by clamping lead shavings onto the palps of 20 individuals and recording the time required for releasal. All 20 palps were released at the point of attachment to the body, either by a jerk or by a long, steady pull. The average time required for release was 4.5 minutes, with a range from 15 seconds to 13 minutes. The force required to release a palp was determined by clamping successive lead shavings onto it, until it was released, The force needed to break a palp was similarly found by holding one end of a palp with forceps and adding shavings to the other end until it broke. The force required for release was 3.5 times greater than the force neede to break a palp (2015 and 70:10 dynes, respectively). Palps are frequently lost in natural populations; 54 out of 300 individuals were found with regenerating palps. Regen- erating palps are translucent and flared until they reach a length of 1-1.4 mm. At this time the ends become knobbed, and the palps themselves take on a brownish hue. Palp regen- Bloom, Bowers, Cullenward Biology of Phyllochaetopterus -14- eration was studied by removing the palps from healthy individuals. This was done both by pulling with forceps to autotomize them and by cutting them with a scalpel to a length of approximately .75 mm. Growth was measured daily. Similar rates of regeneration were obtained for the two different cases; the palps grew about .25 mm per day (Figures 10,11). The growth was fairly linear, and the only difference between the two cases was the initial 3-day period during which there were no visible signs of growth for the palps which were pulled free. * * * Observations on individuals occupying the ends of tubes indicate that the palps are frequently protruded. The rarity of fecal pellet ejection and the frequency of particle capture suggest palps are protruded to feed. This feeding activity is, of course, greatly reduced when an individual is not at the end of a tube. Nevertheless, this phenomenon can serve as a valuable, accessory feeding mechanism. Besides serving to supplement filter-feeding, the palps are important in clearing the tubes, especially in moving debris away from the tube openings. Individuals who had their palps removed tended to keep both ends of their tubes closed with partitions. Feces aggregated about the ends of these tubes, and the partitions were not removed until the palps were long enough to successfully eject debris. This suggests that the partitions were constructed to prevent the reentry of feces into the tubes. The frequency of regenerating palps in natural popula¬ tions leads one to believe that the loss of palps, possibly Biology of - Bloom, Bowers, Cullenward Phyllochaetopterus -15- by predation, may be a common phenomenon. The palp's sensitiv¬ ity to mechanical stimulation and the associated withdrawal response are certainly of adaptive significance to this animal. The gradation in this response permits the animal to react appropriately to its environmental cues. Moderate stimulation, as from particulate matter in the water, causes a slight retraction, whereas the response to a strong stimulus, such as would be inflicted by a predator, is a much greater withdrawal. The relatively weak attachment of the palp to the body as compared to the strength of the palp itself could serve to facilitate the release of an entrapped palp in order to avoid predation. Shedding a palp seems to have no detrimental conse¬ quences for the individual; a new, functional palp can be regnerated in ten days or less, and lost functions of the palps can be compensated for in the meantime. One final point concerning the palps has to do with the ability of the animal to distinguish between fecal pellets and food balls. It is possible that these two types of particles are selectively separated by either of two independent methods. One such method involves particle size; the fecal pellets are too large to be ingested so they remain in the vicinity of the mouth until they are eventually drawn away by the palps. A second possibility is that the mucus in the food balls connects them to the peristomial collar where they are held until swallowed. Having no such mucus, the fecal pellets are caught by the palps and carried away when the animal retracts. Bloom, Bowers Cullenward Biology of Phyllochaetopterus -16- D. Asexual reproduction and regeneration In order to determine various aspects of regeneration, P. prolifica were sectioned with a scapel in different areas of the body: between the anterior and median sections, after the first foliaceous parapodia, after the third foliaceous parapodia, and between the median and posterior sections. Both halves of the sectioned individuals were placed in plastic cap¬ illary tubes and kept in running seawater. Further information on regeneration was obtained by observing regenerating individuals ejected from natural tubes. Results A general growth and time sequence for anterior regeneration in P. prolifica was determined (Figure 12). Frame A shows the healed wound after sectioning an individual with a scalpel. Healing of the wound is done by