Cowan -2 Regeneration in Dorvillea INTRODUCTION Studies on polychaetes have included investigations of many aspects of regeneration. Older papers such as Berrill (1928, 1931, 1936) and Faulkner (1932) are reviewed Many exciting dis¬ in Berrill (1951). coveries have been made, including the elucidation of a neurohormone gradient thought to be responsible for the retention of polarity (Fitzharris, 1973). Nevertheless, there remain large gaps in the infor- mation available on polychaete regeneration. The process has not even been looked at in many species of polychaetes. One such species is Dorvillea moniloceras, family Dor¬ villeidae, which is found intertidally and subtidally on the coast of Monterey, California. In this study, the regenerative capabilities of D. moniloceras were investigated in order to answer the following three questions. Can both posterior and ant- erior regeneration take place? What events take place before full regeneration is attained? Does the position along the body at which a worm is cut affect either the ability to regenerate or the rate of regeneration? MATERIALS AND METHODS This study was done at Hopkins Marine Station of Stanford University in Pacific Grove, California during April and May of 1976. A large population of D. monilo- eras was found at the marina at Monterey Harbor, Regeneration in Dorvillea Cowan -3 underneath the boat docks, specifically among tunicates and Phyllochaetopterus tubes. After collection, the worms were stored in seawater in fingerbowls set in a bath of running seawater at 14-16° C. All sizes of worms were used as experimental animals. After at most a day or two in the laboratory, the worms were cut. The procedure was to place the worms in a solution of MgCl, isotonic with seawater, for 15 minutes prior to cutting. The cut was then made with a razor blade on the posterior side of the desired segment. The position of the cut was recorded. Both front and rear pieces of the worm were stored together for the duration of the experiment. At first the containers used were glass tubes with plankton netting secured at both ends, placed in running seawater. No antibiotics were used. This arrangement allowed an accumulation of phytoplankton and bacteria which resulted in an abnormally high mortality rate. Keeping worms in five inch diameter fingerbowls half filled with seawater proved to be a good alternative. The bowls were set in a bath of running seawater and maintained at temperatures ranging from 14-16° C. The water in the bowls was changed daily. If a worm regenerated to the stage where it could eat and defacate normally, a tunicate and hydroids were placed in the bowl to give it an opportunity to feed and resume a morghatural existence. (Hydroids represent a Regeneration in Dorvillea Cowan -+ common natural food of D. moniloceras according to Marshall, 1976.) The rest of the worms were not fed but it is possible thy could have eaten plankton and diatoms which came in with the unfiltered running seawater. Four groups of worms were divided into anterior and posterior portions according to three plans. In the first two groups, worms were cut transversely in either the anterior region, middle region, or posterior region. The worms in the third group were cut at levels just posterior to 20%, 40%, 60% or 80% of their total number of setigerous segments. Worms in the fourth group were cut just after segment 15, 30, 45, or 60. This cutting strategy yielded 63 anterior and 63 posterior pieces of worms in assorted sizes. The groups were cut at inter¬ vals of one or two weeks so that, at any one time, several stages of regeneration would be observable. Table 1 shows the sizes of the resulting pieces of worms, the day on which they were cut (after "day zero"), and the length of time they survived out of a total of 41 days the the experiment was run. Six worms from the first series of cuts are not included in the calculations due to a lack of accurate data. The worms were observed daily under a dissecting microscope. Data were recorded on each worm, noting the stages to which the worm had progressed. These data appear in Tables 2A, 2B, and Figure 1. C Regeneration in Dorvillea Cowan TABLE 1: Sizes of cut worms used in all experiments. "Day cut" refers tohe the number of days after "day zero" (April 19, 1976). "Time lived" refers to days lived after cutting. All worms in which the sum of "day cut" and "time lived" is equal to 41 were alive at the end of the experiments. 