Ecological Studies on the Associations of Chitons in Puerto Rico, with Special Reference to Sphaeromid Isopods

ECOLOGICAL STUDIES ON THE ASSOCIATIONS OF CHITONS IN PUERTO RICO, WITH SPECIAL REFERENCE TO SPHAEROMID ISOPODS

PETER W. GLYNN Institute of Marine Biology, University of Puerto Rico, Mayagiiez, Puerto Rico

ABSTRACT Five symbiotic isopod-chiton relationships are described, viz., Dyname- nella perforata with Acanthopleura granulata and Chiton tuberculatus, Exosphaeroma (a) with C. tuberculatus, Exosphaeroma (b) with C. marmoratus, and Dynamenopsis dianae with C. tuberculatus. The inhalant chamber of the pallial groove was the site most often frequented by the isopods. Other chiton symbionts described are "Acervulina inhaerens" (Foraminiferida), Harpacticus sp. and Heterolaophonte (Harpacticoida), Parhyale hawaiensis (Amphipoda), and Actaletes neptuni (Collembola). Dynamene moorei is shown to be conspecific with Dynamenella perforata. A widespread occurrence of the Dynamenella-Acanthopleura partnership is indicated by records from Jamaica, Cuba, Florida, the Bahama Islands, and Barbados. It is shown that Dynamenella occurs commonly with chitons living near patches of coarse-grained sediment; the overall character of the habitat and the degree of exposure to wave action do not seem to influence the occurrence of the partnership. A more intimate association of Dyname- nella with Acanthopleura is apparently due to the higher level occupied on the shore by the host and to its more sluggish behavior. The symbiont demonstrated a high tolerance to desiccation under chitons exposed to the atmosphere. Dynamenella orients between individual gill filaments as the chiton browses and it appears that the algal material ingested by the inquiline is initially scraped from the substratum by the host. The isopod is regarded as a facultative commensal with a strong attraction for Acantho- pleura.

INTRODUCTION Monthly collections of the two chitons, Acanthopleura granulata Gme1in, 1791 and Chiton tuberculatus Linnaeus, 1758, carried out over a two-year period (Nov., 1960-Nov., 1962) near La Parguera, Puerto Rico, in order to assess the seasonal breeding condition, consistently revealed an apparent intimate association of sphaeromid isopods with Acanthopleura. At the collecting station on Turrumote Reef, the presence of numerous isopods under the girdle and along the pallial grooves of Acanthopleura was the rule, whereas C. tuberculatus was usually devoid of the isopods. Initially these casual observations evoked two basic questions, namely, why do the isopods live with the chitons, and why do they live preferentially with Acanthopleura rather than with Chiton? Investigation of these questions led to observations and experiments in connection with (a) determination 1968] Glynn: Studies on the Associations of Chitons 573 of the usual species of isopod living with Acanthopleura, (b) description of the association, (c) its occurrence around Puerto Rico and elsewhere in the tropical western Atlantic, (d) determination of other isopod species found with different chitons in the intertidal zone, and (e) influence on the association of environmental and biological factors. Although main consideration is given to the association of Dynamenella perforata (Moore, 1901) with Acanthopleura and C. tuberculatus, three additional species of sphaeromid isopods which live on the undersurface of other chitons are reported. Two of the isopods, Exosphaeroma (a) and (b), are undescribed; their systematic status and ecology will form the subject of a future study. Dynamenopsis dianae Menzies, 1962, was found with C. tuberculatus at only one locality. The ability of the Isopoda to invade diverse environments and the propensity of many forms to occuPy bizarre niches are very much evident within the group. Numerous species have been described from terrestrial, freshwater, and marine habitats, and the success of the Sphaeromidae in the latter two realms is clearly evident. In a study of the littoral marine Sphaeromidae of Brazil, Loyola e Silva (1960) found that mesohaline or brackish conditions are tolerated by five species along the southern coastal belt in this region. One of the species studied, Pseudosphaeroma jakobii, was observed to survive for relatively long periods out of water. The genus Gnorimosphaeroma contains species which live in sea water or in fresh water (Menzies, 1954). Richardson (1904) reported that the number of freshwater Sphaeromidae is large for a marine family. Some of the more specialized niches and the particular species involved are the following: warm spring in New Mexico (Sphaeroma dugesi, S. thermophilum), swamp (S. fossarum), subterranean waters of grottos or caves (Sphaeromides raymondi, Caecosphaeroma virei, C. burgundum). In general, the sphaeromids also exhibit a tendency to occupy secretive or concealed dwellings. Zimmer (1927) noted that some species hide between and under stones, in empty cups of dead Balanus, and in various sponges; certain species of Sphaeroma bore into wood, and other forms even bore into soft stones (tuff and sandstone). In San Francisco Bay, California, the boring activity of Sphaeroma pentodon has resulted in a conspicuous pitting of some rocky surfaces along the shore (Barrows, 1919). Rhizophora, the mangrove tree, represents a natural wood substratum often infected with certain brackish-water species of Sphaeroma and Pseudosphaeroma (Loyola e Silva, 1960). While instances of parasitism within the Isopoda have been well docu- mented, particularly among members of the Epicaridea (Balss, 1956), pertinent review articles and general accounts of the group contain very little information on commensal relationships (Gerstaecker, 1881; Smith, 1909; Caiman, 1909, 1911; Dales, 1957; Patton, in press). Actually, 574 Bulletin ot Marine Science [18(3 ) numerous commensal isopods are known, but the majority of them have been described separately and appear in a widely scattered literature. Sponges with spacious canals and internal cavities, such as species of Spheciospongia, Tedania and lrcinia in the tropical western Atlantic, are notorious hosts for a variety of animals, and frequent but brief mention has been made of the isopods found in them. Three isopod species found in sponges by Pearse (1934) feed on animals also associated with the host: Bopyro and Hemiarthrus are parasites of a synalphid shrimp, and Cirolana {larva is probably a predator of syllid and other polychaete worms. The feeding of C. parva on Porites reefs in Puerto Rico involves ingestion of a considerable quantity of polychaete flesh. The type of food consumed by Paracerceis caudata, also listed from sponges by Pearse, is not known. Potts (19] 5) has described the association of Cirolana lineata with the crinoid, Comanthus annulatum, in Torres Straits. Feeding is executed by diving into the gut of the host where the isopod devours previously ingested food-an interesting deviation from the usual feeding behavior of the Cirolanidae, which normally involves scavenging on dead flesh and free- roving predation. The intimacy of the association is accentuated by the arrangement of chromatophores into two lines and the development of a purplish pigment as an adaptive resemblance to the host species. Other echinoderm-isopod relationships are demonstrated by the idotheid, Coli- dotea rostrata, which lives among the spines of the echinoid, Strongylo- centrotus purpuratus (MacGinitie & MacGinitie, 1949), and the asellote, Antias unirameus, found in pits on the skin of Stichopus moWs (Menzies & Miller, 1955). Isopods living as commensals with other isopods are also known; at least three asellote genera are involved. Species of the genus [ais live on the legs of Exosphaeroma (Giambiagi, 1925), and on the body and between the pleopods of Sphaeroma (Menzies & Barnard, 1951). Jaera has been found on Sphaeroma (Arcangeli, 1934), and Caecijaera has thus far been found only in association with Limnoria (Menzies, 1951). Sphaeromids have been collected in association with a variety of other living forms, including sponges, prosobranchiate (Haliotis) and pelecypod mollusks (Ostrea, Mytilus, and from wood infected by Teredo). Shen (1929) found Dynoides dentisinus living with sea anemones, bryozoans, and barnacles, in addition to some of the animal groups already listed. Cymodopsis gorgoniae, a hemibranchiate sphaeromid, is associated with gorgonians in New South Wales, Australia (Baker, 1926). An additional example of an echinoderm associate within the Sphaeromidae is Exosphae- roma nuttingi, which was collected from among the spines of an unidentified sea urchin at Barbados (Boone, ]921 ) . The association of sphaeromid isopods with chitons was first reported by Richardson (1912). Seventeen specimens of Dynamenella pertorata were 1968] Glynn: Studies on the Associations of Chitons 575 collected from under the girdle of chitons at the tide line in Montego Bay, Jamaica, by E. A. Andrews, and two specimens of Exosphaeroma crenulatum (Richardson, 1902) were found living on the outside of a chiton. The species of chitons were not given, but in the case of D. perforata the host chiton was probably Acanthopleura granulata. Preliminary observations were made by Arey & Crozier (1919) on the presence of Exosphae- roma crenulatum with Chiton tuberculatus at Bermuda. These isopods occupied the ctenidial grooves and the undersurface of the girdle, with as many as 20 or more associated with large chitons. Since Exosphaeroma also occurred on the dorsal surface of Chiton and among algae growing close to the chitons such as Enteromorpha, the commensal relationship was considered more or less facultative in nature by these workers. Arey & Crozier indicated the desirability of obtaining more information on the partnership, but evidently no further studies were published. It is hoped the present findings will demonstrate how common and widespread the association of sphaeromid isopods is with particular chitons along the shore of the tropical waters of Puerto Rico.

ACKNOWLEDGMENTS I am pleased to acknowledge the kind cooperation of many individuals who contributed invaluable labor and knowledge to the present study. At the Smithsonian Institution, U. S. National Museum, Thomas E. Bowman identified Dynamenella perforata, made arrangements for me to work on the collections of Sphaeromidae deposited in the Division of Crustacea, and helped review the manuscript; Harald A. Rehder, Joseph Rosewater, Walter J. Byas, and George Radwin, of the Division of Mollusks, assisted in the search for Dynamenella among the collections of Acanthopleura granulata. A collection of Dynamenella perforata was loaned by Willard D. Hartman of the Peabody Museum of Natural History, Yale University. Dynamenopsis dianae was identified by Jayme de Loyola e Silva of the University of Parana, Brazil. In the Institute of Marine Biology, University of Puerto Rico, Mayagtiez, Luis R. Almod6var identified the various species of algae; Charles E. Cutress supplied a collection of animals from Barbados, and along with Juan Gerardo Gonzalez contributed critical advice on certain aspects of the study and assisted with the photographic techniques. Identifications of other organisms associated with chitons were carried out by: E. L. Bousfield (National Museum of Canada, Ottawa), Amphipoda; Arthur G. Humes (Department of Biology, Boston University), Harpacti- coida; George A. Seiglie (Department of Geology, University of Puerto Rico, Mayagtiez), Foraminiferida; and David W. Walker (Puerto Rico Nuclear Center, Mayagtiez) and David L. Wray (Division of Entomology, North Carolina Department of Agriculture, Raleigh), Collembola. Veri- fication of the location of two collecting stations in the Florida Keys was 576 Bulletin of Marine Science [18(3) made by Anthony J. Provenzano, Jr., Institute of Marine Sciences, Univer- sity of Miami. Rock samples from several localities were identified by John D. Weaver, Department of Geology, University of Puerto Rico, Mayagtiez, and the location of rock formations on Desecheo Island were furnished by Reginald P. Briggs, U. S. Geological Survey, San Juan, Puerto Rico. Considerable help in the field was provided by Francisco Jose Fernandez Irizarry, by Peter Castro (now at the Dept. of Zoology, University of Hawaii), by collecting assistants at the field station, in particular Guillermo Garda Ramos, Gregorio Padilla Rosado, and Pedro Rosado Ramos, and by Captain Trabert M. Felix and the crew of the MRV CARlTE, all of the Institute of Marine Biology. Zoilo Arcelay Cardona, also of the Institute of Marine Biology, constructed the plunger device for measuring the resist- ance of chitons and isopods to wave shock, and a pressure gauge for the apparatus was loaned by Antonio Santiago Vazquez, Water Resources Research Institute, School of Engineering, University of Puerto Rico, Mayagtiez. In the terminal phase of the study, much helpful criticism was rendered by Robert J. Menzies of the Duke University Marine Laboratory. Certain aspects of the work were made possible through National Science Founda- tion Grant GB-888 and a grant awarded by the Research Center, University of Puerto Rico, Mayagliez.

