LIFE CYCLE OF OSTREA PERMOLLTS AND ITS RELATIONSHIP TO THE HOST SPONGE, STELLETT A GRUBIJl
MILTON L. FORBES Lamar State College of Technology, Beaumont, Texas
ABSTRACT Anatomical and functional relationships of the commensal oyster, Os/rea permollis, to its host sponge were studied and common associates, especi- ally predators, were identified. O. permollis larvae were reared for study of responses to: host sponge, non-host sponges, and shell. Recruitment in inshore waters was detected by presence of O. permollis spat on S. grubii during June and November of the years studied. Age-groups were identifi- able in size-frequency histograms for over a year following recruitment.
INTRODUCTION Os/rea permollis Sowerby is a commensal oyster in the sponge Stelletta grubii Schmidt. Adult O. permollis live crowded on the surface of S. grubii or embedded with shell margins protruding. Occurrence of O. permollis in sponges implies a selective advantage for its ability to inhabit sponges. The present study inquires into the existence of dependence on the host sponge and adaptations of the oyster to its host. Review of the Literature.-A previous paper (Forbes, 1964) deals with identification and distribution of O. permollis and S. grubii. The oyster- sponge association is common subtidally in the northeastern Gulf of Mexico. O. permollis has been found only in S. grubii, except once in Halichondria, itself attached to S. grubii. Mantle margins and conchiolin of the relatively fragile shells are golden yellow; otherwise O. permollis resembles other Os/rea species in all essential anatomical features (Fig. 1). General aspects of the biology of Ostrea and the other oyster genera are thoroughly discussed by Korringa (1941,1952), Thomson (1954), Menzel (1955), Nelson (1957), Yonge (1960), and GaltsofI (1964).
PROCEDURES Oyster-sponges (S. grubii with O. permollis) were collected at St. Teresa, Franklin County, Florida; in Alligator Harbor nearby; and near Buoy 26, in the Gulf of Mexico about 10 miles south of Alligator Point. The localities are described elsewhere (Forbes, 1964). Much of the
'Contribution No. 212 from the Oceanographic Institute. Florida State University. From a disser- tation submitted in partial fulfillment of the degree of Doctor of Philosophy at Florida State University. Part of the "ork was done during tenure of a predoctoral National Science Foundation fellowship and part during tenure of a Florida State University Graduate School fellowship. 274 Bulletin of Marine Science [16(2)
I em I i
vm rma h 9
1m
Iv
FIGURE 1. Anatomy of Ostrea permollis: right valve and mantle removed. a, anus; am, adductor muscle; cc, cloacal chamber; ds, dental socket; g, gills, occupying branchial chamber; h, heart; Ii, ligament; 1m, left mantle; Ip, labial palps; lv, left dissoconch valve; pc, pericardial cavity; pi, palliobranchial fusion; r, rectum; rmo, origin of right mantle; um, umbo; vm, visceral mass. 1966] Forbes: Life Cycle of Ostrea permollis 275 research was done at the Alligator Harbor Marine Laboratory of the Florida State University. Oyster-sponges and extracted oysters were kept alive in cages near the end of the Marine Laboratory pier between neap and spring low tide levels. When larvae were needed, 10-20 oysters were cleaned and placed individuaHy in 10 cm finger bowls of filtered sea water in the laboratory. Like other species of Ostrea, O. perm ollis is an incubatory protandric hermaphrodite with alternation of sexual phases. Dishes were checked daily for larvae when the water was changed. Throughout the year, about one in ten large oysters (20-40 mm) liberated a brood of thousands of straight-hinge larvae in about 5 days. Straight-hinge larvae were reared to metamorphosis by methods described by Loosanoff (1954), Davis & Guil1ard (1958), and Loosanoff & Davis (1963). Larvae were diluted to 20 per mI. Sodium sulfametha- zine (Sulmet, American Cyanamid Co.) was added as a bacteriostat in the ratio of 1: 10,000. The chrysomonad Monochrysis lutheri Parke was fed to the larvae at a final dilution of 100,000-200,000 cells per ml. Pediveligers (ready-to-set, or eyed, larvae) were identified by their foot, a pair of ocelJi, and occasionally by their creeping behavior. Pediveligers were selected with a fine pipette under a stereoscopic microscope (45 x) for studies on setting. Substrate samples were prepared for larval substrate specificity studies as foHows. Sponge cuttings of 5-10 mm dimensions were cut from Stelletta grubii, Microciona prolifera Verrill, and an unidentified keratosid that may be a Spongia. Fragments from a clean, dry shell of Crassostrea virginica (Gmelin) were designated "clean shell" and stored dry between experi- ments. Shell fragments with an algal coating were designated "fouled shell," Sponge cuttings and fouled shell were stored in sea water which was changed daily. The growth rate of O. permollis was estimated from size-frequency analysis of periodic collections and verified by periodic measurement of oysters in a sponge. The size-frequency method used here was adapted from the length-frequency method used in fish studies, where each age- group in a population is represented by a separate normal curve around a mean length (Lagler, 1952). Since oysters vary in shape, it seemed desirable in the present study to use a size index based on product of length times height, and then to take the square root to avoid exaggerating older age-groups. The size index, V L X H, approximates length since the length/height ratio is usually close to unity in O. permollis. Measure- ments were made in millimeters to two significant figures with vernier calipers. Length is the distance from the beak of the left valve to the posterior margin; height, the greatest dorsoventral dimension. Five collections were made for the size-frequency study: three from 51. Teresa, 276 Bulletin of Marine Science [16 (2) I em
co --ch
. '. ,... co A
. , .