0 Regeneration in Dorvillea ANTERIOR PIECES (HEAD PRESENT) No. of Time setigerous Day cut lived segments (days) Cowan TABLE 1 POSTERIOR PIECES (PYGIDIUM PRESENT) Time No. of lived setigerou: segments (days) 14 15 15 48 Regen ration in Dorvillea ANTERIOR PIECES (HEAD PRESENT) No. of Time Day cut setigero lived segments (days 60 (co Cowan -7 nued) — — POSTERIOR PIECES (PYGIDIUM PRESENT) No. of Time lived tigero Day cut segments (days) 50 18 Cowar Regeneration in Dorvillea TABLE 24: Probability that worms in each stage will achieve full posterior regeneration. Calculations are based on the percentage of all worms entering each stage that lived to the end of the study and thus would probably have achieved full regeneration. BLE 2B: Probability that worms in each stage will achieve full anterior regeneration. - 8 Regeneration in Dorvillea TABLE 24 STAGES NO. WORMS THAT 51 34 26 REGENERATED T0 EACH STAGE NO. WORMS AT OR BEYONL 20 20 20 EACH STAGE AND ALIVE AT END OF STUDV % OF WORMS WHICH 17% 5 9% 35% COULD REGENERATE FROM EACH STAGE TABLE 28 STAGES NO. WORMS THAT 5 7 32 REGENERATE T0 EACH STAGE NO. WORMS ATOR BEYOND 14 14 EACH STAGE AND ALIVE AT END OF STUDY % OF WORMS WHICH 25% 44% COULD REGENERATE FROM EACH STAGE 22 19 86% 17 14 82% 20 19 95% 14 13 93% 12 11 91% 100% Cowan -9 2 100% 100% 100% Regeneration in Dormillea FIGURE 1: Probability of full anterior and posterior regeneration. Data are from tables 2A and 2B. o--posterior regeneration x--anterior regeneration 100 90 80 10 60 50 40 30 20 10 g OPENING T0 GUT APPEARS STAGES 0 Regeneration in Dorville FIGURE 1 Cowan - Regeneration in Dorvillea Cowan -12 OSTERIOR REGENERATION--Results The process of posterior regeneration was divided into stages marked by distinct morphological events. A description of each stage follows, and sketches are shown on Figure 2. Stage 1. A piece of the orange gut protruded from the cut end; the worms otherwise appeared and behaved normally. The last segment before the cut was constricted, thereby closing down the area of the wound and giving the end a "gathered" appearance. Also because of the constriction the parapodia on the last segment were rotated toward the rear till they occupied the position of the anal cirri which had been removed. tage 2. This stage was marked by a healing of the cut resulting in a flat, colorless posterior end with no gut opening visible. In some cases, food material present in the gut was eliminated by rupture through the lateral body wall; those individuals in which this occurred died within a few days. tage 3. This stage was characterized by the formation of a red pigmented bump on the healed end. Stage 4. The formation of an anus on the dorsalside of the bump usually characterized this stage. Stage 5. Onset of the fifth stage was marked bythe appearance of anal cirri at the posterior end. Two of these colorless projections formed ventrally and two dorsally on the pygidium. c egeneration in Dorvillea Cowan -3 JURE 2: Stages of posterior regener- ation. Stages 1-5 are shown in posterior view. Stages 6-8 appear in dorsal view. Regener STAGE 1 STAGE 3 STAGE 5 ( STAGE 2 STAGE 4 sle figure 2 Regeneration in Dorvillea Cowan -I5 Stage 6. The first clear appearance of a new segment at the anterior end of the regenerating tail marked the beginning of stage six. The new segment was formed posterior to the last original segment, and remained separated from the column of tissue which was externally unsegmented, although sometimes small transverse wrinkles in the cuticle could be seen. Red pigment was found along the dorsal side of most of the new tail, with the greatest concen- tration occurring farthest from the pygidium. Stage 7. This stage was defined by the appearance of parapodia on the first new segment. The segments in the column of tissue between the new segment with parapodia and the pygidium were distinct, and the amount of pigment in the whole new tail increased. Stage 8. In the eighth stage, parapodia were present on all new segments. The characteristic pattern of two transverse red bands per segment appeared on the first several new tail segments while those segments nearest the pygidium appeared solid red. Additional segments were generated immediately anterior to the pygidium. The total mean time to complete posterior regeneration through stage eight was 26.2 days. POS ERIOR REGENERATION--Discussion Some of the physical changes accompanying regeneration warrant special