invagination, and occurs within ten hours after sectioning. Frames B and C show the extension phase of anterior re¬ generation. The new tissue formed is white, and no external segmentation is visible. It should be noted that palps and a mouth form before any external segmentation can be seen (Frame D). Segmentation is an all at once process, as parapodial lobes develop for all segments, then setae appear (Frame E). Frame F shows a fully functional miniaturized anterior re- gion, which is still very white compared to the normal cream color of the anterior region. After an individual has reached this stage, the new anterior region enlarges and regains its normal color over a 2 to 3 week period. Bloom, Bowers, Cullenward Biology of Phyllochaetopterus Varying abilities for anterior regeneration were observed as a function of the location of the artificial sectioning. Those individuals sectioned between the anterior and median region, after the first foliaceous parapodia, and after the third folia- ceous parapodia regenerated at approximately equal rates. In contrast, those individuals sectioned between the median and the posterior sections were observed to take comparatively longer times for similar development. Feeding was observed as early as stage A, but it was not as efficient a process as observed for normal individuals. At stage F feeding was observed to be essentially normal. Feeding was not observed for regenerating individuals lacking foliaceous parapodia. Posterior regeneration A general time and growth sequence for posterior regeneration was also determined (Figure 13). Frame A shows the healed wound after sectioning. In this case, healing is accomplished by evagination of the gut lining. As can be seen in frame C, neuropodia develop before noto¬ podia in the extended region. However, unlike anterior regenera¬ tion, segmentation occurs in a sequential pattern through time. The oldest segments are the most anterior and decrease in age posteriorly. Ppsterior regeneration occurs over an indefinite amount of time. After the initial growth of the tail, the process slows, and segments are then added a few at a time, with no observible pattern. This is evidenced by the finding of individuals in tubes with 20 or more normal sized posterior segments and a very small extension with one or two developing neuropodia. Bloom, Bowers. Cullenward -18- Biology of Phyllochaetopterus As in anterior regeneration, varying abilities for posterior regeneration were observed. Individuals sectioned between the median and posterior regions, after the third foliaceous para- podia, and after the first foliaceous parapodia regenerated at approximately equal rates. Those individuals sectioned between the median and anterior regions were observed to take compara¬ tively longer time for similar development. Foliaceous parapodia regeneration Foliaceous parapodia have been observed to regenerate be¬ tween the anterior and median sections of individuals. Regen- eration between median and posterior segments has never been observed, but the possibility should not be excluded. Time in¬ volved in this process seems to be rather long, and no inherent pattern or criteria for regeneration has been determined. Regeneration in natural tubes Up to 30% of the individuals inhabiting natural tubes have been found undergoing some stage of regeneration. 82% (49 of 60) of the recently budded individuals (frame B in figs. 12813) have foliaceous parapodia directly either anterior or posterior to the budded area; the other 18% are budded directly anterior to a variable number of setigerous segments in the anterior region of the individual. In this case, as with regeneration of whole an- terior sections, the number of setigerous segments in the fully regenerated individual is pre-determined, and enough segments are added to complete the normal anterior region. No indivi¬ duals have been found to be regenerating from anterior or post- erior segments alone. Behavior Biology of Bloom, Bowers, -19- Phyllochaetopterus Regenerating individuals exhibited notable behavior. The anterior halves of those individuals sectioned in the median region set up partitions and built extensions on their tubes, a normal behavior for naturally occurring individuals. These individuals were also observed to have their palps protruding from the end of their tubes. Anterior halves of individuals sectioned between the anterior and median regions exhibited no such behavior, and were often found to have fallen out of their tubes onto the bottom of the finger bowl in which they were stored. This phenomenon was also observed in the case of post- erior halves sectioned between the median and posterior regions, * * * Unlike Chaetopterus variopedatus, where the ability to regenerate is body region specific (Berrill, 1928), Phyllo¬ chaetopterus prolifica has the ability to regenerate anteriorly or posteriorly from any section in the body. It is not known if regeneration can occur both anteriorly and posteriorly from one segment; however, cases have been observed where regeneration is proceeding in both