INTRODUCTION Idotea resecata is a marine isopod found in fairly quiet bay waters along the California coast. Though much work has been done on the appendages for the purpose of taxonomy (e.g. Menzies, 1950), search of the literature reveals little on the functions of the appendages in Idotea. The more ob- vious functions such as the use of the peraeopods for loco¬ motion have been assigned to the appendages (Schultz, 1969). but details of the activities have not been described in Idotea with the exception of feeding activity (Green, 1957 and Cruz, 1963). In the kelp beds adjacent to Mussel Point, Pacific Grove, numerous individuals were found which were missing and regenerating appendages. Regeneration of the appendages in Idotea has been described by several investigators (Boussat, 1958; Varèse, 1960; Glaizal, 1962). However, nothing has been mentioned about compensatory behavior following amputation of an appendage. I decided to examine function of selected appendages in intact animals and in animals with selected appendages removed. Observations on regeneration were recorded as opportunity arose during the study. I. resecata is a very hardy animal, survives well in the lab and is very suitable for studies on appendage utilization and regeneration. The appendages studied were the first and second antennae, the peracopods, the uropods and the pleopods. For this investigation, the peraeopods were considered to fall into groups on the basis of orientation and function: group one includes only the first peraeopods; group two includes the second, third and fourth peraeopods group three includes the last three pairs of peraeopods. MATERIALS AND METHOD Animals to be used for laboratory studies were collected on Macrocystis and maintained on plants held in running sea water aquaria at the Hopkins Marine Station of Stanford Uni- versity. Studies of swimming and of activity on kelp were made in large aquaria. Ventrally placed structures were viewed from below in a glass tube of 1.5 cm in diameter and 1 m in length. Activity of animals on kelp blades was studied under a dissecting microscope as well as in large tanks. Removal of the appendages was accomplished by methods described by Schneiderman (1967), using fine forceps and very sharp manicuring scissors. Animals were first lightly anes- thetized using a solution of MgCl, isotonic with sea water. Their bodies were wrapped in damp cheesecloth to limit movement, leaving only the area to be operated on exposed. All operations were performed under a dissecting microscope. For most appendages studied other than peraeopods (e.g., the first antennae, the second antennae, etc.) the appendage was removed unilaterally in some specimens, bilaterally in others. Three animals were operated on for each condition. For peraeopod groups two and three, either one, two, or all three pairs of appendages were removed. Here, too, operations were performed on three animals for each condition. After surgery, animals were kept in 5 inch finger bowls. three animals to a bowl. Bowls were provided with running sea water at 13-14° C. and covered with cheesecloth to pre- vent the animals from escaping. Macrocystis was added to the bowls for food. Bowls were cleaned out every two or three days to remove wastes and replenish the food supply. UTILIZATION OF APPENDAGES The First Antennae The first antennae are difficult to study because of their small size and rather rapid movements in the normal animal. They are used in chemoreoption and contain aesthetascs on the outer segment which are the sensory structures (Schultz, 1969). Aesthetascs face forward amd the two appendages are moved alternately. In those animals with only one first antenna removed, no change in behavior was noted. Removing both first anten- nae resulted in a decrease in locomotary activity; the animals remained in a stationary position and were non reactive to prodding by the experimenter. The. Second Antennae The second antennae have five peduncular segments and a number of segments in the flagellum which increases in di- rect proportion to the size of the animal (Menzies, 1948). In adult animals 20-25 mm long the number of flagellar seg- ments ranged from 14 to 16. In animals 9-15 mm long there were 8-10 segments. According to Schultz (1969) the second antennae are mainly tactile sensory structures. My own observations affirm they are primarily used for probing the environment. The segments of the peduncle are bent easily; notches in the margins of segments allow for increased movement between adjoining seg- ments (fig. 17). The flagellum, being composed of many seg- ments, has great flexibility which enables