attention. In Stage 1, the last segment constricts, closing down the area of the wound, and in so doing the parapodia are rotated toward the rear, ending up in the orientation of the original anal cirri. Based Cowan -16 Regeneration in Dorvillea on observation of living animals it appears that they temporarily serve the function of those cirri, indicating to the worm whether or not something is behind it when it walks backwards, as it often does. Also deserving of comment is the origin of the tissues which make up the regenerated parts. According to the results of Hill (1969), the regeneration blastema most likely originates from differentiated tissues adjacent to the wound. These tissues representing cells derived from all three germ layers, de-differentiate from their previous state and then re-differentiate into the cell types appro- priate to reconstructing the missing parts. The sequence of events represented by Stages 1-8 was followed in a majority of cases. However, some vari¬ ation was noted, particularly with respect to Stage 3. In 12 out of 23 cases, a bump characterized by pigmentation and lacking an anal opening, formed on the end of the healed segment as outlined. However, in seven cases, following Stage 2 an opening was present on the bump as soon as it appeared at the beginning of Stage 3. And in four instances an opening formed on the end of the about last segment at"the end of Stage 2, where little or no bump at all was present. It seems that the appearance of an anal opening to the gut is an important step toward full regeneration. At this point the posterior end of the gut is functional, although not morphologically complete, Regeneration in Dorvillea Cowan -17 and the worm now has a way to defacate. This markedly increases a worm's chances of survival and full regeneration, as evidenced by the data in Table 2A and Figure 1. ERIOR REGENERATION--Rates All regenerating animals were examined daily five to seven days a week and rated as to stage, so the length of time each animal spent at each stage was a matter of record. The duration of each stage in posterior regeneration for the population of experimental animals proved highly variable (Table 3), but this variation involved only a few individuals (Figure 3). ANTERIOR REGENERATION--Results The process of anterior regeneration (formation of a new head end) was also divided into stages defined by specific events. The stages recognized are outlined below and sketches are shown in Figure 1. Stages 1 and 2. The first two stages of anterior regeneration were morphologicall similar to those recog- nized in posterior regeneration, the differences being behavioral; Stage 1 was characterized by an open wound revealing tissues constricted around the orange gut; in Stage 2 the wound was healed. The hind pieces of cut worms retained their polarity in locomotion (they crawled with their anterior ends first), and they consistently righted themselves when turned on their dorsal sides. 0 Cowan Regeneration in Dorvillea TABLE 3: Duration of each stage in posterior regeneration TABLE 4: Duration of each stage in anteri- or regeneration. Regeneration in Dorvillea TABLE 3 DURATION (in days) STAGES MEAN MIN MAX 0.5* 1 3.7 9 4.3 2 9 2 O.5 * 1.7 8 O.5* 3 1.5 5 12 3.7 O.5 * 6 1.3 2 5.0 8 2 8 5 5.0 5 TABLE 4 DURATION (in days) STAGES MEAN MIN MAX 7 1 2.6 7.3 15 2 3.5 0.5 9 0.5 * 3 4 1.8 0.5 * 5 2.3 6 no data available * O.5 USED FOR VALUES OF LESS THAN ONE DAY Cowan -19 NO. OF WORMS IN SAMPLE 27 27 23 22 10 2 1 NO.OF WORMS IN SAMPLE 29 15 12 2 Regeneration in Dorvillea FIGURE 3: This chart illustrates variation in the duration of the different developmental stages in posterior regeneration. Cowan -20 10 Cowan -21 Regeneration in Dorvillea figure 3 H I ++ ++++ + ++ + +++ ++ — 1+ + + +++ + +++ +— + + +++ — + — — +++ +++ 1++ + + + ++ + +++ +++ ++ 1 + +++ P — ++ + ++ ++ + + ++ + ++ + ++ + ++++ + +++ + + + + + + +++ + +++ + +I +++++ + + +++ + + ++ ++ +++ ++ + t + ++ +++ + + ++++ + + + ++ ++ +++ + +++ + — + + +++ E E + + t +++ ++ +++++ ++ ++ +++ + — + ++ ++ + + + + + + l ++ ++ + — + ++ + ++ + + +++++ + +++ ++ + + ++ ++ +++ +++ + + t — ++ +++ 20 DAYS 41 1 0 neration in Dorvillea URE 4: Stages of anterior regenera- tion. Stages 1-3 are shown in anterior views; Stages 4-6 are shown in lateral views. Stage 6 is cut away on the side to show the position of the developing jaws. Cowan -22 N STAGE 1 STAGE 3 STAGE STAGE 2 STAGE 4 STAGE 6 igur e Regeneration in