STATUS OF THE TAXON Dynamenella perforata (MOORE, 1901) In 1901, H. F. Moore described Dynamene perforata from a sample of about 50 specimens collected from mangrove roots at Culebra Island, Puerto Rico.1 Male and female individuals of different sizes were represented in the collection. Later Richardson (1905, p. 303) examined the presumed female specimens described as Dynamene perforata by Moore and discovered that one individual was actually a male. Two additional specimens presumed by Moore to be females were examined by H. J. Hansen, who found them to be an immature male and an adult male. Although it is possible for a female to possess a penis, as observed in one individual of Dynamenella tropica by Loyola e Silva (1960), it seems that the two adult males determined by Richardson and Hansen were mistakenly identified as females by Moore because they did not display the fully developed pleotelson present in the largest males. Mainly from the obvious morphological development of the pleotelson in large males, Richardson concluded that other male individuals lacking a prominent notch and heart-shaped opening represented a distinct species which she named

1 It is likely that the isopods were not collected from mangrove roots, since the species has never been found in this niche after extensive collections at numerous localities around Puerto Rico. Moore did not personally collect the type material, but obtained it from the FISH HAWK Expedition to Puerto Rico. ]968] Glynn: Studies on the Associations of Chitons 577

FIGURE 1. Three individuals of Dynamenella perforata collected from underneath Acanthopleura: a, mature male with elaborately notched pleotelson; b, earlier stage male with incipient cleft on pleotelson; c, mature female with plain, ovate pleotelson. Photographs of alcohol-preserved specimens mounted in glyc- erin and pressed down lightly under a cover slip.

Dynamene moorei (1905). Thus Richardson concluded that the sexes are similar in gross morphological appearance in the genus Dynamene. Hansen ( 1905, p. 107) established the genus Dynamenella on the basis of the following characters relevant to the present discussion: "Both sexes rather similar in aspect, without real processes; abdomen with a notch which is semicircular or oblong in the female, in the male narrow in the distal part, while the proximal part constitutes a transverse foramen; .... " According to this definition, the synonymy of Dynamene moorei with Dynamenella perforata was established. At the outset of the present study, individuals of D. perforata and D. moorei were treated as a single species because of the great similarity in morphology (Fig. 1), discounting the differences in the pleotelson which were attributed to sexual dimorphism, a frequent condition in the Sphae- romidae (Beddard, 1886; Hansen, 1905; Baker, 1908), and because of their frequent occurrence together in the highly specialized niche of the undersurface of Acanthopleura. Subsequent sex determinations and studies of the development of the pleotelson in a series of animals of known sex and considerable range in size (Fig. 2), as well as rearing studies (Table 2) reveal that the males of D. moorei are smaller, less mature males of D. perforata and that the females of both species are indistinguishable. Examination of the external anatomy of D. perforata and D. moorei with 578 Bulletin of Marine Science [18(3) reference to the descriptions of the two species (Richardson, ] 905) demon- strates no significant difference in the number of articles on the flagellum of the first and second antennae or in the appearance of the segments of the thorax. Aside from the differences noted in the pleotelson, all the other characters listed do not seem to warrant a separation of the species. In addition, a female individual of D. perforata with a pleotelson resembling the male condition has never been observed by the author. The hundreds of sex determinations made in this study were always based on the presence in males of a penis on the ventral surface at the midline of the last thoracic segment and an accessory stylet on the endopod of the second pleopod, or on the presence of an incubatory pouch in females. Although Richardson (J 905, p. 301) maintained that both sexes are known, I found that six specimens of the material examined by her (Dynamene perforata, Cat. No. 3204, Peabody Mus. Nat. Hist., Yale Univ.) were males; they ranged in length from 2.56 mm to 3.81 mm. Three specimens were in possession of both a penis and stylets; two animals had a penis, but the second pJeopods were absent; a sixth specimen had a well-developed penis, but the animal was badly crushed and stylets were not visible on the second pleopods. The co-type designation of this collection probably refers to Dynamene cordata, a provisional name assigned to this species by Richardson. There is no evidence that Moore examined this material. Differences in the appearance of the pleon in males appear to represent morphological changes which accompany maturation (Fig. 2). The posterior border of the pleotelson in young males, best observed from a ventral view, is ovate or slightly indented. Only a short, erect penis is present on the sternite of the last thoracic segment at this stage. In this size range, small and intermediate-sized females (2.20-2.70 mm) superficially re- semble the males (Fig. 1, c). A distinct indentation of the pleotelson (Fig. 1, b) and incipient foramen are evident as translucent areas visible through the exoskeleton in the more mature males; a long penis which tends to stand erect in alcohol-preserved specimens and an incompletely formed stylet united along its length with the endopod of the second pleopod were present at this stage. The largest males, ranging from about 2.80 mm to 3.40 mm in total length and comprising nearly 60 per cent of the males in the sample, displayed an elaborately indented pleotelson. Even at this stage, however, the exact configuration varies in different individuals. Moore (1901, p. 173) appreciated this variation in the pleotelson in males as evidenced by the following statement: "in some the groove is shallow, and in some it is deep, and in one it is clearly an incision connecting the terminal notch with the foramen." The measurements in Figure 2 also show that mature females are as large as mature males. Seventeen, or 53 per cent, of the females were gravid. Information on the number and size of developing eggs or embryos, 1968] Glynn: Studies on the Associations of Chitons 579 TABLE I INFORMATION ON THE DEVELOPMENTAL STAGES IN 17 GRAVID FEMALE SPECIMENS OF Dynamenella PRESENT IN THE SAMPLE ANALYZED FROM STATION No. 22 Description of Length of Length of developmental Number eggs or embryos female isopod stagel developing (mm) (mm) 7 0.3 2.90 10 0.4 2.93 14 0.4-0.5 2.77 Egg 19 0.4-0.5 3.33 7 0.5 2.91 13 0.5-0.6 2.69 13 0.5-0.6 3.00 12 0.5 2.70 Embryo elongate only; segmentation, 9 0.5-0.7 2.61 eyes and appendages absent 12 0.6 2.78 13 0.6-0.7 3.00 Elongate and segmented 13 0.6 2.66 11 0.6-0.7 2.69 Elongate and segmented, with II 0.6-0.7 2.84 crescentic eyes 15 0.6-0.7 2.96 8 0.7 2.60 Nearly full-term juvenile 11 0.7 2 0.8 2.92

1 Individuals are ranked according to the maturity of the embryonic stage, j.e., from the earliest to the latest. and the particular stages observed is summarized in relation to parental size in Table 1. These data show that females of about 2.60 mm and larger produce from seven to 19 young at a time when in an active breeding condition. One individual brooding young of two distinct developmental stages points up the possibility of occasional, multiple fertilization and probably sequential release of young. Animals less than about 2.10 mm in length, which amounted to 21.3 per cent of the total number in the sample, could not be sexed on the basis of the structures examined, and were thus presumed to be in an immature condition. A chi-square test failed to show any significant difference in a sex ratio of 1 : 1 at the 0.500 level for the 59 individuals that could be definitely sexed. Although this is admittedly a small sample, casual sexing of larger numbers of individuals in other collections tended to corroborate these findings. Confirmation of the notion that D. perforata is an older, morphological variation of D. moorei came from rearing studies, where some individuals of the D. moorei species developed into the D. perforata species with an elaborately notched pleotelson. 580 Bulletin of Marine Science [18(3)

SEX d'd' 9~ IMMATURE long absent PENIS prostrate 10"9, erect short. erect a bs ent fully STYLET developed united with pleopod oblent absent obi en r PLEO - TELSON ,-' ... _ .. ' , , Q V'- ' 0'-' g, -- V V 0'-- + 3.40 3.40 + •.. +t+ + + 3.20 +++ 3.20 + +

3.00 + ~+t: 3.00 $ ~ ~ <- 2.BO + + 2.BO + +t+ to ? + + -> *+=1=+ *"•... 2.60 + * *' 2.60 LENGTH + (MM) + + + 2.40 + 2.40 + f 2.20 $ 220 + t 2.00 2.00 + :j: I.BO + LBO +

1.60 :j: 1.60

+ 1.40 + 1.40 ++

NUMBER 16 I 3 2 5 32 16 % d'd' 59.3 3.7 11.1 7.4 IB.5 TOTAL % 21.3 1.3 4.0 2.7 6.7 42.7 21.3

FIGURE 2. Relation of maturity of the sexes and morphological development of the pleotelson to size in 75 individuals of Dynamenella perforata collected from underneath Acanthopleura at Mona Island, March 21,1966 (Sta. No. 22). The total length of each animal was measured with an ocular micrometer under 30x magnification from the anterior margin of the head to the posterior margin of the pleotelson while pressed flat to approximate the crawling posture. Sketches of the pleotelson portray this structure when viewed from above and 1968] Glynn: Studies on the Associations of Chitons 581 Animals were isolated within an hour after collection from Turrumote Reef and introduced into a glass jar of four liters capacity. A second similar jar was later added to accommodate animals which had developed into the "intermediate" of D. perforata species. Constant aeration and illumination (40-watt fluorescent lamp) were provided, and fine cotton cloth was tied over the mouth of each jar to exclude dust and artifacts. The sea water was filtered through a Millipore filter (type AA, pore size 0.80 p.) and renewed whenever the animals were examined. The following species of filamentous, green algae were introduced as food on pebbles to which they were affixed: Cladophora de/icatula Montagne, Enteromorpha flex- uosa (Wulfen) J. Agardh, and Rhizoc/onium hookeri Kiitzing. Certainly other species of algae were present, but these forms predominated. The culture was begun on March 6, 1966, with the introduction of 26 large individuals of D. moorei (Table 2). At intervals of time ranging from three to seven days, the isopods were pipetted from the jars and examined under 20x magnification for any changes in the pleotelson. Determination of sexes was not carried out for fear of injuring the animals. That fairly healthy conditions prevailed in the culture is evidenced by an overall, non- accidental mortality of six individuals out of a total of 29 introduced. Over a period of three days the pleotelson in three isopods changed from an ovate form to an "intermediate" condition with a notch or a notch and foramen showing through the exoskeleton. One additional "intermediate" type developed over the next four-day period. By March 25, three "intermediate" types had developed a pleotelson with a prominent notch and foramen in open communication between the upper and lower surfaces of the abdomen. Since three "intermediate" types were introduced on March 18 (seven days earlier) it is not possible to conclude that a minimum of 16 days was required for this transformation. Neither is it possible to determine if the" "intermediate" stage is always preceded by an exuvia from the number of exuviae recovered in the culture jars. It is quite likely,