directions. As found for C. variopedatus, no dedifferentiation of existing segments seems to occur during regeneration. The total number of segments in the anterior region of an individual is constant. This number is achieved in regeneration whether complete or partial regeneration is occurring. Complete anterior regeneration is a faster process than posterior regeneration. There seems to be a need for a fully functional anterior section to regenerate fairly quickly. This assumption is supported by the development of sensory and feeding organs (palps, eye spots, and a mouth) before the development of Cullenwar Bloom, Bowers, Cullenward Biology of Phyllochaetopterus -20- segmentation. Also, a fully functional anterior region is formed in miniature, and then the enlargement occurs. This method of regeneration is completely different from the sequential poster¬ ior method of regeneration, which, as shown, occurs over an in¬ definite amount of time. In support of asexual reproduction by autotomy in the median regiom, the following points should be noted: 1) Anterior and posterior regeneration from median regions is faster than the same processes from posterior or anterior seg¬ ments alone. 2) 82% of the recently budded individuals found in natural tubes have done so in the median region. The other 18% can be explained by the loss of anterior segments due to predation. It is unlikely for autotomy to occur in the anterior region, as behavior patterns have been shown to be particularly maladaptive in the case of lone anterior segments. 3) No anterior or posterior regions alone have been observed undergoing regeneration in natural tubes. 4) Barnes (1965) observed that the foliaceous parapodia are the major food gathering segments, and provide water currents through the tubes. It has been observed that regeneration of the foliaceous parapodia is a slow process. Thus, if autotomy were to occur outside of the median body region, one half of the split individual would have to go without these essential segments for an extended period of time. This process would be maladaptive for the survival of the half without foliaceous parapodia, and therefore the advantage of asexual reproduction by autotomy would be lost. The preceeding four points clearly support and document the Bloom, Bowers, Cullenward Biology of Phyllochaetopterus -21 fact that asexual reproduction by autotomy and regeneration is taking place in the median body region of P. prolifica. E. Aspects of sexual forms Investigation of Phyllochaetopterus colonies revealed the presence of two different tube sizes. The smaller tubes ranged in diameter from 0.5-1.0 mm, while the larger tubes ranged from 1.2-1.8 mm. Whereas the smaller tubes contained the expected multiple asexual forms, the large tubes were, singly inhabited by a much larger fonm of P. prolifica. Table 5 compares average number of segments per body region in the two forms. The differ¬ ences in all means are significant at the.OOl level. The posterior segments of the large forms are either orange or cream colored, associated with the presence of female and male gametes respectively. The orange color in the female is due pri¬ marily to pigmented spheres; the eggs themselves are brown, often not quite spherical, and range in size from 65-70 1. Coloration in the male is due to the sperm, which are slightly oval with a diameter of 3 /. Attempts to fertilize eggs were made in the latter part of May, but were unsuccessful. Twenty colonies of P. prolifica were examined, all containing at least four sexual forms in individual large tubes. Each large tube was enmeshed with small tubes, forming a dense, flat mat which was attached to a styrofoam float or a cement pillar. Integration of a large tube into the mat occurred either at one end with the other end of the tube hanging freely, or in the middle of the tube with both ends hanging freely. Tubes containing sexual individuals are very rarely branched (3 tubes out of 123 investigated), and if so, only in the last few centimeters of the free-hanging end. No connections between Bloom, Bowers, Biology of Phyllochaetopterus -22- large tubes or between large and small tubes were observed. Large tubes are of a uniform diameter throughout their individual length, and have an average length of 16.2 cm (n=43, range= 8-23 cm). Upon investigation of a 10 ft“ area under the floats at the Monterey marina, sexual forms were found, each occupying their own tube, in the absence of asexual colonies. Of the 69 in¬ dividuals found in this area, 33 were male, 34 were female, and two contained no gametes. Although containing no gametes, the latter two closely resembled the sexual forms in size and appear- ance. Individuals that contain orange pigment and eggs have been found living in association with, and in the tubes of asexual forms of P. prolifica. However, these individuals resemble the asexual forms in size and appearance, and the relative amount of pigment and gametes present is greatly reduced. Differences were found in the average size of asexual in¬ dividuals found in different colonies. In