Dorvillea Cowan However, they were generally much less active than the anterior pieces. The parapodia of the segment just back of the cut were rotated to the front by the constriction of the anterior segment and occupied somewhat the posi¬ tions of palps and antennae. Stage 3. In the third stage of regeneration a re- tractable bulge appeared on the healed surface. As in the original prostomium, there was no pigmentation. The worms jerked back quickly when poked in this region, and the level of activity observed in the worms increased at this atage. Stage 4. The distinctive feature of this stage was an anterior opening to the gut, a mouth, formed in the lower portion of the bulge. When the bulge was extended, the opening was at the tip. At this stage, the worm regained its normal level of activity and resumed the characteristic behavior of an entire worm. Worms in this stage were often observed to drag the bulge, fully extended and pointed downward, along the bottom of the fingerbowl. One gained the impression that feeding could be taking place. Stage 5. This stage was defined by the appearance of an invagination formed in the ventral half of the bulge at the opening to the gut. At this stage the bulge was large enough so that the parapodia resumed their original orientation and the "gathered"appearance disappeared. tage 6. This stage was characterized by the forma¬ tion of rudimentary jaws posterior to the opening. The -24 Regeneration in Dorvillea Cowan -25 red-brown cylindrical jaw buds moved in and out of the small mouth opening in the same manner as the fully developed jaws in a normal worm. Furthur stages of prostomial development were not available for observation at 34 days. The total mean time for regeneration up to Stage 6 was 19.4 days. ERIOR RFCENET TION--Discussion EGENEF As in posterior regeneration, it seems that the para¬ podia on the last segment anterior to the cut, temporarily serve the function of the missing appendages. In this case, they are oriented in the former positions of the palps and antennae, and apparently serve as sensory appendages. In support of this statement, these parapodia were out of synchrony with the rest of the parapodia while they occupied the anterior position. The appearance of the opening in the head bulge presented an interesting possibility. It seemed that with this development the worm was able to ingest small food particles. Although worms were never directly observed taking in particles, they exhibited apparent feeding behavior by dragging the bulge, pointed downward and fully extended, along the bottom of the fingerbowl. The increased probability of regeneration after this stage supports the possibility that the worms were no longer relying on reserves, but that they were capable of ingesting food. (Table 2B and Figure 1.) C Regeneration in Dorvillea Cowan -26 REGENERATION--Rate ANTERIOR I As in the case of posterior regeneration, daily observations of the stage of development of each regen¬ erating fragment allowed calculation of rates of anterior regeneration. Means and ranges for the duration of each stage appear in Table 4, and variation appears in Figure 5. RELATION BETWEEN REGENERATION RATE AND NUMBER OE SEGMENTS It seemed possible that capacity to regenerate and speed of regeneration might be related to the size of the regenerating fragment. With this in mind data were analyzed to determine the duration of Stage 1 in relation to fragment size. Data are shown in Table 5. The results in Table 5 suggest an inverse correlation between the number of segments in a piece of worm and the time it takes for regeneration to occur. Worms with 1-20 segments developed significantly more slowly (p..05) than those with 45 or more segments, for the first stage of regeneration. The relationship of the subsequent stages could not be deduced because of the large die-off of worms containing 1-20 segments. A determination of the limiting number of segments necessary for regeneration to occur was not the objective of this study, although results bearing on this question were obtained. At the time of writing, posterior regenera¬ tion was observed through the eighth stage in a worm containing the head and only nine setigerous segments. Cowan- 27 n in Dorvillea JURE 5: This chart illustrates variation in the duration of the different developmental stages in anterior regeneration. 