~ overlapped by the preceding pleonal segment. The different morphological types are slightly exaggerated as illustrated in this figure. In males with a long penis the notches and foramen present on the margin of the pleote]son are not in communication between the upper and lower surfaces, but simply visible through the thin, translucent exoskeleton. Broken lines delimit the lateral extension of the convex upper surface of the pleotelson. One fully developed male, noted by a question mark, broke in half and therefore could not be measured accurately. Arrows indicate females carrying eggs or embryos. The number of all isopods and their percentage contribution to each category, as well as the percentage distribution of the different male stages, are summarized at the bottom of the figure. Although an attempt was made to collect an unbiased sample representing all sizes, it is likely that some of the smallest individuals were not taken since the isopods were removed from the chitons in the field. 582 Bulletin of Marine Science [18(3) TABLE 2 SUMMARY OF THE DIFFERENT MORPHOLOGICALSTAGESOBSERVED IN A CULTURE OF Dynamenella perforata MAINTAINED OVER A 19-DAY PERIOD

Numbers of different morphological types Date Total during D. "Inter- D. per- Inde- Total number Mar., moorei mediate" forata termi- number sur- 1966 (3 & !t) (3 ) (3 ) nate! dead viving Notes2

6 26 0 0 0 0 9 19 2H 3 0 2 2 24 One isopod died in container, other escaped and died from exposure. 13 18 2 4 0 0 2 22 Two D. moorei-type isopods died, one in container and other from exposure after es- caping. 18 17 I 3 I 0 0 2 20 One D. moorei-type isopod died in container, one "intermediate" type escaped and died from exposure. Two D. moorei exuviae shed. Three additional "intermediate" types were introduced. 25 12 2 2 I 3 3 3 20 Two died in D. moorei container, one in "intermediate" type container. One D. moorei exuvia shed in D. moorei container; three "intermediate" exuviae shed in "intermediate" container.

1 Indeterminate individualswere those which decomposedbeyond recognitionafter death. 2 Additional pertinent information, obtained mainly from the periodic examinations of the cultures, is listed under notes. 3 Boldfacenumerals denote number of dead within a group. however, that a single exuvia preceded the assumption of the characters of the D. perforata species, as three intact "intermediate" exuviae were recovered along with the development of three individuals of D. perforata. At the end of the growth studies the 20 surviving animals were sexed and found to agree in every particular with the scheme presented in Figure 2. All 14 individuals with a smooth or slightly emarginate pleotelson were 1968] Glynn: Studies on the Associations of Chitons 583 females (10, or 71 per cent, were gravid); the three "intermediate" types were males, with a long, erect penis and stylet still fused along its length with the endopod of the second pleopod; and the three individuals with a fully developed pleotelson were males, with a long, prostrate penis and free stylet attached to the endopod only by its base. These results illustrate the care that must be exercised in order to determine the first appearance of secondary sexual characters and their subsequent development in the life history of a sphaeromid species. In spite of the opinions of Holmes (1904) and Moore (cited in Richardson, 1905, p. 315), Richardson reluctantly synonymized females of Dynamene tuber- culosa with Cilicaea cordata and of Dynamene bermudensis with Cilicaea caudata. She thought that several males and females of the genus Dyna- mene were alike in many respects. Rearing studies are likewise needed to resolve the systematic status of these species.

DESCRIPTION OF THE ASSOCIATION Various Species of lsopods and Chitons lnvolved.-While main consideration is given to the association of Dynamenella perforata with Acantho- pleura granulata and Chiton tuberculatus, collecting information summarized in Table 3 illustrates that other sphaeromid-chiton relationships are quite common. First, it should be noted that D. perforata was the only isopod found living with Acanthopleura. Chiton tuberculatus, however, played host to three other sphaeromid species besides D. perforata, viz., Exosphaeroma (a) and (b), and Dynamenopsis dianae. (In this paper, host will refer to a chiton which affords shelter to isopod symbionts.) The C. tuberculatus-Exosphaeroma (a) relationship was observed at four different localities (Sta. Nos. 8, 15, 19, and 22; see Table 3), with an incidence which ranged from 8 per cent to 42 per cent. Exosphaeroma (b) was found with only one chiton at Pta. Maldonado on the northeastern coast of Puerto Rico (Sta. No. 24). At the northern Mayagtiez seawall (Sta. No. 12), Dynamenopsis dianae (Fig. 3) occurred with C. tuberculatus with an incidence of 6 per cent and 20 per cent on two different occasions. This relationship was also noticed at other times at the same locality. Dynamenopsis was recently described from San Quintin Bay, Baja California, Mexico (Menzies, 1962). It was collected from a formalin wash of intertidal rocks, apparently apart from any chitons. The relatively high abundance of Dynamenopsis under small boulders and in empty barnacle tests at the Mayagtiez seawall suggests that its association with chitons is not very persistent. At two different times of the year, about 40 per cent of the individuals of Chiton marmoratus Gmelin, 1791, examined at Pta. Maldonado contained Exosphaeroma (b). This was the only locality where Exosphaeroma (b) was found associated with chitons. Although collections of Chiton 584 Bulletin of Marine Science [18(3)

FIGURE 3. Photograph of an alcohol-preserved male specimen of Dynamen- opsis dianae collected from underneath Chiton tubercuLatus. Mounted in glyc- erin and pressed down lightly under a cover slip. squamosus Linnaeus, 1764, were made at eight different localities, only at Mona Island (Sta. No. 22) did the chitons contain any isopods, and these were all D. perforata. The density of D. perforata with Acanthopleura was very high at Sta. No. 22, and the C. squamosus-Dynamenella partnership seemed most prevalent where the two chiton species lived adjacent to one another at the same vertical level on the shore. A total of 37 individuals of Ceratozona squalida C. B. Adams, 1845, was sampled from four different localities and all were devoid of symbiotic isopods. Numerous individuals of the following chitons, which occur subtidally on the dead, basal corallum of Porites porites var. furcata, also were devoid of free-living isopods: Acanthochitona hemphilli Pilsbry, 1893; Acanthochitona pygmaea Pilsbry, 1893; Cryptoconchus {ioridanus Dall, 1889; Tonicia schrammi Shuttleworth, 1856.

Location of lsopods on Host.-Although D. perforata was originally reported free of Acanthopleura in Puerto Rico, ostensibly from mangrove roots at Culebra Island (Moore, 1901), and I found it infrequently on algae-coated coral rubble from reefs and on bare boulders imbedded in sediment along the shore line (Table 5), present findings indicate a more frequent association of the isopod with chitons, apparently wherever environmental conditions are mutually favorable. Some collecting records outside of Puerto Rico, however, show that D. perforata can occur abundantly in other habitat niches along the shore. For example, one hundred specimens, in the U. S. National Museum, collected by J. P. E. Morrison at Homestead Bayshore Park, Dade County, Florida, were found under 1968] Glynn: Studies on the Associations of Chitons 585

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-o .....- 588 Bulletin of Marine Science [18(3)

,-.. o

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00 ] 968] Glynn: Studies on the Associations of Chitons 589

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0\ o •....• l"l 590 Bulletin of Marine Science [18(3)

, nJ .§

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,-... Q (l) (l) u.l r:: r:: ::> CQU U o o z Z Z i= 00 o z o U 0"""001/")0""" 0001/")00 Lr) v)v)N~OO ("t')("t')('1")N,...... ""'" ~CQ"c5o~CQ"c5 -<"u"o~c50

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I/") N 592 Bulletin of Marine Science [18(3)

., 1 em I 1968] Glynn: Studies on the Associations of Chitons 593 rocks at high tide along a breakwater. Also, Monod (1933) noted that D. perforata was collected from the cavities of barnacles in the harbor of Rio de Janeiro many years ago. Where Acanthopleura and Dynamenella occur together, and when the chiton is relatively inactive (not feeding), the isopod is concentrated mainly in and along the entire length of the pallial groove, and in lesser numbers along the pallial fold and in the inhalant and exhalant respiratory canals communicating with the exterior. A marked orientation is maintained by the isopods along the pallial groove when the chiton is feeding and moving about. At this time nearly all the isopods insert the head end of the body between the individual gill filaments, often as far medially as the exhalant chamber. The projecting rear portion of the pleotelson is frequently about the only part of Dynamenella visible when in this posture. The undersurface of a specimen of Acanthopleura crawling on plate glass in the light showed that the isopods either ride along with the chiton by clinging to its girdle or swim in the film of water carried forward by the chiton. An overall impression of the numbers present, size relations of host and symbionts, and the different regions of the chiton occupied can be gained from Figure 4, a and b. Figure 5 illustrates in more detail how Dynamenella is oriented along the gill as Acanthopleura browses. Dynamenella occurs in and along the whole length of the pallial groove in Chiton tuberculatus as well. Indeed, all sphaeromid isopods found living with chitons in this study showed a decided preference to orient themselves within the comparatively voluminous space provided by the inhalant chamber of the pallial groove. Incidence of Association, Number of Isopods per Chiton, and Relation to Size of Host.-The presence of Dynamenella in samples of Acanthopleura ranged from 3 per cent to 100 per cent. At certain localities, where environmental factors seemed to favor the occurrence of both species, the

~ FIGURE 4. a, Enlarged view of the undersurface of Acanthopleura showing specimens of Dynamenella still adhering to a chiton immediately after dislodgment from a dead coral rock (Turrumote Reef, La Parguera, October 11, 1965). As many as 30 isopods are visible in this photograph. Arrows 1, 2, and 3 point to two isopods in a respiratory canal in the girdle, to the inhalant chamber of the pallial groove, and to the pallial fold, respectively. The exhalant chamber of the pallial groove is not visible, but lies medial to the inhalant chamber.-b, Enlarged view of specimens of Dynamenella adhering to the surface of a dead coral rock immediately after dislodgment of Acanthopleura (Turrumote Reef, La Parguera, October 11, 1965). Most of the animals were scurrying for cover in the interstices and concavities of the rock at the instant the picture was taken. As many as 35 isopods are visible in the photograph. The chiton under which these isopods were living was not the same individual illustrated in part a, above. An arrow points to six isopods clumped together. 594 Bulletin of Marine Science [18(3)