. .
':::'::':~ ••...... /. ,':'..:.:'" ":':'~.:.: ~. ,".',. co B
FIGURE 2, Ostrea permollis in sagittal section, right valves in situ in the host sponge. Arrows indicate inferred path of excurrent stream, am, adductor muscle; b, bill of sheIl; ea, canal in sponge choanosome; eh, choanosome; co, sponge cortex; g, gill; n, nacre; ss, surface of host sponge; V III , visceral mass. 1966] Forbes: Life Cycle of Ostrea permoLlis 277
and two from the north shore of Alligator Harbor. The two stations were about a mile apart. Results from the size-frequency study were verified by measurements on individual O. permollis in a sponge over a 10-month period. Length was used for the latter determination since height could not be measured without extracting oysters.
OBSERVATIONSANDRESULTS Anatomical and Functional Relationships of O. permollis and S. grubii.- The specimens of Stelletta grubii were massive, usually spherical or elongated, and up to 20-30 cm in diameter. The surface was hispid, often strongly wrinkled, and usually encrusted with sand, broken shells, and algae. The cortex was 1-3 mm thick and densely packed with spicules (predominantly triaenes). The choanosome was relatively soft and full of canals. O. permollis and its empty valves were invariably located in or at the surfaces of oyster-sponges. All sponge centers were free of oysters and valves. Ostrea perm ollis specimens grew normally when isolated from S. grubii and maintained in Alligator Harbor or in sea water tables. Stomach contents consisted of phytoplankton, and O. permollis did not appear to depend on S. grubii for nutrition. ORIENTATIONOF OYSTERSIN S. GRUBH: Oysters appeared to be crowded haphazardly at the surface of the host sponge. Beaks lay near the sponge surface and pointed in all directions with respect to gravity. A stacking effect, several oysters lying parallel next to one another, was a frequent result of crowding. Orientations of 152 O. permollis in S. grubii were analyzed to determine whether oysters were arranged in any consistent way. Orientation was analyzed in terms of four variables: the angle of the oyster's longitudinal axis to the sponge surface; the angle of the dorsoventral axis to the sponge surface; whether the inhalant (ventral) region protruded or was embedded; and whether the exhalant region was exposed or embedded. These variables were estimated by eye. The longitudinal axes of all O. perm ollis were approximately paraJJel with the sponge surface. The dorsoventral axes of ] 38 oysters were 450 -900 to the surface. The dorsoventral axes of 14 oysters were 00 _450 to the surface. The inhalant regions of all oysters protruded from the sponge. Exhalant regions of 146 oysters were fully embedded; four were partly embedded; two were fully exposed. Hence, the essential factor in orientation of O. permollis is access of its inhalant region to the sea. WATER FLOW THROUGHO. PERM OLLIS ANDS. GRUBH: Sagittal sections of 30 preserved O. permollis and surrounding sponge were cut with a razor blade. Canals were clearly present in the sponge at the exhalant region of 278 Bulletin of Marine Science [16(2) TABLE 1 COMMON ASSOCIATES OF astrea permollis AND Stelletta grubii =·======-=.c= _ Alligator Harbor Gulf of Mexico (Buoy 26)
CRUSTACEA CRUSTACEA Neopanope texana texana Stimpson Balanus amphitrite Darwin Menippe mercenaria (Say) Synalpheus sp. Pilumnus dasypodus Kingsley Pilumnus sayi Rathbun Balanus amphitrite Darwin PeUa mutica (Gibbes) Balanus eburneus Bruguiere Petrolisthes galanthinus (Bose) Cilicaea caudata (Say) Zoeae and megalops larvae, Brachyura
POLYCHAETA POLYCHAETA Leodice rubra (Grube) Sabel/aria fioridensis Hartman Lepidonotus variabilis Webster Lepidonotus variabilis Webster EulaUa myriacyclum (Schmarda) Sthenlais articulata Kinberg Polydora websteri Hartman Poly dora websteri Hartman Hydroides sp. Eupomatus sp. Terebella sp.