the antennae to explore the environment very efficiently. Physical inter- faces, e.g. the air-water interface, are detected by the second antennae. When the interface is contacted, the animal alters its behavior, i.e. in the case of a swimming isopod, the animal changes direction (fig. 1). When the animal is stationary on a kelp leaf, the anten- nae are held in a forward position but angled approximately 40° from the anterior-posterior axis. At rest on the stipe of Macrocystis the second antennae straddle the tubular stipe for balance or for gripping the stalk (fig. 2). When the animal is walking on kelp, the antennae swing about irregularly in an arc (fig. 34). There is no set pattern relating the behavior of the antennae to one another in the probing, but they never contact each other. An animal about to go around the edge of a kelp blade from the upper to lower surface, first bends the antennae to probe the under surface (fig. 4); following this it may move. In swimming, the anten- nae are extended straight forward, allowing for smoother fluid flow about the body. Sometimes the extreme outer segments of the flagellum are pointed outward. The animal may be sonsing the presence of a solid surface and is poised for contact (fig. 5). The second antennae are fastidiously groomed by the first peraeopods (fig. 6). Material on the antennae of I. resecata living on Macrocystis consists of mucus from the kelp and adhering debris. These appear to be acceptable food sub- stances, and the second antennae thus play a role in feeding by serving as collecting devices. Both antennae are regularly cleaned and the material is brought to the mouth parts. Fur- ther details are discussed in relation to the first peraeopods. A specific aspect of behavior was seen in a laboratory aquarium which indicates the second antennae may participate actively in another way in bringing detritus to the mouth parts. Much detritus, mainly Macrocystis debris, had accum- ulated on the bottom of the tank. On several occasions when an I. resecata came to rest on the bottom in the detritus, the second antennae which weré initially extended were pulled in toward the cephalon as in grooming. A mound of debris was accumulated in one stroke, and the first peraeopods grabbed portions of this and transported it to the mouth parts. The second antennae serve as a pivot point for the body in certain situations as in righting. An isopod lying on its back arches its body until only the telson and the tips of the second antennae remain in contact with the substratum. The arched position attained is unstable and usually the animal is able to flip over to one side and right itself (fig. 7). The second antennae also act as a pivot point when an animal alights on a stipe of kelp. As the animal swims the second antennae are extended anteriorly. Upon contacting a solid surface the antennae are spread apart and provide a pivot point as the anterior part of the body swings toward the surface. The first three peraeopods are used to grab the substratum and establish a hold (fig. 8). Removal of one antenna affects the behavior of the ani- mal but not drastically. Interfaces are still detected. In walking, the animal compensates by increasing the arc through which the remaining antenna is swung (fig. 3B). In swimming, animals with one antenna missing swim in a curving course toward the side of the remaining antenna in 80% of the cases observed. The intact antenna and the stump are groomed. In righting themselves the animals arched their bodies and flipped over to the side lacking the antenna. Removal of both antennae had more noticeable effects. In swimming, the air-water interface was not perceived until the cephalon of the isopod was almost entirely out of the vater. There is less variation of swimming patterns in these animals. They tended to swim in straight lines, exhibitihg little maneuverability. Righting was difficult to achieve. The Peraeopods Each peraeopod is composed of six segments. All per- aeopods are subchelate and biunguilate. Except for the first peraeopods, they are mainly locomotary and clinging appendages and the biunguilate condition is a modification for walking. The first peraeopods are used more like hands, and are closely associated with the mouth parts. The number of setae decreases as one goes from the first to the seventh peraeopods. The first peraeopod has three main clusters of setae, each centered around a large file seta. The second, third, and fourth peraeopods each have two clusters of setae and the last three pairs of legs have only one main cluster. As mentioned previously; the first peraeopods are used in grooming the