2 em

I 2 mm

FIGURE 5. Lateroventral view of the right side of Acanthopleura showing the orientation of Dynamenella between individual gill filaments along the pallial groove. The enlarged sketch illustrates in more detail how one isopod situated between two gill filaments has inserted its head into the exhalant chamber of the pallial groove. incidence of the association was usually in the range 90 per cent to 100 per cent. Even in commonly frequented habitats, though, the isopod occurred less often with the chitons near their lower limit of vertical distribution in the intertidal zone (see Fig. 11 and Table 9). On the three occasions when Dynamenella was found with C. tuberculatus, the incidence of the association was only 10 per cent, 35 per cent, and 56 per cent. Of 50 specimens of Acanthopleura collected from an intertidal level subject to nearly uniform conditions of submergence, splashing, and exposure, Dynamenella was found in 100 per cent of the chitons. The number of isopods per chiton ranged from 1 to 77. Twelve chitons, with a sample frequency of 24 per cent, contained between 10 and 19 isopods, and a relatively large proportion of the chitons sheltered numerous isopods, up to 40-49 per individual. The number of symbiotic isopods decreased rapidly at 50-59 individuals per chiton, and only 4 per cent of the sample was represented in the class 70-79 (Fig. 6). When Dynamenella occurred with C. tuberculatus in the field, the number of individuals per chiton was generally low, usually between one and three, and occasionally as high as five. In one instance, 13 specimens of Dyna- menella were present under a single chiton. 1968] Glynn: Studies on the Associations of Chitons 595

20 OIl C .•..0 .J::. 0 15 >. 0 c Q) :J fJ 10 Q)•... Ii..

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0-9 20-29 40-49 60-69 10-19 30-39 50-59 70-79

Number of Isopods FIGURE6. Frequency distribution of the number of specimens of Dynamenella associated with individuals of Acanthopleura in a sample of 50 chitons collected at Laurel Reef, La Parguera (Sta. No.7). All isopods were enumerated under lOX magnification in the laboratory, including those adhering to the substratum after removal of the chitons.

A plot illustrating the number of isopods per chiton versus weight of the host demonstrated a more or less random distribution (Fig. 7). The three highest densities (one instance of 59 isopods, and two instances of 77 isopods) occurred in hosts of intermediate size, with a range in weight of 17.82 gm to 22.20 gm. Many other chitons of the same size contained from about 50 symbionts to less than 10. Small chitons with weights of 8.53 gm and 9.58 gm contained 38 and 40 isopods, respectively, while the two largest chitons with weights of 37.00 gm and 41.09 gm contained only 31 and 15 isopods, respectively.

OTHER SYMBIONTS ASSOCIATED WITH Acanthopleura AND Chiton tuberculatus Not only is the undersurface of chitons a frequent abode of certain sphaeromid isopods, but also the pallial groove and the smooth surface of the girdle, which is usually more or less firmly appressed to the substratum, provide living space for a foraminiferan, two species of harpacticoid cope- 596 Bulletin of Marine Science [18(3)

c 80 2 0 0 .<: u 70 •... 60 0 ••Q. 0 ...,III 0 00 50 0 0 0 0 Q. 0 40 00 0 0 0 !2. (}($)O 0 0 •.. 30 0 0 0 0 0 •... 0 0 20 cfJ 00 00 0 0 .Cl•• o 0 0 o E 0 0 ::J 10 0 Z 0 0 0 0 0

10 20 30 40

Weight of Chi t on (gm) FIGURE7. Scatter diagram of the number of isopods per chiton versus weight of chiton in the same collection illustrated in Figure 6 (Sta. No.7). Weights are of alcohol-preserved specimens blotted dry with paper towel. Individual measurements lost from a grouping of the data in Figure 6 can be approximately retrieved from this graph by visual inspection. pods and a springtail. On occasion, amphipods were also observed with chitons, particularly with individuals located at a relatively low level on the shore. "Acervulina inhaerens" Schulze, 1854, which attaches firmly to the girdle of C. tuberculatus, occupies a very unique niche when compared to other members of the Foraminiferida. "Acervulina" was tentatively distinguished from the closely related Planorbulina acervalis on the basis of its smaller chambers, which are not so coarsely perforate as in Planorbulina. The inner walls of the early chambers in "Acervulina," however, are chitinous as in Planorbulina. Cushman et al. (1954) have pointed out the difficulty in establishing a clear-cut separation of the two species. Tn any event, the description of the genus "Acervulina" is not yet clear; a study of topotypes of "A. inhaerens" must be made before the name can be applied with a reasonable degree of confidence. The shape of "Acervulina" attached to C. tuberculatus is oblong, with the body oriented in a direction perpendicular to the long axis of the chiton's mantle. The attached side of the test is concave and striated, matching the striations present on the fleshy girdle of the chiton. It is firmly attached to the girdle, being difficult to dislodge in an unbroken condition. "Acervulina" was found most commonly on C. tuberculatus, with an incidence of about 5 to 10 in 100 chi tons. When present, one 1968] Glynn: Studies on the Associations of Chitons 597 foraminifer per host was the rule; on only one occasion were two foramin- ifers found on a single chiton. Chitons from Turrumote Reef in La Parguera (Sta. No.8) and from the seawall in Mayagiiez Bay (Sta. No. (2) showed the highest incidence of infestation. Only one "Acervulina" was found on Acanthopleura, out of a sample of 270 chitons collected at Pta. Brea on Sept. 13, 1966. The two harpacticoids, Harpacticus sp. (Harpacticidae) and one species tentatively assigned to the genus Heterolaophonte Lang, 1948 (Laophon- tidae), were often found clinging to the girdle of Acanthopleura, especially in proximity to sandy areas and on semiprotected shores. Both species of harpacticoids were sometimes found together under the same chiton. No attempt was made to count the numbers present on individual chitons, though casual observation showed this to lie in the range of three to about 50 harpacticoids per individual host animal. On occasion, C. tuberculatus was also observed to harbor these symbionts. This relationship represents but one additional record of numerous harpacticoid-invertebrate associations reported on in recent years (Good- ing, 1957; Humes, 1958). A host site commonly occupied by harpacticoids in crustaceans is the gill chamber. Very often past workers have tacitly assumed some kind of parasitic role in such a niche, but Gotto (1957) found that the feeding activities of Ascidicola rosea, a notodelphyoid copepod inhabiting the esophagus of the ascidian Corella, remove only a small and insignificant fraction of material from the food string. A study of the feeding behavior of harpacticoids living on chitons is clearly needed. A rarer occupant of chitons was a member of the amphipod family Hyalidae, Parhyale hawaiensis (Dana, 1853), encountered as an associate on only a few occasions, but otherwise one of the most dominant species among algae in the shallow littoral zone. As this amphipod seems to prefer wet surroundings, it was found most commonly with C. tuberculatus which occupies a lower level on the shore than Acanthopleura. Only two individuals at most were ever recovered from a single chiton host. From the few observations made, the inhalant chamber of the pallial groove and the inhalant and exhalant respiratory canals on the girdle seemed to be the preferred sites occupied by the amphipod. Some amphipods observed less commonly with Acanthopleura, but which escaped, were possibly species other than P. hawaiensis. Actaletes neptuni Giard, 1889 (Actaletidae), a rare collembolan until now known from France and other European shorelines, is very abundant on limestone rocks in the spray zone at Pta. Brea (Sta. No. 21) and also under Acanthopleura at the same locality. Many chitons present at the higher levels of vertical distribution often contained one to four of the springtails, which like Parhyale and Dynamenella were confined largely to the more voluminous spaces of the girdle and pallial groove. Since most 598 Bulletin of Marine Science [18(3)

ATLANTIC OCEAN

o 0 25-0 24-0 IBO 2 3 IBo 30' I 30' Culebrq I. S t. Thoma. Sa n J •••an .. q:O .:~ 10-0 ~, ~ 23-. RICO 5-0 6-()

Io Mono I. ~ \ 22-. V ieques I. 21 i \ IT-O () 20 19-0 0 St. Croix 170 CARIBBEAN 5 EA 30' 17° 30'

61°00· 66°00· 6~oOO'

FIGURE 8. Map of Puerto Rico and westernmost Virgin Islands showing the distribution of the Dynamenella-Acanthopleura relationship. Acanthopleura was present at all stations indicated; an open circle denotes the absence of Dynamenella, a partly occluded circle denotes the presence of the partnership in at least one part of the locality collected, and a fuJly occluded circle indicates a general presence of the partnership everywhere within the area of the station. This map was adapted from Coast and Geodetic Survey chart 920 of Puerto Rico and the Virgin Islands, West Indies (1963). species of Collembola live on decaying matter (Comstock, 1949) and many of the marine littoral species on plant debris (Delamare Deboutteville, 1954), the attraction of Actaletes to Acanthopleura might be due to the presence of plant debris resulting from the chiton's feeding activity.

OCCURRENCE OF Dynamenella WITH Acanthopleura IN RELATION TO ENVIRONMENT AL FACTORS Because the Dynamenella-Acanthopleura partnership was observed with greatest regularity over a rather broad spectrum of physical conditions within the wave-washed, rocky habitat, a study of its occurrence in relation to environmental factors was carried out. The association was investigated primarily with reference to (a) character of the habitat (e.g., coral reef versus rocky promontory), (b) degree of exposure to wave action, and (c) nature of the substratum. In its distribution on the shore around Puerto Rico, Acanthopleura typically demonstrated a high tolerance to a relatively wide range of conditions, providing a solid substratum was present, period- ically washed by the sea. The eurytopic habit of Acanthopleura proved to 1968] Glynn: Studies on the Associations of Chitons 599 be well suited for an analysis of the occurrence together of symbiont and host under varying environmental conditions. The Habitat.-Near La Parguera the wave-washed foreshore of the coral reefs, Laurel (Sta. No.7) and Turrumote (Sta. No.8), is composed of dead coral rubble which supports a dense population of Acanthopleura. The incidence of the isopod-chiton relationship is 100 per cent on these semiprotected reefs (Fig. 8, Table 3). Similar habitats in La Parguera (eastern end of Enrique Reef; 17°57'32"N, 67°02'39"W) and elsewhere (south coast of Culebra Island, Sta. No.5; Berberia Reef, Sta. No. 19) characteristically contained high population densities of Acanthopleura without Dynamenella. Rocky, limestone headlands on the southwestern coast of Puerto Rico demonstrated a high incidence of the association (91 per cent and 98 per cent at Pta. Periones, Sta. No.9) or its complete absence (Cabo Rojo Lighthouse, Sta. No. 11). Although the numerous calcarenite beachrock and cemented dune formations along the north coast of Puerto Rico support a high density of Acanthopleura, not a single record of the partnership was obtained from this area. At Saba Island (Sta. No. 6), however, 60 per cent of the chitons examined from beachrock on the lee side of the island contained Dynamenella. A high incidence of the association was observed on the elevated reef pavement at Mona Island in March, 1966 (Sta. Nos. 22 and 23). Both collecting localities were in the lee of high seas during that time of year. These observations indicate that the presence or absence of Dynamenella is probably not determined alone by the overall character of the habitat. Exposure to Wave Action.-In a general way the field data initially sug- gested that the occurrence of the partnership might be correlated with the degree of exposure to surf action. It was hypothesized that the two species live together in areas protected from relatively severe wave shock. All localities collected which border the coast of the Atlantic Ocean showed Dynamenella to be absent (Fig. 8). The north coast is usually subject to greater water turbulence than the south coast, although local topographic features such as lunate bays and offshore islands can provide sheltered niches along the north shore (Kaye, 1959b). At Saba Island, the isopod and chiton were found together where wave action was slight, but only Acanthopleura was present where the wave height and surge increased substantially. The partnership occurs with a high frequency near La Parguera, which is usually protected from high seas, but one locality sampled at Mona Island in March showed an incidence of 100 per cent (Sta. No. 22). This locality is subject to high surf and surge during the summer months when the trade winds trend more from the southeasterly quarter (U. S. Coast & Geodetic Survey, 1962). A possible influence of wave shock in limiting the occurrence together 600 Bulletin of Marine Science [18(3)