MOLLUSCA MOLLUSCA Acanthochitona pygmaea Pilsbry astrea equestris Say astrea equestris Say Chama congregata Conrad Brachidontes exustus L. Pododesmus r!ldis Broderip j'vlodiolus americanus Leach Arca zebra Swainson Musculus lateralis Say Engina turbinel/a Kiener Diodora cayenensis Lamarck Mitrella lunata Say Urosalpinx perrugata Conrad Petaloconc/1us sp. Urosalpinx sp. Crepidula aculeata Gmelin Pleuroploca gigantea Kiener Crepidula fornicata Say Murex fiorifer Reeve Crepidula plana Say Murex pomum Groelin Diodora cayenensis Lamarck Crepidula plana Say Unidentified mytilids, juvenile Crepidula maculosa Conrad Unidentified venerids, juvenile Vermicularia knorri Deshayes
ECTOPROCTA ECTOPROCTA Bugula sp. Bugula sp.
ECHINODERMATA ECHINODERMA TA Unidentified ophiuroids Unidentified ophiuroids Arbacia punctulata, juvenile
SIPUNCULTDA Dendrostoma alutaceum Grube
COELENTERATA Cladocora arbuscula (Lesueur) Astrangia solitaria (Lesueur) Unidentified actinarians 1966] Forbes: Life Cycle of Ostrea permollis 279 each oyster (Fig. 2). Openings into the sponge were also revealed in the exhalant region by spreading the valves of preserved oysters with a razor blade. Dyes pipetted into the ventral gapes of live oysters in situ indicated the water flow through the oysters and sponge. The entire gape was inhalant in each case. Oysters collected carmine from a sea water suspen- sion and rejected it in mucus strings (pseudofeces). Sponge tissues sec- tioned at exhalant regions of three oysters so treated contained carmine and showed that the exhalant stream entered canals in the sponge. Neutral red (1 per cent) introduced into the ventral gape of an oyster in situ was discharged via the osculum 8-10 sec later. The treatment was repeated on three oysters embedded singly in pieces of S. grubii whose cut surfaces had healed. In each case neutral red entered the ventral gape and emerged from canals through the sponge. It is apparent that circulation of water in the usual ostreid fashion is possible in O. permollis because its inhalant area is open to the sea. GROWTH OF O. PERil10LLlS IN S. GRUBII; The initial stage of new shell formation in O. permollis is the deposition of a thin, flexible, yellow bill around the edge of the valve (Fig. 2). The bills consist of prismatic layer on the right valve and periostracum on the left. The bill is subsequently strengthened by addition of subnacreous deposits. The completed shell thus has the same structure as in O. edulis (Korringa, 1951a). Most of the yellow conchiolin of O. permo/lis is in the periostracum on the left valve and in the outer region of the prismatic layer of the right valve. The bill breaks off and adheres to the sponge when O. permollis is removed from its host. It seems remarkable that the host sponge does not interfere with addition of shell. The oyster's gaping probably spreads and gradually divides the sponge's choanosome. The exhalant stream probably follows any canals present. Shell growth is greatest posteriorly where the gape is greatest and at the protruding ventral margin. O. permol/is differs from other ostreids in that the left valve is rounded and without a scar of attachment to a substrate. Small oysters (to 15 mm) are usually round or oval and resemble O. edulis L. or O. lurida Carpenter in general shape. Larger O. permollis are usually extended ventrally (e.g., Fig. 2B). The ventral shell extension probably facilitates communication of the inhalant region with the sea when the sponge might otherwise overgrow the shell and is comparable to flared shell margins produced by ostreids in shifting mud. Unequal lengths of the dorsal and ventral rows of hinge teeth are also thought to reflect somatic adaptation of the oyster to its host. Hinge plates of 20 oysters (10-25 mm) contained 0-22 denticles (mean 9.0) in ventral rows, 0-9 (mean 3.6) in dorsal rows. Relict dorsal rows were often embedded in the translucent subnacreous deposits. 280 Bulletin of Marine Science [16(2)
Iv
es rm vc h ve m
FIGURE 3. Pediveliger of Ostrea permollis. Length 400/-to es, eyespot; t, foot; h, heel; lv, left definitive prodissoconch valve; m, mouth; ps, primary prodisso- conch; rm, right mantle; rv, right definitive prodissoconch valve; ve, velar cilia; ve, velum.