10 41 Cowan- 28 tion in Dorvillea Regenerat figure 5 — H I +t + ++ + — ++++ ++++ +++ + ++++ + ++ + ttt +++ ++ +t + 1 ++ t +t — ++ ++ + ++++++ ++ +t + ++++ + +++ t + + tt ++++ ++ t E ++ +++ +++ tt +++ ++ +++ + + +++ +++++ ++ —— + + +++ ++++ + + 1 + +++ ++ t ++++ + ++++ ++ + + + + + ++ + + + + t + ttt ++ pt ++ + ++ +++ +++ + + +++ ++ + ++ + 1 ++ + + ++ + + + + t + ++ + + — — +++++ + ++++ ++ + ++++ — + +t + + ++ t tt + +++ +++ pt + ++ + ++ + +++ + lttttttt —+++ + ++ ++ +++ + l + + ++++ ++ t ++++ + +++ ++ +++ ++ + ++ +++ +++ — + +++ ++ ++ +++ + + + + + 1 ++ ++ ++ + lt + ++++++ + t ttt +++ —+ ++ ++ + ++++ ++ +++ ++ + +++ ++ +t 1++ +++ ++ + + +++ +++++ +++ +++ + + ++ + + + ++++ + ++t + ++++ +++ t ++ +++ lt t +++ ++ DAYS Regeneration in Dorvillea TABLE 5: Correlation between number of segments and the time necessary for posterior regeneration through Stage 1. Cowan -29 Regeneration in Dorvillea table 5 no. of segments in 1-20 anterior piece ofworn no. of worms duration of mean 5.7 stage 1 0.5 -10 range (days 21-40 8 3.3 0.5-7 Cowan -30 41-n 11 3 1-9 Regeneration in Dorvillea Cowan -31 Anterior regeneration through stage four occurred in a worm containing only 19 segments. A middle section of worm containing 38 segments had, at the time of writing, regenerated a pygidium, one new segment, and an opening to the gut on the prostomial bulge in a period of 34 days. DISCUSSION It seems possible that the rate-size correlation is maintained throughout the regeneration process. Data are sparse, but two individuals, 9 and 25 segments long, that were cut on the same day had, at the time of writing regenerated 6 and 11 segments,respectively. EVIDENCE OE INJURY AND REGENERATION IN A NATURAL POPULATION The preceeding study indicates that D. moniloceras regenerates quite effectively. A survey of a natural population of 103 worms revealed that 13 of them (12.6%) were regenerating or showed signs of previous damage and repair. Six worms had regenerated a pygidium, two were regenerating a palp, two were regenerating an antenna, and threehad irregular body markings which suggested the possibility of previous regeneration. SUMMARY Regeneration, both posterior and anterior, are 1) observed in D. moniloceras. 2) The continuous process of regeneration, both anterior and posterior, can be broken up into various Regeneration in Dorvillea Cowan -32 changes in stages, marked byAthe appearance of the cut end. Follow- ing are the stages recognized in posterior regeneration: 1) wound open; 2) wound healed, but flat; 3)central protrusion, pigmented; 4) anal aperture forms; 5) anal cirri appear; 6) new segment delimited; 7) parapodia form on new segment, other smaller segments form; 8) para- podia on all new segments. Somewhat similar stages can be recognized in anterior regeneration: 1) wound open; 2)wound healed; 3) non-pigmented bulge forms on cut end; 4) mouth opening forms in bulge; 5) invagination in bulge forms at opening; 6) jaws appear. 3) Posterior regeneration through Stage 8 occurred in a worm with a head and 9 chaetigers. Anterior regener¬ ation to Stage 4 was observed in a worm with 19 segments and a pygidium. A midsection of a worm 38 segments long regenerated a pygidium and one new segment posteriorly, and a beginninghof a new prostomium anteriorly. 4) An inverse correlation seems to exist between the number of segments in a piece of worm and the length of time necessary for regeneration. Regeneration in Dorvillea Proper acknowledgements should go to Dr. Donald P. Abbott, without whose help this paper would still be a rough draft, and to Mr. Chuck Baxter for his sympathy on the second day when I had still seen no sign of regeneration. Cowan -33 Regeneration in Dorvillea Cowan -34 1) Berrill, N.J. 1928. Regeneration in the polychaete Chaetopterus variopedatus. J. Mar. Biol. Ass. U.K. 15: 151-158. Berrill, N.J. 1931. Regenerationin Sabella pavonina 2) (Sav.) and other sabellid worms. J. Exp. Zool. 58: 195-523. Berrill, N.J. and D. Mees 1936. Reorganization and 3) regeneration in Sabella. I. Nature of gradient, summation, and posterior reorganization. J. Exp. Zool. 73: 67-83. 4) Berrill, N.J. 1951. Regeneration and budding in worms. Biol. Rev. 27: 401-438. 5) Faulkner, G.H. 1932. The histology of posterior regeneration in the polychaete Chaetopterus variopedatus. J. Morphol. 53: 23-58. Fitzharris, T.P. 1973. Control mechanisms in regenera¬ 6) tion and expression of polarity. Amer. Scientist 61: 156-462. Hill, S.D. 1969. Origin of the regeneration blastema 7) in polychaete annelids. Am. Zool. 10: 101-112.