N 1

1 km-

t ',

FIGURE9. Aerial photograph of Pta. Brea, Puerto Rico (Sta. No. 21), illustrating the location of nine substations. A clear diminution in the height of swells is evident along the northern, landward side of the point. The photograph was taken on Feb. 16, 1963, at a flying height of 10,000 ft above average ground level. Courtesy of U. S. Department of Agriculture, Soil Conservation Service, Hyattsville, Maryland. of the isopod and chiton was tested at Pta. Brea (Sta. No. 21), where the degree of exposure to the open sea varies significantly over a relatively short distance (Fig. 9). Acanthopleura occurs abundantly on limestone rocks around the entire headland. The extent of exposure to wave action around the point is well illustrated by the "white water" visible in Figure 9 and by a plot of the wave heights (estimated visually from the shore) in the lower portion of Figure 10. Substations 1 through 4 and 9 were subjected to waves of less than one meter in height, whereas substations 5 through 8 were more exposed, with a wave height usually in excess of one meter. The prevailing direction and force of the sea, and its direct influence on the shore, do not deviate markedly throughout the year. The number of chi tons which contained the isopod at the nine different substations are summarized in Table 4. Fair agreement of the incidence of the relationship within a particular substation on different occasions should be noted. Only substations 1 and 4 showed an appreciable deviation from the mean. Excluding substation 2, the total percentage of Acantho- pleura which contained Dynamenella decreased steadily from an incidence 1968] Glynn: Studies on the Associations of Chitons 601

Juaw!paS + ++ I

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..., '" Q)

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o 000 0 o m to .,. N 1%) ~ouenbeJ~ (WJ·I~ &ADM 602 Bulletin of Marine Science [18(3 ) TABLE 4 INCIDENCE OF THE Dynamenella-Acanthopleura PARTNERSHIP AT NINE SUBSTATIONS LOCATED AROUND PTA. BREA (STA. No. 21)

Substations 2 3 4 5 6 7 8 9 Collections (1966) Jan. 25 - 27 14 5 2 0 Aug. 4 0 28 26 26 Aug. ]0 12* 28 19 11 0 Sept. ] 3 27 28 22 21 0 0 30 29 27 Total number chitons sampled 60 90 90 90 90 105 60 60 60 Total number chitons with isopods 39 83 55 37 3 0 58 55 53 Total % chitons with isopods 65 92 61 41 3 0 97 92 88

* Numbers refer to the chitons which contained isopods out of 30 animals examined at each sub· station, except at substation 6 on Aug. 4, when only 15 chi tons were examined. of 65 per cent at substation 1 to a per cent at substation 6. Contrary to an expected outcome of a low incidence of association, the exposed substations 7 and 8 showed total incidences of 97 per cent and 92 per cent, respectively. These observations, coupled with a 100 per cent occurrence of the partnership at Mona Island, show that Dynamenella is probably not particularly sensitive to wave shock. Further support of the notion that the occurrence of Dynamenella is not limited by pounding surf was obtained by subjecting the partnership to a graduated series of shocks generated by thrusting a piston suddenly against a seawater surface confined in a cylinder. With the exception of a stainless steel rod, those parts of the plunger which came in contact with the sea water or animals were constructed of non-metallic materials as follows: the cylinder was polyvinyl chloride pipe; the piston was hard wood; the piston valve was leather. The piston was 8.0 cm in diameter and was provided with a flat piece of coral rock to which the chitons could attach. The maximum pressures generated were read from a pressure gauge con- nected to the base of the cylinder. Chitons containing isopods were removed from their natural sites of attachment on coral rock, placed immediately on the piston and allowed to adjust to the new surface for 15 minutes. The piston was then plunged sharply against the water ten times at 30-second intervals at consecutive pressures of 10, 20, 30, and 40 lbsj in2• Most chitons succeeded in maintaining a grip up to 30 Ibsjin2 or 2.4 kgjcm2, equivalent to the pressure generated by a wave of 5.5 meters height (Thorn, 1960). Some individual isopods were observed to cling tenaciously to the piston during ten thrusts at a pressure of 40 Ibsjin2 or 3.1 kgjcm2, equivalent to a wave height of approximately 7.0 meters. It 1968] Glynn: Studies on the Associations of Chitons 603 seems that the few isopods which were found free of the chitons after exposure to a shock of 30 Ibs/in2 were not forcefully dislodged, but moved away voluntarily during the brief time allowed for readjustment of the animals on the piston or from other unnatural disturbances. Certainly the results obtained under these artificial conditions are at variance with the usual response of the animals under natural conditions. The high pressures resisted by some of the test animals, however, are indicative of a tolerance to rough seas which batter unprotected coasts. If wave shock and severe turbulence acted as limiting factors, one would expect a significant diminution in both the incidence of the association and the number of isopods per chiton host following the effects of a hurricane. The influence of such a disturbance on the partnership was assessed at Turrumote Reef on Oct. 4, 1966, six days after the center of Hurricane Inez passed 60 miles south of La Parguera. Turrumote Reef was subject to a pounding surf of 3-4 meters height from the southerly quarter and a storm surge of about 1 meter height. Virtually all herbaceous vegetation was removed from the reef island and the coral rubble shifted and re- deposited to such an extent that the physical features of the littoral zone were completely disrupted. Counts of the number of specimens of D. perforata per Acanthopleura, made in the field following the storm, are summarized in the following distribution: Numberof isopods Frequency Numberof per chiton (0/0 ) chitons o 0 o < 10 16 4 10-20 48 12 > 20 36 9 All twenty-five chitons sampled contained isopods. Moreover, comparison of these results with data obtained on two different occasions at Turrumote Reef prior to the storm (Fig. 12) show no significant effect of the disturbance on the association of the two species. The Substratum.-A coarse-grained sediment characterized all localities where the isopod-chiton partnership occurred (Table 3). Calcareous sand provided a base for the coral rubble on reefs where the two species were associated (Sta. Nos. 7 and 8), was present in the immediate area as patches (Sta. Nos. 23 and 24), or formed an extensive sandy bottom which bordered the rocky coastline (Sta. No.9). Sixty per cent of the chitons sampled from a beachrock formation adjacent to a small sandy beach at Saba Island contained the isopod, whereas it was absent from Acantho- pleura collected on an outcrop of volcanic flow rock located 25-50 meters from the beach. At Pta. Brea, the association was observed with consistent regularity at all substations where calcareous sand was present (Fig. 10, Table 4). 604 Bulletin of Marine Science [18(3 ) Dynamenella was encountered at substations 5 and 6 only rarely or not at all. The promontory in this vicinity is characterized by a gradually sloping, limestone substratum which has its origin relatively far from the shore. A continuation of the solid substratum was visible seaward to a distance of at least 10 meters and no sign of recent transport of sand from deeper water was evident. Counts of the number of isopods per chiton showed the < 10 class to be the most frequent in collections where sediment was present. Dynamenella showed the greatest density at substation 9, where the modal class, which represented 10-20 isopods per chiton, made up 33 per cent of the sample. Two other prominent classes were the < 10 and the> 20, at frequencies of 30 per cent and 27 per cent, respectively. No explanation is evident relative to the comparatively high, overall mean density of 16 isopods per chiton at this substation. Actually, even this sample was not exceptionally high compared with densities of 28 per cent for the 10-20 class and 56 per cent for the> 20 class observed at Laurel Reef (Fig. 6). An apparent dependence of Dynamenella on sandy areas, given a chiton host, is not out of line with the habitat requirements of numerous other isopod species. For example, Hult (1941) demonstrated a strong influence of the type of substratum on the distribution of certain sublittoral isopod assemblages. One such assemblage of stenoedaphical, soft-bottom forms included eight species. Several species of minute isopods, particularly microparasellids and anthurids, inhabit marine beaches in the Mediter- ranean Sea (Chappuis & Deboutteville, 1954), and Dahl (1953) has included cirolanids as a characteristic faunal element of the midlittoral zone of temperate and tropical beaches. Menzies & Frankenberg (1966) recognized a sand-beach isopod fauna in Georgia; two sphaeromids found in the habitat were Ancinus depressus and Exosphaeroma diminutwn. Analysis of the field data in the present study indicates that D. perforata is most abundant under Acanthopleura which live adjacent to sandy patches or more extensive areas of coarse-grained sediment. Absence of the partnership from several localities where these conditions prevail, however, indicates a possible influence of additional, unknown factors. In general, one would expect a more widespread occurrence of chitons owing to a greater dispersal of their planktonic larvae, as opposed to isopods which undergo development within incubatory pouches and are set free as immature adults. Perhaps chitons free of isopods at certain presumed favorable localities is a result of a slower rate of dispersion of the inquiline. Some workers have demonstrated a very close association of certain shore isopods with specific algal types. In the upper intertidal zone in the Sea of Japan, "Dynamenella" glabra is found in the small, bushy, red alga, Gloiopeltis capillaris (Mokyevsky, 1960), and on the California coast the same species of isopod lives in Endoc/adia muricata, also a small, dendritic, 1968] Glynn: Studies on the Associations of Chitons 605 TABLE 5 PRESENCE OF Dynamenella per/orata LIVING INDEPENDENTLY OF Acanthopleura granulata AT SELECTED COLLECTING LOCALITIES

Number Station Date of isopods number ( 1966) Material washed with formalint collected