ASSOCIATESOF O. PERMOLLIS ANDS. GRUBIl: Associates were collected from an oyster-sponge from Alligator Harbor on July 15, 1958, and from ten oyster-sponges collected near Buoy 26 at 10m on July 2. Dr. Meredith Jones, Smithsonian Institution, kindly identified most of the polychaetes and several other animals. The more common associates are listed in Table 1. Nestling animals (mytilids, Crepidula) occupied nooks and crevices among the shells on sponges and probably do not affect setting or survival of O. permollis. Encrusting animals (barnacles, corals, various pelecy- pods) coated many of the shells and in some instances seemed to overgrow sponge surfaces from points of initial attachment. Encrustation over sponge surfaces seemed more common at the offshore station than in Alligator Harbor. Vermicularia was commonly found at the surface of the sponge and was sometimes abundant. Ophiuroids, errant polychaetes, and actinarians may well eat swimming or setting larvae but probably do not affect older oysters. Xanthid crabs, predaceous snails, and Polydora are well known oyster pests and are treated further. 1966] Forbes: Life Cycle of Ostrea permollis 281
CRAB.PREDATIONON O. PERMOLLIS: Of 82 oyster-sponges collected at St. Teresa or in Alligator Harbor, ten were hollowed out from the base. Two such sponges contained crabs (one Pilumnus sayi Rathbun and one Menippe mercenaria [Say]) of 5 em carapace width. The crabs probably entered openings in S. grubii when small and excavated the choanosome as they grew. Broken valves that projected into the burrows from the cortex evidenced predation from the interior of the sponges. Crabs prob- ably attacked O. perm ollis from burrows more easily than from the exterior since the sponge cortex is tough and the narrow protruding edges of oysters offer no grip. Large crabs apparently removed large pieces of sponge to attack oysters from the exterior in a few instances. Two Menippe carried out extensive predation in separate cages of oyster-sponges kept for 5 months. The two crabs (5-10 em in width) occupied burrows excavated after the sponges were put in the cages. Many of the O. permollis were destroyed as well as numerous adherent Crassostrea virginica. The remaining individuals of O. permollis were well embedded in the sponges and probably survived for that reason. Shell fragments bore numerous small pieces of regenerating sponges. The healthy condition of the sponge fragments indicated that predation on O. permollis indirectly aids in propagation of the host sponge. SNAIL.PREDATIONONO. PERMOLLIS: Drill holes were common in empty O. permollis shells. The most common drill on oyster-sponges in the Alli- gator Harbor area was Murex fiorifer. In a test of its ability to attack O. perm ollis, seven Murex were placed with two oyster-sponges in a sea water table. The sponges contained 19 O. permollis. Four C. virginica and one O. equestris were on O. permollis shells. The Murex attacked three O. permollis and three C. virginica in 2 weeks. All attacks were on shell areas not covered by the sponge. A concealing or protective effect of a layer of sponge over O. permollis seems probable. OBSERVATIONSONO. PERMOLLIS ISOLATEDFROMS. GRUBlI: A group of Os/rea permollis was isolated from sponges on June 5, 1958, and kept in a cage at the Marine Laboratory pier. By July 26, the O. permollis were heavily encrusted with O. equestris, C. virginica, and Balanus sp. Twenty O. permo/lis were cleaned and returned to the cage. They were densely overgrown by C. virginica and four were recently dead when examined October 23. The mudworm Polydora websteri and its burrows were very evident on all valves. Valves were so thoroughly riddled with Polydora burrows that some of them fragmented when opened, and internally they were covered by large "mud blisters." Such extensive damage was never seen in C. virginica or O. equestris. By contrast, the only evidence of Polydora in 20 O. permollis of comparable size (20-50 mm) growing in S. grubii was the presence of small mud blisters in four oysters. Mudworm infestation on O. perm ollis is not surprising since they are well known as 282 Bulletin of Marine Science [16(2) ps pS d ds
A c