second antennae (fig. 6). The animal places one of its antennae betwéen the dactylus and the propodus of the two first peraeopods and then pulls the antenna through from base to tip. The three large file setae and their clusters may be acting as food strainers. Mucus and debris are scraped off and brought to the mouth parts where the material is mas- ticated. This behavior may be regular, with the animal repeatedly cleaning off first one antenna and then the other. If the food is rejected by the animal the material is removed from the mouth parts by the first peraeopods. The first peraeopods also actually hold chunks of food for the mouth parts to 10 chew on. If one of the first peraeopods is removed, the second peraeopod on the same side takes over the job of groom- ing the antennae, though this is done somewhat inefficiently, often leaving the outer segments of the flagellum untouched. Removing both first peraeopods is compensated by the second peraeopod pair taking over the role of the first. The second peraeopods are well endowed with setae and are used in grooming the first peraeopods when the latter are burdened with debris. In an animal missing one of the second peraeopods, e.g. the left, the animal compensates by bringing the left first peraeopod to the right second peraeopod to be cleaned. The second, third, and fourth peraeopods are grouped to- ion. The gether in part because of their anter other basis faces forward in the restin segments are bent at an angle towards the surface of the kelp. The fifth, sixth, and seventh peraeopods, on the other hand, are oriented posteriorly. The basis faces toward the rear and the other segments are usually aligned with the basis in a resting animal. On a stipe of Macrocystis the fifth through the seventh peraeopods are used mainly for gripping the stipe. Often the fourth pair joins the other three pairs. This leaves the first three peraeopods free to groom and carry out feeding activity. This separation of function brings to mind the more extreme case of the arcturid isopods. 11 The second through the fifth peraeopods are the major ambulatory legs. Locomotion is ditaxic with the body remaining fairly rigid (fig. 9). The unguis is used to establish anchorage in ambulatory movement. If the unguii are removed from all of the peraeopods the animal cannot carry out nor- mal walking, and only inefficient slow gliding movement re- sults. While the animal is walking, the first peraeopods are extended in quick, alternating movements in front of the body, acting like probes. The sixth and seventh peraeopods are normally inactive in slow walking. Their activity increases with the speed of movement. Monotaxic movement is sometimes exhibited by the appendages, with the frequency of this in- creasing as one goes from the seventh to second peraeopods. The first legs have never been observed moving monotaxically. In backward motion the sixth and seventh peraeopods play more of a role, moving ditaxically; their posterior orientation The animal also employs allows this to be done efficiently. these two pairs of legs extensively in turning around. In fast swimming the peraeopods are all pulled in close to the body giving a streamlining effect (fig. 10). The first three pairs are oriented forward, dactylus out, poised for grasping. The fourth through the seventh legs are oriented posteriorly with the fourth pair bent at the basal-ischium joint and the last three pairs aligned straight back. To slow forward movement in swimming the last three peraeopods are extended laterally. Similarly in slow swimming the last three pairs of peraeopods are extended laterally perpendicular to the body where they act as water brakes, disrupting the flow of water about the body (fig. 11). The braking phenomenon is also observed when an isopod falls freely through the water. This was seen in isopods placed in the glass observation tube and also in aquaria. The animals are not long term swimmers and after a few swimming excursions they appear to tire. When a tired animal is placed in the water it falls, making no effort to swim, but the fall is retarded, and balance is maintained, by the lateral perpendicular exten- sion of all the peraeopods except for the first pair (fig. 12). Once the animal has rested, swimming my be resumed again. The first, second, and third peraeopods are used in a swimming animal to grab an algal stipe when the animal initially contacts it. As soon as the hold is established, resulting in a stable position with the last four peraeopods firmly gripping the stipe, the first peraeopods are pulled up to assume the role of aiding the mouth parts once more. Removal