7 Aug. 27 Algae-coated coral rubble from mid-intertidal zone. 3 Dominant algae: Cladophoropsis memhranacea (c. Agardh) B¢rgesen (C) *, Laurencia papillosa (Forssklll) Greville (R), Bryopsis pennata Lamou- roux (C), A I11phiroa /ragilissima (Linnaeus) La- mouroux (R). 8 Sept. 7 Same as Sta. No.7. 6 21 Sept. 13 Substa. No.1: bare boulders embedded in sand. 1 Substa. No.2: algae-coated rocks. Dominant alga: 3 Laurencia papillosa. Also boulders embedded in sediment. i" Chitons were excluded from all of the collections to avoid the presence of symbiotic isopods. • The abbreviations Rand C refer to the major algal groups, Rhodophyta and Chlorophyta, respectively. red alga (Glynn, 1965). A similar relationship was noted on English shores by Colman (1940). There, the sphaeromid, Campecopea hirsuta, is associated with the lichen, Pygmaea pumila. It should be noted, however, that Morton (1954) found the same isopod in the vacant tests of Chthama- Ius stellatus. The possibility that Dynamenella per/orata occupies an additional habitat niche, other than the undersurface of Acanthopleura, was investigated by washing (with 5-10 per cent formalin) boulders and rock fragments over- grown with algae, as well as the thalli alone of a variety of algae, which were taken from different levels in the intertidal zone (Table 5). The various species of algae examined, other than those included in the table, were the following: Rhodophyta-Digenia simplex (Wulfen) C. Agardh, Laurencia obtusa (Hudson) Lamouroux, Ceramium nitens (c. Agardh) J. Agardh, Centroceras clavulatum (C. Agardh) Montagne, Polysiphonia howei Hollenberg, Gelidiella acerosa (Forsskill) Feldmann & Hamel; Chlo- rophyta-Valonia ocellata Howe, Cladophora fuliginosa Ktitzing; Phae- ophyta-Padina sanctae-crucis B¢rgesen; Myxophyta-Hydrocoleum co- moides (Harvey) Gomont. A total of 13 isopods was found in four of 18 different collections, but Dynamenella never occurred free of Acanthopleura where the partnership had not been observed previously. For example, Dynamenella was not present in the various habitats examined at Station Nos. 1, 2, 24, and 25 along the north coast, nor did Acanthop/eura ever play host to the isopod at these stations. At certain localities where the 606 Bulletin of Marine Science [18(3)

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o 00 tr) '

t'- t'- 00 00 00 00 t'- N - - III0 Ir) tr) ", . .D .D 1:1- 00 00 III III ;::0\ 00 00 U. U...... - - z~ z~ z~ Z~ Z~ z~ oin oin r.,OONt--<"l<"l 00 r') tr) r') tr) ON '

OCCURRENCE OF THE Dynamenella-Acanthopleura PARTNERSHIP WITHIN THE TROPICAL WESTERN ATLANTIC PROVINCE Besides a common occurrence of the Dynamenella-Acanthopleura partnership in Puerto Rico and nearby islands, examination of chitons from other localities within the same zoogeographic province has revealed a common and wide-ranging distribution of the two species (Table 6). A total of 14 separate collections of A. granulata was examined in the Division of Mollusks at the Smithsonian Institution. The undersurfaces of dried and liquid-preserved chitons were searched for Dynamenella; also, any sediment present in wet collections was sieved. Eight of the collections so examined (USNM Cat. Nos. 113346-113353) contained Dynamenella. In spite of having been collected and preserved with a sole interest in the mollusk, and in certain cases cleaned (soft parts cut out), even the oldest chiton collections contained a fair number of the isopods. One such collection from Knights Key, Florida, made around 1885, showed an average density of 3.7 Dynamenella per individual host chiton. Two additional sources of information on the partnership, obtained from Richardson (1912, USNM Cat. No. 43221) for Jamaica and from a recent collection made by C. Cutress (personal communication) at Barbados, are collated with the museum records in Table 6. Two collections from the Swan Islands (likely from the same locality), northeast of Spanish Honduras, and one from Montego Bay, Jamaica, show that the two species live in association in the western Caribbean Sea. The partnership was observed in the Lesser Antilles at Barbados, too, eastern- most of the Windward Islands. Both species have been found together in four collections from the Florida Keys, and their presence in one collection each from the Turks Islands, Bahama Islands, and from Baracoa, Cuba, indicates a common occurrence in the tropical Atlantic Ocean as well. Dynamenella was not present in chiton collections from Pelican Island (two different samples) and Lower Matecumbe Key, Florida; Mariel, Cuba; Cubagua Island, Venezuela; and Cura~ao. However, failure to find D. perforata in six collections does not definitely rule out an absence of the partnership from a particular region. Two factors responsible for negative evidence of this kind are a possible accidental loss of the isopod through improper handling, and the presence of diverse microhabitats in 608 Bulletin 0/ Marine Science [18(3) TABLE 7 NUMBER OF SPECIMENS OF D. perforata WHICH SOUGHT HABITATION WITH Acanthopleura AND C. tuberculatus OVER A 4.5-HOUR PERIOD*

Acanthopleura Chiton Number of Number of isopods Number of Number of isopods chitons per chiton host chitons per ch iton host

3 0 2 0 2 2 I I I 4 2 2 1 5 I 4 2 7 3 5 1 8 1 30 Total 10 35 10 54

• The chitans employed ranged in wet weight from 10.12 gm to 30.02 gm for A call1hopiellra and from 7.63 gm to 25.51 gm for Chiloll. the same general vicinity, which may selectively favor a high density of the host species only.

OCCURRENCE OF Dynamenella WITH Acanthopleura IN RELATION TO BIOLOGICAL AND ECOLOGICAL FACTORS The Host Species.-A fundamental question that had to be answered regarding the preferential association of Dynamenella with Acanthopleura was whether or not the isopod possessed a greater affinity for the more frequently inhabited host. The method adopted to study this problem was designed to simulate as closely as possible conditions thought likely to influence the animals in their natural surroundings. Ten average-sized individuals each of Acanthopleura and Chiton tuberculatus were allowed to attach and accustom themselves for one-half hOllr on boulder-sized pieces of coral rubble submerged to a depth of 25 cm in a fiberglass tank of 80 liters capacity. Two hundred individuals of D. per/arata, representing all size groups, were then isolated from Acantho- pleura, introduced into the tank, and allowed an adjustment period of two hours. Water was siphoned from the tank at a constant rate of 10 em/hour, roughly equivalent to the maximum rate of tidal recession at La Parguera (D. S. Dept. of Commerce, 1965), until all the water was drained off. The entire operation was carried out during the daylight hours in the shade out-of-doors. The number of isopods per host which sought refuge under the two chiton species over the 4.5-hour period are listed in Table 7. A chi-square test for significant differences failed to show any particular preference by Dynamenella for either of the chitons, at least over the interval of time 1968] Glynn: Studies on the Associations of Chitons 609 TABLE 8 COMPARISON OF THE VOLUMES OF THE PALLIAL GROOVES IN Acanthopleura AND C. tuberculatus ON THE BASIS OF A VOLUME TO WEIGHT RATIO

AcalltllOpleura Chitoll Weight Mean Vol. to Weight Mean Vol. to (gm) vol. (ml) wt. ratio (gm) vol. (m1) wt. ratio 4.33 0.07 ± 0.04 0.02 3.81 0.31 ± 0.04 0.08 9.81 0.28 ± 0.11 0.03 4.56 0.42 ± 0.10 0.09 ]0.05 0.29 ± 0.09 0.03 7.27 0.37 ± 0.08 0.05 15.94 0.55 ± 0.21 0.03 10.14 0.77 ± 0.06 0.08 17.30 0.51 ± 0.24 0.03 13.83 0.77 ± 0.16 0.06 ] 7.73 0.49 ± 0.18 0.Q3 16.01 1.39 ± 0.20 0.09 22.45 2.11 ± 0.30 0.09 18.87 0.98 ± 0.09 0.05 25.58 0.74 ± 0.]7 0.Q3 25.3] ] .86 ± 0.45 0.07 32.86 1.80 ± 0.3] 0.05 33.08 2.16 ± 0.47 0.07 44.]6 2.92 ± 0.60 0.07 46.11 4.04 ± 0.29 0.09 tested.2 Of the 200 isopods released, 89 were recovered from under chitons, one on the outer surface of the girdle of Acanthopleura, and the remainder from the coral rocks. A total of 35 isopods was present on Acanthopleura, and 54 on Chiton. A very high density of 30 isopods per host was observed on one individual of Chiton, in contrast to 13 specimens of Dynamenella per C. tuberculatus, which was the highest density ever observed under natural conditions. Further observations on the ability of isopods to remain with the two chiton species when actively moving (see below), coupled with the occurrence together of Dynamenella with both chiton species in the field, clearly show that some factor or factors other than a preferential attraction to the host must be responsible for the greater density and more frequent association of the isopod with Acanthopleura. Pallial Grooves.-Gross appearance of the pallial grooves in the two different chiton species sometimes gives the impression that this structure is more capacious in Acanthopleura than in Chiton. Thus it was tentatively hypothesized that Acanthopleura can accommodate a greater density of isopods by virtue of its larger pallial grooves which accordingly provide a more voluminous living space. This possibility was examined by compar- ing the volume of water present in the pallial grooves (actually on the entire undersurface of the animal) in the two species (Table 8). The tabulated mean volumetric values, with their corresponding standard deviations, are actually a measure of the liquid retained in the pallial grooves plus a lesser amount which adhered to the undersurface of the chiton. Since variation was high within a set of measurements, five separate

2 Statistical analysis here and throughout the remainder of the paper is based on a probability level of p ~ 0.95. 610 Bulletin of Marine Science [18(3)

ACANTHOPLEURA CHITON 10 ••• •• • +5 •••• •

I mean sea level 0 •••• • • ::E •• C,) -5 •••• ~ ;1 J: c.:l ••••• W :I: 10 •• ...I • • • • 0 0 • • o • 0 0 0 o 0 o 0 20 • 0 0 0 o 0 0 0 o • 0 25 o 0 0

0 30

Legend: 0 Dynomenella absent • < 10 Oynomenel10 • 10-20 Dynomenello • > 20 Oynom.e nella