one colony, tube diameter was approximately 0.5 mm and the average length of asex¬ ual individuals was 7 mm (n=20, range- 5-9 mm). In another colony, tube diameter was approximately 1.0 mm, and the average length of an asexual individual was 14 mm (n=65, range- 10-25 mm). No differences were noted between the sexual individuals of these two colonies. * * * Berkeley and Berkeley (1952) first reported the presence of sexual forms of P. prolifica. However, they were only described c Cullenward Biology of Bloom, Bowers, Cullenward -23- Phyllochaetopterus as having up to 40 or more posterior segments, and no description of different sexes was given. The striking difference of the ex- ternal morphology between sexual and asexual forms shown previous¬ ly, coupled with the finding of egg-bearing individuals in asexual tubes, leads us to believe that the sexual forms described by Berkeley and Berkeley are the egg-bearing individuals in asexual tubes. The fact that sexual individuals are always found associated with sexual individuals, and the discovery of sexual forms in isolation suggests that the asexual colonies arise from larvae which settle on sexual individuals. This assumption is supported by the finding of uniform asexual tube diameters in a colony, while variations occur between colonies. This variation is most probably not due to environmental factors, as colonies of varying tube di¬ ameters were found in close proximity of one another. One possi¬ ble explanation for this variation may be genetic similarity within a colony, and genetic dissimilarity between colonies. The possi¬ bility exists that an asexual colony can arise from a single asex¬ ual individual. A possible model for asexual versus sexual development is differential development of settled larvae. Larvae that settle solitarily develop directly into sexual individuals, while those that settle in close proximity of mature, sexual individuals are inhibited from maturing and develop asexually. This is supported by the finding of single immature individuals in large tubes, and the constant association of sexual individuals with asexual individuals. Since sexual individuals are found near one another, this differential settling may be very specific for the presence of already mature sexual individuals before asexual Bloom, Bowers, Cullenward Biology of Phyllochaetopterus -24- development occurs. Larvae may be searching for mature sexual tubes to settle on, and, if unsuccessful, they settle solitarily and develop into sexual forms themselves. It is doubtful that the individuals found with gametes in asexual tubes ever develop into fully mature, sexual individuals. For maximum genetic input, these 'maturing' asexual individuals would have to grow to a size paralleling that of the mature sex¬ ual individuals. This also seems unlikely as the average asex- ual individual would have to increase its length at least tenfold, This increase in size would require a large tube to accomadate the individual. However, the total lack of connections between asexual and sexual tubes and the uniform diameter of large tubes indicate that no enlargement is taking place. It is also highly improbable that an individual would leave its tube to start building a new one, due to the presence of predators associated with the colony, and an individuals inability to maneuver effi¬ ciently outside of a tube. A possible explanation for the presence of egg-bearing individuals in asexual tubes may be the decrease in effect of the sexual maturation inhibitory process, permitting limited gamete production. This decrease in effectiveness may be due to the colony size or certain environmental factors. It is sus¬ pected that the gametes produced are eventually reabsorbed by the asexual individual. Bloom, Bowers, Cullenward -25- Biology of Phyllochaetopterus UMMARY 1. P. prolifica communally inhabits tubes in densities of one animal per 4 centimeters of tube. 2. Rate of mucous ball formation is a function of particle concentration. 3. P. prolifica does not create a unidirectional water flow through the tubes; instead, each animal periodically comes to the mouth of the tube to feed. 4. Rate of particle movement through the gut corresponds to the amount of time spent away from the tube mouth. 5. The palps are used in feeding, voiding feces, and as sensory appendages. 6. Palps are extended from the tube opening for approximately 50% of the time, in both field and lab observations. 7. Palps are capable of autotomy, and 18% of the population is found to be in the process of regeneration. 8. P. prolifica has the ability to regenerate anteriorly or posteriorly from any body region. 9. Anterior regeneration occurs faster than posterior regen¬ eration and is achieved by a different process. Anteriorly, seg¬ mentation and further development of the segments takes place over the whole new body region uniformly, as cpposed to the more sequential segment-by-segment process of posterior regeneration. 10. Asexual reproduction by autotomy and regeneration occurs in the median body region. 