of the first peraeopods does not affect ambulatory motion. When the second peraeopods are removed, the animal compensates by using the first peraeopods as locomotary ap- pendages. The first peraeopods become much more important for locomotion if peraeopods two and three, or two to four inclusive, are removed. The animal also compensates for lack 13 of support in the peraeon segments by arching its body. In w-alking there is increased pleopod action to give a forward thrust. The animals have much difficulty in moving around the edge of a kelp blade from the upper surface to the under- side; the anterior appendages are clearly needed to establish the initial hold. Removal of the fourth or the fifth peraeopod results in difficulty as far as turning around on a stipe is concerned. These center peraeopods are used as pivot points in an intact animal, and the animal must compensate for their absence by moving the pivot point forward or backward to a less favorable location on the body. Removal of two of the last three pairs of legs impairs gripping of the substratum, but the animal can still hold on reasonably well with just the seventh pair. If the last three pairs are removed the animal has difficulty in holding onto a stipe or blade of kelp. In swimming, the animal compensates for the loss of the last three pairs of peraeopods by having the fourth peraeopods assume the role of a water brake (fig. 13). One peculiar use of the fifth, sixth, and seventh per- aeopods is their role in the removal of the anterior molt. When an isopod molts, the posterior half of the exoskeleton is molted first; then after a period of approximately 24 hours the anterior molt is discarded. In removing the pos- 14 terior shell the animal merely attaches to the kelp with the last three pairs of legs and then pulls itself out of the old exoskeleton, segment by segment, in a series of undulating, worm-like movements. Removal of the anterior shell is quite different. The animal does not attach its anterior legs and then walk backward out of its shell using the last three pairs of legs. Instead, it bends its body sharply ventrally and then the last three peraeopods grasp the anterior exo- skeleton and are used to pull it off. This may demonstrate the animal's tendencies always to move forward in locomotion and also the importance of the second through the fourth per- aeopods in accomplishing any ambulatory locomotion. Surgical removal of the last three peraeopods causes the animal great difficulty in removing the anterior molt. One such animal took 6 hours to remove the anterior shell, whereas normally it only takes 15 minutes. The Uropods Each uropod consists of two recognizable segments, a proximal protopodite and a distal endopodite. The uropods form valves covering the ventral side of the branchial chamber. They serve to protect the pleopods, which are very delicate structures and, in addition, they direct water flow through the pleotelson. Proper regulation of water flow in the branchial chamber increases the efficiency of the pleopods in respiration and 15 swimming (fig. 14). Water currents are drawn into the chamber when the pleopods are raised and water is forced out when they are lowered. The uropods can regulate the aperture through which the stream of water flows, thus controlling the speed of locomotion by the Venturi principle. The endopodite of the uropod is free to move separately from the protopodite and thus is able to narrow or widen the posterior aperture of the pleopod chamber. It is pos- sible that the endopodites help steer the body in swimming by acting as rudders. However, the right and left endopodites have never been observed to move independently of each other in a way suggesting steering control. The animals with one uropod removed always swam on a course curving toward the side with the remaining uropod. The water stream is deflected off the uropod and toward the opposite side, creating a force pushing the animal to the side with the uropod remaining (fig. 15). In animals lacking both uropods, the two rami of each pleopod were more widely separated than usual, so the exopodites extended laterally out of the normal chamber. This resulted in less water being jetted out posteriorly, and the animals without uropods swim more slowly. 