FIGURE 11. Vertical distribution of 37 individuals of C. tuberculatus and 50 individuals of Acanthopleura in relation to the presence or absence of Dyna- menella perforata. Four distinct symbols indicate the number of Dynamenella associated with each chiton. These observations were made at Punta Peiiones, La Parguera (Sta. No.9, Table 3) during the placid morning of Sept. 22, 1966. At the outset the water level stood at 15 cm below MSL. Heights of individual chitons were obtained from their measured distance above or below a line held horizontally at the surface. All measurements were corrected with reference to the permanent tidal trace obtained at the U. S. Coast and Geodetic Survey tide station operated by the Institute of Marine Biology on Magueyes Island. The tide ascended only 3 cm over the period of observation. 1968] Glynn: Studies on the Associations of Chitons 611 determinations were carried out on each animal according to the following procedure. Animals were placed on the concave surface of a watch glass (100 mm diameter) submerged in water in an inverted position so as to expel all air bubbles. Usually 3-5 minutes were required before the pallial grooves became fully expanded and began to circulate seawater. The girdle was then tapped gently all around so that the water taken up by the chiton would be retained once the animal was exposed to the air. The upper surface of the chiton, as well as the entire exposed glass surface, was immediately blotted dry with a cloth towel. The chiton was then slipped off the edge of the glass and the undersurface allowed to drain into the concavity by tapping the animal while held in a vertical position for 10 seconds. The weight of the liquid retained was then quickly determined. Before each animal was slipped off the glass it was made certain that the pallial grooves were filled with water so that no loss in volume would occur from the blotting. All animals were collected at La Parguera except for the three heaviest individuals of Chiton, which were obtained from the rock seawall at the north end of Mayagi.iez Bay. Contrary to the expected outcome, the ratios of the mean volume of water retained to the weight of each chiton reveal unequivocally a greater living space in Chiton. The null hypothesis of an equal volume/weight ratio in the two species was rejected on the basis of a calculated T-statistic of 3.4, compared with the tabular value, To.o2r;(18) = 2.101. In Chiton, five individuals demonstrated a quotient of 0.08 to 0.09, whereas only one individual of Acanthopleura had a value in this range. The mean quotient within each group of Chiton and Acanthopleura, respectively, was 0.07 and 0.04. Therefore the notion that insufficient living space on the undersurface of Chiton acts in limiting the presence of Dynamenella must be discarded. It is also evident that the volume-to-weight ratio does not vary with size. Height on Shore Occupied by Host Species.-Although both chiton species inhabit a rocky substratum on the wave-swept shoreline, a distinct difference does exist in the intertidal height occupied by each species and the consequent time relations of submersion and exposure. In general, Chiton occupies a lower level on the shore, subject to only infrequent and brief periods of exposure. The center of distribution of Acanthopleura is located at a higher level on the shore; at low water, the bulk of the population is fully exposed to the atmosphere. To determine the effect of vertical height on the partnership, two collections were made at one locality along a protected shore (Pta. Peiiones, Sta. No.9) where the height occupied by each of the two chiton species was measured in relation to the presence or absence of Dynamenella (Fig. 11; observations made earlier, on Nov. 2, 1965, are not shown). The overlapping but different levels maintained by the two chiton species 612 Bulletin of Marine Science [18 (3) in the intertidal zone are well illustrated. The highest level occupied by Acanthopleura on Sept. 22, 1966, was 10 em above MSL (mean sea level). Of particular interest is a tendency for the isopod-chiton partnership to occur more frequently at the higher levels occupied by each chiton species. On Nov. 2, 32 Acanthopleura showed a vertical range from +35 em above MSL to -16 em below MSL. The three chitons which lacked isopods were located at -2 em, -4 em, and -12 em below MSL. Thirty-eight individuals of Chiton ranged intertidally from + 3 em to -30 em below MSL. The four Chiton infested with Dynamenella were located at -6 em (two animals), -7 em and -12 em below MSL. The data in both collections showed a rather close agreement, although the location of Acanthopleura at a lower level on Sept. 22 probably resulted in part from the displacement of chitons sampled previously. A tendency for a greater density of isopods per chiton at the higher levels can be seen in the collection made on Sept. 22 (Fig. 11). Eleven Acan- thopleura collected between -10 em and +5 em contained more than 20 Dynamenella each. Of the 17 Acanthopleura taken at or below the -1O-cm level, only one contained as many as 20 isopods, and eight, or 47 per cent, contained less than 10 isopods each (Dynamenella was altogether absent from one of these). No specimen of C. tuberculatus was observed with 10 or more isopods. The higher distribution on the shore of the 13 specimens of Chiton which possessed Dynamenella is evident. These results indicate that Dynamenella prefers a slightly higher intertidal level than that occupied by the center of distribution of Chiton. Height on Shore Occupied by Symbiont.-A gradual disassociation of Dynamenella from Acanthopleura which had been moved from the usual wave-splashed habitat to a depth of 60-70 em below MSL lends further support to the idea that there is a preference by the isopod for a comparatively high level on the shore. Frequency distributions of the number of isopods per relocated chiton, over a total period of 16 days, are illustrated in Figure 12. Seventy-two per cent of the 25 individuals of Acanthopleura initially sampled in the normal habitat contained more than 20 specimens of D. perforata. The contribution of classes depicting more than 20 isopods per chiton decreased considerably by the second and fourth days. By the eighth and sixteenth days the modal classes of less than 10 isopods per chiton displayed frequencies of 75 per cent and 67 per cent, respectively. Chitons similarly relocated at a subtidal level on Sept. 7, 1966, essentially corroborated the above results. Twenty-five chitons were examined for the symbiont at the outset (0 days) and 15 chitons on each occasion after 2, 4, 8, and 16 days. The modal class within each distribution gradually shifted from> 20 to < 10 isopods per chiton over the 16-day period. The actual percentage contributions of the modal classes to each distribution demonstrated the following chronological series: 44 per cent (> 20), 47 1968] Glynn: Studies on the Associations of Chitons 613 TABLE 9 NUMBER OF SPECIMENS OF D. perjorata WHICH BECAME ASSOCIATED WITH Acanthopleura AFTER A PERIOD OF 16 DAYS (J AN. 11-27, 1966) *

N umber of chi tons Number Numerical classes sampled after of isopods (isopods per Frequency 16 days per chiton chiton) (%)

2 1 2 2 2 3 1 4 <10 56 2 5 2 6 2 8 1 9 2 12 1 13 10-20 20 I 17 1 18 3 22 1 33 > 20 24 1 47 1 48

• Forty chitons free of isopods were transplanted from a rocky promontory near the Cabo Rojo Lighthouse (Sta. No. 11) to the coral rubble shoreline on Turrumote Reef (Sta. No.8). Chitons indigenous to the study area at the western end of the reef, and the stones to which they were attached, were moved about 10 meters down the shore. Enumeration of isopods per chiLon was carried out in the field. per cent (> 20), 67 per cent (LO-20), 40 per cent (10-20), 67 per cent «10). The opposite effect, resulting from the displacement of Chiton tuberculatus to a higher level on the shore, was difficult to observe owing to the rapidity with which this chiton attains its former, lower level. After eight days, only 10 chitons were recovered out of 35 that were initially detained a few cm above MSL with cotton netting. Three of the chitons contained a single isopod each, and seven were free of isopods. Transplantation of specimens of Acanthopleura free of isopods to a locality known to possess a dense population of Dynamenella showed a rapid rate of association if placed at the usual level occupied by the chitons in the intertidal zone (Table 9). Every chiton examined after a period of 16 days contained at least one isopod. Fourteen chitons, or 56 per cent of the total sampled, contained less than 10 isopods per individual, but six chitons, or 24 per cent, possessed more than 20 isopods, and as many as 47 and 48 isopods were associated with two chitons within this class. Modal classes of more than 20 isopods per chiton made up 72 per cent 614 Bulletin of Marine Science [18(3)

DAYS

(25) 80 60 0 40 20

80 60 2 40 20

~0 ...... • 80 >- 4 () 60 c: CIl 40 ~ l:T 20 CIl•.. I.L. ((2) 80 60 8 40 20

(12) 80 60 16 40 20

o <10 10-20 >20

Number of Isopods

FIGURE12. Frequency distributions of the number of Dynamenella found with Acanthopleura at the level on the shore it normally occupies (0 days), and after 2, 4, 8, and 16 days at a subtidal level (Turrumote Reef, La Parguera; Oct. 16- Nov. 1, 1965). Number of isopods per chiton are grouped into the four classes indicated at the bottom of the figure. The number of chitons sampled on the five different occasions is indicated in the upper right-hand corner of each distribution. 1968] Glynn: Studies on the Associations of Chitons 615 (Fig. 12) and 44 per cent of two samples examined previously from the same locality. The isopods associated with six of the transplanted chitons were recounted after 48 days with the following results: 4, 10 (in each of two chitons), 12, 20, and 32. Parallel observations on 20 individuals of Acanthopleura introduced into deeper water (about 3 meters seaward and 0.5 meter deep) failed to provide additional significant information because only three individuals were recovered after 16 days. One chiton was devoid of Dynamenella, and the other two contained one and five isopods each. Although the isopods which became associated with the chitons were not measured, comparison with earlier observations on the sizes of Dyname- nella living under unmolested chitons (e.g., Fig. 2) clearly indicates that all size groups were represented. This leads one to the assumption that all members of a Dynamenella population are quite mobile and possibly exchange hosts at frequent intervals. At least this was the case with animals maintained in captivity. On Dec. 10, 1965, 100 specimens of Acanthopleura devoid of Dyname- nella were again transplanted from the Cabo Rojo Lighthouse area, but on this occasion to the eastern end of Enrique Reef, La Parguera. The coral rubble shoreline at this locality has a similar appearance to that at Tur- rumote and Laurel Reefs, where the isopod-chiton partnership always showed a high incidence. One striking difference, however, was a near absence of Acanthopleura from the eastern end of Enrique Reef. Only two small individuals were found after a thorough search of the area. Seventy- five of the chitons were placed on rocks in the splash zone, and 25 subtidally to the lee of the rubble islet on rocks at a depth of about 0.5 meter. Twenty-seven chitons were examined from the splash zone after 16 and 32 days, and none was found with the isopod. Only nine chitons, all devoid of the isopod, were recovered from the subtidal habitat after 16 days. It seemed likely that Acanthopleura suffered from the large amount of fine sediment present, since five dead chitons were found and those alive appeared in an unhealthy condition with much particulate matter deposited in the pallial grooves and on the gill filaments. The reasons for the absence of Dynamenella from the wave-washed, coral rubble habitat are not readily apparent. A possible explanation might be related to a dependence of the isopod on a relatively abundant host population, which was absent from this locality. Throughout this study Dynamenella has never been collected from an area devoid of Acanthopleura, although a few of the isopods have been collected independently of the chiton from the same locality. In summary, the following may be concluded in connection with the above observations: (a) The density of isopods per chiton had attained a level of about half the number usually present after 16 days. (b) Asso- ciation of small and large isopods with Acanthopleura after only a few 616 Bulletin of Marine Science [18(3) TABLE 10 TABULATION OF HIGHEST VELOCITIES ATTAINED BY CHITONS ON A WET, SLOPING (15° ANGLE FROM THE HORIZONTAL), CONCRETE SURFACE IN FULL MIDDAY SUNLIGHT