B FIGURE 4. Definitive prodissoconch of cultured Ostrea perm ollis larva. Length 400p.. A, left valve, interior; B, left valve, exterior; C, right valve, interior; D, right valve, exterior. d, denticle; ds, denticle socket; ps, primary prodissoconch. serious pests of oysters and contribute heavily to mortality (Korringa, 1951b; review in Hopkins, 1958). It is concluded that S. grubii is impor- tant in protection of O. permollis from Polydora and probably also from 1966] Forbes: Life Cycle of Ostrea perm ollis 283 fouling organisms. Protection probably led to degeneration of the protec- tive value of the oyster's shell through relaxation of selection. O. permollis shells are rather fragile. Development of O. permollis.-O. permollis ova are 60-80p. in diameter and hence much smaller than O. edulis, O. lurida, and O. denselamellosa ova (105p. [Seno, 1929]). Cleavage is total, unequal, and spiral in O. permollis as also shown in O. edulis (Horst, 1883, 1886), O. densela- mellosa (Seno, 1929) and O. lurida (Hori, 1933). Embryos develop on parental gills until liberated as straight-hinge larvae 108-127p. in length. Veligers cultured at 22°-24°C attained the pediveliger stage and began setting 30-33 days after liberation. The pelagic stage was long compared with 15-16 days in O. edulis at 18°-20°C (Loosanoff & Davis, 1963), probably due to differences in cultural conditions. The pediveliger stage of O. permollis closely resembles that of O. edulis, described by Erdmann (1934), but with some differences in coloration (Fig. 3). The digestive gland is yellow with spots of black pigment. A black band covers the base of the foot, belly, and base of the velum. The rest of the body is white to grey. The bilobed byssal gland was made visible with methyl orange after fixation. The byssal gland is spherical in O. edulis (Erdmann, 1934). A fine byssal thread often issues from the heel and trails behind swimming larvae. Ranson (1960, and discussed by Galtsoff, 1964) found the definitive prodissoconchs (shells of pediveligers) to be of taxonomic value in ostreids. O. permollis prodissoconchs were cleaned with sodium hypo- chlorite (commercial bleach) and examined in glycerine. Prodissoconchs (Fig. 4) were indistinguishable from O. equestris prodissoconchs reared under similar conditions. Samples of pediveligers averaged 300 to 360p. in length. Prodissoconchs on shells taken from oyster-sponges were smaller (average, 290p.). BEHAVIOROF O. PERMOLLIS PEDIVELIGERS:Pediveligers taken out of culture vessels were observed to swim about, lie on the bottom, or creep upon debris or clumps of algae. Larvae exhibited a weak photonegative kinesis in watch glasses. Creeping larvae frequently paused and contacted the substrate with the tip of the foot as though testing it. Behavior of O. permollis pediveligers placed in a watch glass was not affected by addition of a cutting of S. grubii. If pediveligers were placed on S. grubii, about 10 per cent began to creep and set within a few minutes. The remainder eventually swam away. Placing a cutting of S. grubii near a creeping larva had no effect unless it touched its foot to the cutting. The setting process consisted of three phases as also observed in C. virginica (Nelson, 1924; Prytherch, 1934) and in O. edulis (Cole & Knight-Jones, 1939). Duration of the creeping phase on S. grubii was 284 Bulletin of Marine Science [16(2)
50
ST ELLE TT A
SHELL 40
30 GLASS I- « Q.. If) 20 •...... ··Choice
10
o o 2 3 4 5 0 2 3 4 5 0 2 3 4 5
DAYS
FIGURE 5. Setting rate of Ostrea permollis larvae on Stelletta grub ii, fouled shell, and interiors of glass containers. See text for experimental conditions.
5-30 min (mean, 14 min). Byssal threads laid down on the substrate were detected by disturbing the larvae with needles. The foot became shorter and thicker as the larva entered the second phase. The foot was alternately extended to the left and contracted to produce a roughly counterclockwise movement. Nelson (1924) stated that the heel gripped the substrate, while Prytherch (1934), and Cole & Knight-J ones (1939), said that the tip held to the substrate. Both tip and heel appeared to grip the substrate in O. permollis. This phase varied from 2 to 15 min (mean, 5 min). In the final phase of setting the pediveliger tilted its body to the left, and the foot protruded slightly from the ventral gape of the shell and became spatulate. After a few seconds to a minute, the entire body contracted spasmodically several times and the foot was withdrawn. Nelson (1924) and Prytherch (1934) concluded that the byssal gland discharged cement between the left valve and substrate during the contractions. In O. perm ollis a byssal secretion likewise cements the left valve to the substrate. The cement was seen with difficulty but appeared to be a whitish material. The prodissoconch appeared to be cemented to the soft tissue of the 1966] Forbes: Life Cycle of Ostrea permollis 285 TABLE 2 COMPARATIVE SETTING RATES OF Ostrea permollis LARVAE UPON Stelletta grub ii, FOULED SHELL AND GLASS CONTAINERS
ax" = 11.75; P<.OI; b~" = 8.53; P<.OI; eX' = 1.39; P>.05; dX" = 5.67; P<.OS. Chi-square test for association applied to setting on Stellella grub;; and fouled shell and calculated in a single-classification table (1 d. f.).