16 The Pleopods The pleopods are the five pairs of appendages of the anterior pleonal segments. They are biramous, each pleopod having a free endopodite and exopodite coming off the basis. The second pleopod in the male is sexually modified, having a style. This modification is common to all members of Valvifera (Schultz, 1969). In addition, the first and second pleopods and the exopodite of the third pleopod have hairs around the periphery. This is the case in Sphaeromatids, also (Schultz, 1969). Size of the pleopod increases going from the first to the fifth pair and the length of the hairs decreases in going from the first to the third pair. The presence of the hairs suggests that the first and second pleopods are natatory. The hairs themselves are na- tatory in structure. In an individual hair, there is one main root with small bristles branching off from it (fig. 16). The fourth and fifth pleopods lack hairs and have very thin membranes, suggesting that they play more of a role in respir- ation than locomotion. Animals lacking the first and second pleopods are not able to swim. The remaining pleopods beat but no motion is generated. Animals missing the fourth and fifth pleopods are able to swim but the incidence of death is high (50%). Thus it seems that there are specialized functional modifications in the pleopods of I. resecata. 17 The pleopods also may be associated with circulation. There is a direct correlation between the rate that the pleopods beat and the rate of heart beat. When the heart con- tracts the pleopods are extended ventrally and when the heart expands the pleopods are brought back into the branchial chamber. This implies that the pleopods are either aiding in pumping the blood are moving as a consequence of the ex¬ pansion and contraction of the heart. When the pleopods stop beating, the heart stops momentarily. The heart starts beating again at a constant rate if the pleopods remain stationary. This rate is maintained until the pleopods start beating again. Then the heart beat rate speeds up or slows down to coincide with the pleopod beat rate. The pleopods may recommence beating before the heart does. This suggests that the two structures are not enervated by the same nerve. 18 REGENERATION The sequence of events during regeneration of appendages varies with the type of appendage, the level at which the original appendage was lost, the extent of damage to the tissues remaining within the stump and the stage of the an- imalin the intermolt cycle. Since it was not possible to monitor these factors in the time period of this study, only a general descriptionof the process will be made. Regen- eration was studied up to the third molt for the first antennae and up to the second molt in the other appendages (see fig. 17-36). A typical sequence is given by a description of the events in the regeneration of a peraeopod. The ischium of the peraeopod is the amputation site. Immediately after the operation bleeding occurs. This stops shortly thereafter. A dark green scab is sometimes present. Three to four days later an exoskeletal covering is formed over the wound. The area beneath the wound site is white. After the first molt a stump is formed. It is rounded in appearance, unsegmented and colorless. The second molt produces a peraeopod with the same number of segments as in a normal limb. However, the segmentation is not complete, the joints are not entirely movable and the setae are absent (fig. 31). The regenerate résembles the juvenile peraeopod described by Menzies (1948). Removing a limb may have an effect on the molting cycle of the animal. It was observed that in large animals 25-30 mm long the period between the first and second molts was much shorter than the period between the operation and the first molt. This suggests that amputation alters the molting rhythm. Regeneration was abnormal in the uropods and the pleopods. Both were structurally distorted relative to normal appendages. The uropod had setae on the posterior part of the protopodite and were thickened (fig. 36). This thickening has also been reported in Idotea baltica by Glaizal (1962). The regenerated pleopods differed from the normal paddle shape in having a very irregular surface (fig. 33). 20 SUMMARY 1. The marine isopod Idotea resecata is found in the fairly quiet waters of Monterey Bay living on the brown kelp Macro- cystis. The utilization of selected appendages of I. resecata was studied. The second antennae probe the environment and may be used in feeding in conjunction with the first peraeopods. They act as pivot points in righting and in alighting on kelr while swimming. The first peraeopods are used in grooming the sécond 3. antennae in a manner suggesting a feeding mechanism. They are associated with the mouth parts and are used for locomotion only in compensation behavior. 4. The second, third, and fourth peraeopods are oriented forward. Along with the fifth pair, they are the main ambu- latory legs. The first three peraeopods are used for grabbing kelp 5. when the animal is swimming. 