A canthop/eura granu/ata Chiton tuberculatus Length of mov- Speed* Length of mov- Speed ing animal (em) (em/min) inganimal (em) (em/min) 5.4 1.0 4.5 10.1 8.0 2.2 5.5 0.9 4.9 6.1 0.9 4.7 5.7 2.3 5.4 12.9 5.8 2.2 5.5 5.5 2.0 4.5 2.3 18.9 7.0 1.8 5.6 .10.2 0.9 9.7 • Devious courses were traced with a string which was then straightened out and measured with a centimeter rule. The speeds were usually determined over one-minute intervals of time. As many as three trials were recorded for some of the chitons. days indicates that the young released by a female are not necessarily restricted to a single chiton throughout their life span. (C) The isopod is a facultative symbiont with a strong preference for living in association with the chiton when it is present. Locomotory Behavior.-Chiton is a much more active species than Acan- thopleura. It browses at a relatively fast pace and executes a rapid escape response when subjected to an abrupt change of conditions, for example, a sudden exposure to bright light. Measurements of the maximum rates of progression of the two species of chitons during a typical escape response illustrated the more sluggish behavior of Acanthopleura (Table 10). Crawling velocities of Dynamenella (Table 11) were nearly equivalent to the speeds attained by Chiton. While uninterrupted swimming speeds along a straight course could not be obtained, extrapolations based on brief movements demonstrated a rate of around 200 to 300 em/min. Once Dynamenella had become associated with either chiton species it would seem that quick maneuvering of the host could possibly result in an expulsion of the isopods. The following experiment was designed to test this hypothesis. Isopods were allowed to associate with chitons in wide-mouthed glass bowls filled with sea water to a depth of 1 em. Thirty isopods were introduced into each bowl containing a chiton and were allowed two to six hours 1968] Glynn: Studies on the Associations of Chitons 617 TABLE 11 TABULATION OF HIGHEST VELOCITIES ATTAINED BY INDIVIDUALS OF D. perforata INTRODUCED ONTO A DRY GLASS SURFACE IN A DROP OF WATER

Length of animal (mm) Speed* (em/min) 1.6 4.4 1.9 4.9 2.0 9.2 2.1 4.5 2.2 7.2 2.3 10.5 2.4 8.0 2.6 5.2 2.7 6.8 2.9 7.5 * Each animal was allowed to run for one minute, the sinuous trail was marked with a wax pencil, and the distance covered was traced with a string which was placed on the trail and then straightened out and measured with a centimeter rule. The one-minute run was short enough so that the animals were not noticeably slowed down from loss of moisture; actually, considerable adhesive water was always carried along. Most of the isopods stopped momentarily for a few seconds during the one- minute period. to take up refuge under the host. The water was then drained from the bowl, and the partnership exposed to direct sunlight for one-half hour in order to encourage a quick escape response. The numbers of isopods disassociated from the host, as a result either of voluntary movement away by the isopod or of abandonment of the isopod by the chiton, are listed in Table 12. All animals were collected the day before study at Turrumote Reef, La Parguera. An attempt was made to distinguish between isopods disassociated from chitons through their own activities, since these appeared to be unrelated to expulsion brought about by the accelerated behavior of the host. Isopods so listed often moved away from the host quickly, and during the initial few minutes of observation. Isopods actually abandoned by the host were usually less active and simply remained behind in the film of water left by the chiton. First of all, it was once more demonstrated that no preferential selection of either chiton species as a host was exercised by Dynamenella. A total of 101 isopods became associated with Acanthopleura and 108 with Chiton. Also, it is clear that isopods which moved away voluntarily from the two chiton species did so with equal frequency. A total of 13 individuals of Dynamenella disassociated voluntarily from Acanthopleura, as opposed to 14 which moved away from Chiton. Those isopods remaining with the host hung on to the mantle with their forward pereopods, or moved along in the water film retained on the undersurface of the chiton. Of greatest interest was a complete lack of involuntary abandonment of Dynamenella from Acanthopleura. In Chiton, however, from one to six 618 Bulletin of Marine Science [18(3 )

TABLE 12 COMPARISON OF DISASSOCIATION RATES OF Dynamenella WITH Acanthopleura AND C. tuberculatus UNDER DIFFERENT CONDITIONS OF ACTIVITY OF THE HOSTS

Isopods disassociated from host Total number Voluntarily Involuntarily isopods under chiton No. % No. % Host activity 24 1 4 0 0 Inactive. 12 0 0 0 0 Inactive. 16 2 12 0 0 Inactive. 7 1 14 0 0 Inactive. 4 0 0 0 0 Inactive. Acanthopleura 7 0 0 0 0 Active, rotating. 11 0 0 0 0 Active, rotating. 6 1 17 0 0 Active, slowly moving forward. 6 3 50 0 0 Active, slowly moving forward. 8 5 62 0 0 Very active, rapidly moving forward. Total \01 13 0

27 2 7 0 0 Slightly active. 5 2 40 0 0 Slightly active. 5 2 40 0 0 Slightly active. 4 0 0 0 0 Slightly active. Chiton 5 0 0 I 20 Active, moving forward. 12 3 25 3 25 Active, moving forward, rotating. 18 2 11 0 0 Active, moving forward, rotating. 11 0 0 6 55 Very active, rapidly moving forward, rotating. 12 8 6 50 Very active, rapidly moving forward, rotating. 9 2 22 2 22 Very active, rapidly moving forward, rotating. Total 108 14 18 1968] Glynn: Studies on the Associations of Chitons 619 isopods were left behind by five of the most active hosts. Further, none of the four inactive individuals of Chiton abandoned the isopod, but a total of 18 isopods was abandoned by the six active animals; one active Chiton did not leave any isopods behind. These results demonstrate a significant influence of the locomotory behavior of the host species on the density of isopods associated with the two chitons. It appears that the fewer symbionts associated with C. tuberculatus is due to the lower, vertical level maintained on the shore by the host and to its greater activity.

SYMBIOTIC RELATIONSHIP Protection.-Association of Dynamenella with Acanthopleura located relatively high in the intertidal zone must play a beneficial role in easing the predation pressure of aquatic carnivores. The only animal ever found feeding on Acanthopleura, including such land forms as birds and crabs, was the gastropod mollusk Nitidella ocellata Gmelin, 1791, which had attacked the mantle of small chitons located under cobbles near the water- line. Because Dynamenella can survive only brief periods of exposure in the absence of shelter, the extent to which association with Acanthopleura enhances survival from extreme physical factors is of importance in per- mitting the isopod to occupy a higher level on the shore. In La Parguera, where the tides are chiefly of an irregular, diurnal character (Coker & Gonzalez, 1960), Acanthopleura is normally wetted at least once a day, or exposed and subject to drying for a maximum period of about 20 hours (individuals located near the upper limit of vertical distribution). An experiment was undertaken to test the tolerance of specimens of Dynamenella present under chitons that were exposed to drying. The procedure was as follows. Dead coral rubble containing chitons at rest was transported above the splash zone from the wave-swept shoreline in the daylight hours of the morning at Turrumote Reef, La Parguera. The rubble was placed as it was found at the lower level on the shore so as not to disturb the relative position of the chitons. Most of the chitons were located on the undersurfaces of the rocks. The chitons ranged in wet weight from 6.88 gm to 26.45 gm. Black Flag insecticide was used to encircle the rubble (at a minimum distance of about 1 meter from the chitons ) to prevent scavenging by terrestrial forms. At the end of each period of exposure the animals were immersed in seawater and allowed to recover for six hours. Lack of movement of pereopods and pleopods was employed as the criterion of death for the isopods; death in chitons was judged from lack of movement of the foot and mantle. Observations were carried out from July 29 to Aug. 15, 1966, during which time normal weather conditions prevailed. Cloud cover was usually less than 20 per cent, maximum wind velocity was about 40 km/hr from the southeast, and the maximum, midday temperature recorded was 32.4 °C. The relative humidity was not 620 Bulletin of Marine Science [18(3)

~ "0 N -de 0 I£) 0\ 0\ \D \D --0 n.l 0 r- 0 00 ;;iE"" Cl '" \D ....., ....., N \D r- oo Cl N u" 0 \0 - .- ~ - "'0 0\ .5~ ~ 00 "-l n.l ....., OJ ~ .:: N 00 """000 000 "< ._;~0. •• n.l Uo. Cl .•.. :J;" 0 "0 "-lCIJ ~~ ~ (\j 0 00- N N- ....., r- ....., 0\ r--: ....., 0", n.l N ....., "," . Q.. .9 -0"''' >< 0 0°0. X 'oj "., 0 '"~ :Ooe 0.. ::l.<: Q 0_ ~° 0 E ,g •...... 0 (l) "' ... ~ .~ •...... c c ,,0'"- •.•... ;; 0 4-< U .0 dl .- "0 u "'~o ...I 0 o;j E •.... ,,"'J: '-•.... .0 C;; C;; o='~ >' E 0 :~ .c!:~ ~ :l U "0 u"u :l

SUMARIO

ESTUDIOS ECOLOGICOS DE ASOCIACIONES DE CHITONES EN PUERTO RICO CON REFERENCIA ESPECIAL A ISOPODOS ESFAEROMIDOS Se describen cinco asociaciones simbi6ticas is6podo-chit6n, ej.: Dyna- menella perforata con Acanthopleura granulata y con Chiton tuberculatus, Exosphaeroma (a) con C. tuberculatus, Exosphaeroma (b) con C. marmoratus y Dynamenopsis dianae con C. tuberculatus. La camara inhalante del surco paleal fue el lugar mas frecuentado por los is6podos. Otros organismos descritos en simbiosis con chitones son: "Acervu/ina inhaerens" (Foraminiferida), Harpacticus sp. y Heterolaophonte (Harpacti- coida), Parhyale hawaiensis (Amphipoda) y Actaletes neptuni (Collem- bola). Se muestra que Dynamene moorei es conespecifica con Dynamenella perforata. Los reportes de Jamaica, Cuba, Florida, Islas Bahamas y Bar- bados indican que la asociaci6n Dynamenella-Acanthopleura esta muy extendida. Se muestra que Dynamenella ocurre comunmente con chitones 1968] Glynn: Studies on the Associations of Chitons 623 que viven cerca de manchas de sedimentos de grano grueso; el canicter general del habitat y el grade de exposici6n a la acci6n de las olas, no parece influenciar la ocurrencia de la asociaci6n. Una asociaci6n mas intima de Dynamenella con Acanthopleura se debe aparentemente a que el huesped ocupa un nivel mas alto en la orilla y su comportamiento es mas perezoso. Los organismos simbi6ticos demostraron una alta tolerancia a la desecaci6n bajo chitones expuestos a la atm6sfera. Dynamenella se orienta entre fila- mentos branquiales individuales, mientras el chit6n se alimenta y parece que las algas ingeridas por el inquilino son inicialmente raspadas del substratum por el huesped. Los is6podos son considerados como comensales facultativos con una fuerte atracci6n por Acanthopleura.

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