sponge rather than to spicules. The setting reaction in O. permollis was in all respects as in other ostreids. SETTING ON SUBSTRATESOTHER THANS. GRUBII: A few larvae set upon inner surfaces of plastic culture vessels. Larvae also set in numbers in glass culture vessels when a coating of slime had built up after a day or two. O. permollis spat on glass were al10wed to grow to a size of about 8 0101. Their valves were circular and flat against the glass; they closely resembled O. equestris and C. virginica spat but for their golden color. Some larvae adhered loosely to glass vessels by an organic deposit thought to be the byssal secretion covered with algae. If so, they had not tilted to the left as in normal setting but remained in a vertical position. Such spat became detached within a few days but continued their growth. Larvae were placed on sand to observe their reactions to this very wide- spread substrate. They attempted to crawl over the grains, but rolling of the grains impeded their movement, and they soon swam away. SUBSTRATE SPECIFICITY-COMPARATIVE SETTING RATES ON S. GRUBII, SHELL, ANDGLASS: An experiment was performed to compare setting rates on Stelletta and fouled shell under free-choice and no-choice conditions. Four Stender dishes of 30 mOl diameter were used. In dish 1, no substrate was added; dish 2, two S. grubii cuttings; dish 3, two pieces of fouled shell; dish 4, one piece of fouled shell and one S. grubii cutting. Fifty pediveligers, 15 011 sea water, and Monochrysis were added to each dish. Spat were counted and dishes were cleaned daily for 5 days. Results of three trials are listed in Table 2 and summarized in Figure 5. Setting was heavier on S. grubii than on fouled shell. The difference in setting rate on Stelletta and shell was greatest the first day under no-choice conditions and indicates postponement of setting in absence of Stelletta. Setting was heavier on no- 286 Bulletin of Marine Science [16(2)
TABLE 3 TEST FOR ATTRACTION OF Ostrea permollis PEDlVELIGERS TO SUBSTRATES
Experimental Sectors Control Sector (Stelletta) Number of Value of x." Substrate larvae in Number of (1 d. f.) five sectors larvae Microciona 22 7 1.20; P>.05 Keratosid 16 6 1.72; P>.05 No substrate 44 8 .0002; P>.05 Expected ratio 5 1 choice shell than on free-choice shell. A low rate of setting occurred on dish interiors under all conditions. SUBSTRATE SPECIFICITY IN SELECTED PHASES OF THE SETTING PROCESS: The setting process is a sequence of responses to substrate characteristics that culminates in the cementation reflex. A series of experiments was performed to determine specificity of responses at the following phases of the setting process: (I) the possible attraction of larvae to favorable substrates prior to contact; (2) stimulation of creeping when larvae are placed on substrates; (3) resumption of creeping after creeping larvae are transferred to another substrate; (4) persistence of creeping behavior once resumed after transfer; and (5) cementation. Preliminary observations indicated no tendency for larvae to move toward Stelletta. To test for attraction of larvae from a distance, the bottoms of 10 cm finger bowls were marked into six sectors around a 30 mm center. Test substrates were placed in the radial sectors. Sea water (l00 ml) was added and 20 pediveligers were placed in the center. After I hour, distribution of larvae was random with Stelletta in one sector and either the keratosid, Microciona, or no substrate in five sectors (Table 3). In other trials larvae did not discriminate between fouled shell and empty sectors but preferred them to Stelletta (P<.05). To test whether larvae respond specifically on their first contact with
TABLE 4 RESPONSES OF Ostrea permollis LARVAE AT FIRST CONTACT WITH SUBSTRATES
Number of larvae Number of larvae creeping on substrate placed on each Value of x.' (\ d. f.) substrate Slellelta Fouled Shell Keratosid 356 48 28 5.75; P<.05 82 19 7 5.53; P<.05 1966J Forbes: Life Cycle of Ostrea permollis 287
TABLE 5 SETTING OF Ostrea permollis LARVAEAFTER TRANSFER DURING CREEPING PHASE
Condition of Substrates larvae after 30 minutes Stelletta Fouled shell Clean shell