11. Sexual forms of P. prolifica are larger and inhabit bigger tubes than the asexual forms. Bloom, Bowers, Cullenward Biology of Phylloch -26- 12. All asexual colonies contain sexual forms inhabiting large individual tubes. Sexual forms are also found in the ab¬ sence of asexual colonies. 13. Differential larval development is suggested as a pos¬ sible mechanism for asexual versus sexual development. * * * ACKNOWLEDGEMEN We would like to thank the faculty and staff at Hopkins Marine Station for their guidance and assistance. In addition, our thanks go to Thomas Barmeyer and Michael Imperato for their assistance in field collections. We are especially grateful to Chuck Baxter for his time, effort, and generousity throughout the duration of this project. m, Bowers, Cullenward 227 Biology of Phyllochaetopterus RTRT T BIBLIOGRAPHY Barnes, R. D. 1964. Tube-building and feeding in the Chaetopterid polychaete, Spiochaetopterus oculatus. Biol. Bull. 127:397-412. Barnes, R. D. 1965. "Tube-building and feeding in Chaetopterid polychaetes. Biol. Bull. 129:217-233. Berkeley, C., and E. Berkeley. 1952. Canadian Pacific Fauna 9b: 63-64. Dales, R. P. 1963. Annelids. Hutchinson and Co. LTD., London. 200 p. Faulkner, G. H. 1932. The histology of posterior regeneration in the polychaete Chaetopterus variopedatus. J. Morphol. 3: 23-58. Hartman, O. 1969. Atlas of the Sedentariate Polychaetous Annelids from California. A. Hancock Foundation, Los Angeles. 812 p. MacGinitie, G. E. 1945. The size of the mesh openings in mucus feeding nets of marine animals. Biol. Bull. 88: 107-111. Potts, F. A. 1914. Polychaeta from the N. E. Pacific: The Chaetopteridae. Zool. Soc. Lond. Proc. 67: 955-994. Sokal, R. R. and F. J. Rohlf. 1969. Biometry. W. H. Freeman and Co., San Francisco. 776p. Bloom, Bowers, Cullenward Biology of Phyllochaetopterus -28- ABLE LEC Table 1 Time for particle uptake by each of three animals in six 15 cm. long plastic tubes. The same six tubes were used for all three trials. Time spent in various positions in tubes. Zero position Table 2 represents the open end. The 15 cm. position represents the end covered with phytoplankton netting. Table 3 Percent time that individuals protruded or withdrew palps from ends of tubes. Lab: n=10, observed 5 hours. Field: n=15, observed 2 hours. Table 4 Comparison of distance individuals retracted when stim¬ ulated with 1 and .1 gm weights. n=10. Table 5 Comparison of number of segments per body region and average lengths for asexual and sexual forms. Biology of Phyllochaetopterus Table 1 Tubes with pinholes Tubes with¬ out holes Particle type Dunaliella Dunaliella Nitzschia Dunaliella Dunaliella 1 Nitzschia Bloom, Bowers Cullenward -29- Number of animals trapping particles at 10 min. 20 Observations 2 animals at tube end animals dispersed dispersed 2 animals at tube end dispersed 1 dispersed dispersed all moved towards end 1 dispersed dispersed 3 animals in irst 5 cm. dispersed dispersed dispersed dispersed 2 animals at tube end dispersed dispersed Biology of Phyllochaetopterus Table 2 Individuals Tube AO Tube B □ Tube CO Mean Standard deviation Table 3 Minutes spent: furthest from in the middle opening 162 108 126 45 144 117 132 108 102 171 81 150 48 168 115.7 103.3 43.8 39.1 Palps % Time out % Time ir 48 Lab 52 Field 53 47 Bloom, Bowers, Cullenward closest to opening 120 75 120 144 66 105 84 99 96.0 29.7 Biology of Phyllochaetopterus Table 4 .1 gm 1 gm Table 5 No. of orms Asexual 61 Sexual 67 Ave. Distance Retracted Range .5-4 mm 2.5 mm 2-13 mm 8.5 mm No. of Segments Post. Med. Ant. 26 11 (3-41) (3-17) (10-12) 21 74 (9-13) (12-32) (29-126) Bloom, Bowers, Cullenward 31 Length 10 mm Average (5-16 mm) Range 11 cm Average Range (4-19 cm) Biology of Bloom, Bowers, Cullenward oterus ochaet -32 FIGURE LEGEND Figure 1 Dorsal view of P. prolifica. Figure 2 Ventral view of anterior end, showing ciliated grooves on palps. Figure 3 Linear correlation between number of individuals and tube section length. (r=.84, pf.Ol, slope=.20) Figure 4 Linear correlation between number of pores and tube section length. (r=.73, pf.O1, slope=.30) Figure 5 Linear correlation between number of individuals and number of pores in a given section of tube, (r=.74, pf.Ol, slope=1.69) Figure 6 Rate of mucus ball formation. Analysis of variance shows p.001. n=3. Figure Schematic dorsal view of P. prolifica. Dotted areas are ciliated. Arrows show directions of water currents around the body. Figure 8 Positions of three individuals in A, B, and C. each of three 15 cm. plastic tubes. Animals were ob¬ served at 30 or 45 minute intervals. Symbols repre¬ sent the individuals; arrows represent the orientation of the prostomium. Tube growth. Points are means; bars represent standard Figure 9 errors. Figure 10 Palp regeneration. Palps were pulled free with forceps. n=20. Figure 11 Palps were cut with a scapel to Palp regeneration. 0.75 mm. n=15. Figure 12 General growth and time sequence for anterior regen¬ eration after sectioning. Figure 13 General growth and time sequence for posterior regen¬ eration after sectioning. 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