6. The last three peraeopods are used for gripping the substrate and are used as water brakes in swimming behavior. They are also used for removing the anterior molt. The uropods direct water flow through the pleotelson. The pleopods show functional differentiation. The first two pleopods possess hairs and are natatory. The fourth and fifth pleopods are probably used mainly for respiration. 9. The correlation between the rate that the pleopods beat and the rate of heart beat suggests that the pleopods play a role in circulation. 10. Observations on regeneration were done. 0 ACKNOWLEDGEMENTS I would like to thank Dr. Donald P. Abbott for his invaluable guidance on this project. 21 22 LITERATURE CITED Boussat, M. 1958. Territoire de régénération des antennes de l'Isopode Idotea baltica (Aud.). Compt. rend. soc. biol. 246: 2530-2532. Cruz, A. de la. 1963. Observations on the feeding activity of the isopod, Idothea baltica (Pallas). Publ. Seto ma. biol. Lab. 11: 165-170. Glaizal, M. 1962. Sur la régénération de l'uropode du Crustace Idotea baltica (Aud.). C. R. Acad. Sci. Paris. 254: 2452- 2453. Green, J. 1957. The feeding mechanism of Mesidotea entomon (Linn.) (Crustacea: Isopoda). Proc. Zool. Soc. Lond. 129: 245-254 Menzies, R. J. 1950. The taxonomy, ecology and distribution of northern California isopods of the genus Idothea with the description of a new species. Wasmann Jour. Bio. 8(2): 155-195. Menzies, R. J. and Waidzunas, R. J. 1948. Postembryonic growth changes in the isopod, Pentidothea resecata (Stimpson) with remarks on their taxonomic significance. Bio. Bull. 95(1): 107-113. Schneiderman, H. A. 1967. Insect surgery, p. 753 to 765. In Wilt, F. H. and Wessells, N. K. (ed.) Methods in developmental biology. T. Y. Crowell Co., New York. 23 Schultz, G. 1969. How to know the marine isopod crustaceans. Wm. C. Brown Co., Dubuque, Iowa. 359 p. Varèse, J. 1960. Sur la régénération des antennes de l'lsopode Idotea baltica (Aud.). C. R. Acad. Sci. Paris. 250: 3399- 3340. FIGURES Fig, 1: Detection of the air-water interface by the second antennae and consequent change in the direction of swimming. Fig. 2: Position of the second antennae of an isopod resting on an algal stipe. Fig. 3A: Arc of second antennae in walking isopod. Fig. 3B: Arc of single antenna of walking, unilaterally amputated isopod. Fig. 4: Antennae probing the underside of a kelp leaf. Fig. 5: Antennae position of swimming isopod. Fig. 6: Grooming of the second antennae by the first peraeopods. Fig. 7: Righting of isopod lying on its back. Fig, 8: Sequence of movements involved in alighting on stipe. Fig. 9-13: Positioning of peraeopods. Fig. 9: Walking Fig. 10: Fast swimming. Fig. 11: Slow swimming, showing water braking by the last three peraeopods. Fig. 12: Free fall through water. Fig, 13: Slow swimming in isopod missing the last three peraeopods. Fig. 14: Water flow through the pleotelson. Arrows indicate flow. Fig. 15: Water flow through the pleotelson in isopod with only one uropod. Arrows indicate water flow. FIGURES: Fig. 16: Natatory hair of first pleopod. Scale in mm. Fig. 17-19: Regeneration in the second antennae. Scale in mm. Fig. 17: Normal left antenna. Fig. 18: After one molt, 11 days. Amputated at fifth peduncular segment. Fig. 19: After two molts, 20 days. Fig. 20-23: Regeneration in the first antennae. Scale in mm. Fig. 20: Normal left first antenna. Fig. 21: After one molt, 5 days. Amputated at third segment. Fig. 22: After two molts, 26 days. Fig. 23: After three molts, 37 days. Fig. 24-26: Regeneration in the first peraeopods. Scale in mm. Fig. 24: Normal first peraeopod (left). Fig. 25: After one molt, 6 days. Amputated at merus. Fig. 26: After two molts, 21 days. Fig, 27-31: Regeneration of the peraeopod. Scale in mm. Fig. 27: Normal sixth peraeopod. Fig. 28: After one molt, 16 days. Amputated at the merus. Fig. 29: After two molts, 21 days. Fig, 30: After one molt, 16 days. Seventh peraeopod amputated at the ischium. Fig. 31: After two molts, 21 days. Fig. 32-33: Regeneration of the first pleopod. Scale in mm. Fig. 32: Normal pair of first pleopods. Fig. 33: After two molts, 33 days. Amputated at the basis. First molt, after 25 days produced no change. FIGURES Fig. 34-36: Regeneration in the uropods. Scale in mm. Fig. 34: Normal left uropod. Fig. 35: After one molt, 15 days. Right uropod. Essentially unchanged since operation. Fig. 36: After two molts, 31 days. Note two setae. — — — aaa- Ster . .. - - ... 6 . . -*.. —.. - 8.4 A 10 L 12 — — — L 11 L 13 — — — S 1 — — — t f-+ --- +- Ef- —— — — + — 15 v- — . . 1 16 . ke A r 4: . 4 4 2 18 32 21 27 Ar 33 23 28 30 36 ..... ... 29 31 .