Set 13 6n Qb Not set 4 11 17 nX" = 4.25; P<.05. bX" = 17.23; P<.01. Chi-square values calculated for StelIetta grlllJii against experimental substrates (l d. f.). substrates, pieces of fouled shell, Stelletta, and the keratosid were placed in separate Stender dishes. Pediveligers were placed on substrates usually 20 at a time. Those creeping within 10 min were recorded (Table 4). Larvae commenced creeping on Stelletta more frequently than on the keratosid or fouled shell. The first effective contact with the substrate appeared to be with the foot rather than with the shell. Creeping larvae usually resumed the setting process after transfer from one substrate to another. More larvae selected for creeping behavior set on Stelletta than on fouled shell; none set on clean shell (Table 5). Next, two phases of presetting behavior were explored: resumption of creeping after transfer to substrates, and subsequent persistence of creeping. In each of 22 trials one creeping larva was placed on each of five substrates: Stelletta, Microciona, the keratosid, fouled shell, and clean shell. Larvae which failed to touch the foot to the substrate within 5 min were replaced. Larvae resumed creeping most often on Stelletta, the keratosid, and fouled shell, and least often on clean shell (Table 6). Response to Microciona was intermediate. More larvae persisted in creeping on Stelletta than upon the keratosid or clean shell. The difference in number of larvae creeping 1 min or more on Stelletta and fouled shell was not significant. None crept for more than a few seconds on Microciona. Apparently the relatively numerous, sharp spicules rendered the surface of Microciona unsuitable for creeping. In 14 trials of the experiment above, larvae were further observed for setting on Stelletta, fouled shell, and the keratosid. More larvae set on Stelletta than on fouled shell in 30 min (Table 7). Additional setting on fouled shell within another 30 min indicated more rapid completion of the setting reaction on the host sponge than on non-host substrates. The experiments described above show that O. permollis larvae responded preferentially to S. grubii in the various phases of the setting process. Larvae swam off clean shell after the first contact with the foot. 288 Bulletin of Marine Science [16(2)
55 6
I V
rv
FIGURES 6, 7. 6, Unmetamorphosed Ostrea perm ollis spat on Stelletta grubii. Length 350M, /, foot; SS, surface of host sponge. 7, Early stage of embedment of metamorphosing Ostrea permollis on Stelletta grubii. Length 400p.. hsp, hillock of proliferating sponge tissue; lv, left prodissoconch valve; rv, right prodissoconch valve; ss, surface of host sponge. 1966] Forbes: Life Cycle of Ostrea permollis 289 TABLE 6 RESUMPTION AND PERSISTENCE OF CREEPING OF Ostrea permollis LARVAE ON SELECTED SUBSTRATES ------Substrates Activity of larvae Sielletta Keratosid Microciona Fouled Clean shell shell
1a Did not resume creeping 2" 1" 41> 2" 10 1b Resumed creeping 20" 21 " 181> 20" 12 2a Crept for less than one minute 1 lIe 18d 3" 6f 2b Crept one minute or more 19 10e Od 17e 6f ---~ --~-----~~ "x" = 5.61 or more, P<.05; I>x"= 2.62, P>.05; tested against clean shell. eX" = 0.28, P >.05; Larvae usually resumed creeping on Microciona but ceased within a few seconds. Larvae resumed creeping as frequently on the keratosid as on Stelletta but initiated creeping at first contact less often, left sooner, and failed to set. Larvae resumed and persisted in creeping on fouled shell as well as on Stelletta but initiated creeping less often, prolonged the setting process, and set less often. S. grubii and films of microorganisms probably contained factors similar in effect on larvae but differing in degree. Biological films on substrates increase settlement of O. edulis (Cole & Knight-Jones, 1949) and many other sessile invertebrates (Zobell, 1935; Scheer, 1945; Knight-Jones, 1951; Woods Hole Oceanographic Institution, 1952; Wilson, 1954, 1955; Scheltema, 1961). Metarnorphosis and Embedment of O. permollis Spat.-Metamorphosis of O. permo/lis was similar to that of O. edulis (Cole, 1937, 1938) and C. virginica (Jackson, 1888) in rotation of body within the shell; loss of velum, foot, eyespots, and anterior adductor muscle; and growth of posterior adductor muscle and gill filaments, Metamorphosis was iden- tical on glass and host sponge. Metamorphosis was complete within 3 days in finger bowls at 23 °-24°C but had not occurred in most spat after 6 days in flowing sea water at 13°-20°C (Fig. 6). Growth of the host sponge over four O. permollis spat was observed on sponge cuttings maintained in a sea water table at 13o_20°C. Twenty-four hours after setting, the sponge adhered sufficiently to the left (lower) valves of two spat to anchor them (Fig. 7); the other two spat were held only by their byssal cement (Fig. 6). By the sixth day, sponge adhered to left valves of all four spat and covered the right valves of two (Figs. 8, 9). In a later stage (length 1 mm, Fig, 10), prodissoconchs were 290 Bulletin of Marine Science [16(2) hsp '. ... "",,, I \ Isp . \ l . I ,• I ,I I I I. \ \ \ \ rv ", 8 es ...... go . rv '...... ".•.I.. - .... - .. 9 hsp FIGURES 8, 9. Ostrea permollis spat in later stages of embedment on Stelletta grubii. Length 350-400J.t. es, eyespot; ga, gape; hsp, hillock of proliferating sponge; lsp, layer of proliferating sponge over shell; rv, right prodissoconch valve. 1966] Forbes: Life Cycle of Ostrea permollis 291 TABLE 7 SETTING OF Os/rea permollis LARVAE WHICH PERSISTED IN CREEPING Substrates Activityof larvae Sielletta Keratosid Fouledshell Ia Did not set in 30 minutes 2" 5 lOll Ib Set within 30 minutes 10" 0 I" 2a Did not set in 60 minutes 2 5 6 2b Set within 60 minutes 10 0 5 "x' = 9.88; P<.Ot in a test for association applied to selling on Stellelta and fouled shell. Other frequencies too small for a statistical test. entirely covered by sponge, but inhalant and exhalant regIOns of the dissoconch were not covered. Water currents were traced with neutral red in eight spat (2-3 mm) on sponge cuttings about 10 days after setting. None was fully embedded in the host sponge, and cloacal chambers were located by positions of the clearly visible gills and adductor muscles. Three spat had no sponge growth over their right valves. Their exhalant streams discharged over the sponge surface. Five spat were well covered by sponge in the exhalant region and their exhalant regions clearly entered the sponge (e.g., Fig. 11). Embedment of O. permollis in its host sponge therefore results from the growth of the sponge. S. grubii tends to adhere to and overgrow objects that are in contact. The exhalant margin of oyster spat lies next to the substrate so that the sponge can readily incorporate the exhalant stream into its own canal system. Stimulation of sponge growth by conchiolin is a possible effect that was not apparent under laboratory conditions. Recruitment and Growth of O. permollis.-Study of temperature relation- ships to breeding is complicated in Ostrea by its alternation of sexual phases and incubatory habit. Ripe stages were identified in sections prepared from oysters collected February 21-November 29, 1958, in the Alligator Harbor area and probably occur throughout the year. Incubating embryos were found April 5-December 7, 1958, and indicated spawning throughout the range 15°-30°C (Forbes, MS). Relationship of spawning and recruitment is obscure when source and age of setting larvae are unknown (discussion by Korringa, 1941). Hence recruitment and growth rate are more useful in understanding population dynamics of oysters than are dates of spawning. Time of spatfa]) and growth rate of O. permollis were studied from oyster-sponges collected at St. Teresa and Alligator Harbor. RECRUITMENT: Whole sponges or pieces of large ones were exam- ined for spat (to 1 mm) under a stereoscopic microscope (45 x). O. 292 Bulletin of Marine Science [16(2) rv isp-f- 9 om II FIGURES 10, 11. 10, Metamorphosed Ostrea permollis spat on Stelletta grubii in a hillock of proliferating sponge. Length 1 mm. be, branchial chamber; ee, cloacal chamber; 1m, left mantle; lv, left dissoconch valve; pt, palliobranchial fusion; rm, right mantle; rv, right dissoconch valve; lsp, layer of proliferating sponge over shell. 11, Ostrea permollis spat, length 4 mm, discharging effluent into host sponge. Spat lies flat against Stelletta grubii; dotted outlines are those overgrown by sponge. am, adductor muscle; g, gill; lsp, layer of proliferating sponge over shell; p, definitive prodissoconch; rv, right dissoconch valve. permollis spat were identified by their yellow color; white spat were iden- tified as O. equestris and were included for comparison (Table 8). All O. permollis (45 spat) occurred on or in the host sponge; all O. equestris (38 spat) occurred on shells. This distribution of the two species is clear evidence of specific differences in larval reactions to substrates. O. permollis spat occurred each June and November of the years that collections were made. O. equestris spat appeared in June of 1957 and 1958. One cannot conclude that spat settled only at those times since total numbers were small and newly settled spat would be counted as such for only a few days. Mortality would also obscure spatfalls. A more com- plete study of O. permollis spatfall should utilize weekly counts on a large number of sponge cuttings. More complete data would probably show some setting from spring through the fall. Spatfall maxima occurred in June and November of the years studied, and that is why spat were noticed during those months. In comparison, Menzel (1955) found spat of C. virginica and/or O. equestris during 8 months at Port Aransas, Texas, with a major peak in late spring-early summer and a minor peak in October. Butler (1965) found ostreid spatfall to begin March 15-May 20 and end 1966] Forbes: Life Cycle of Os/rea permollis 293 TABLE 8 OCCURRENCEOF OYSTERSPAT UP TO 1 MM IN LENGTH ON OYSTER-SPONGES ------Number of sponges Number of spat Date Locality Examined With Spat Os/rea Os/rea perm ollis equeslris 6-28-57 S1.Teresa 5 1 14 7 7-20-57 S1.Teresa 1 8- 3-57 S1.Teresa 4 8-20-57 S1.Teresa 6 8-21-57 S1.Teresa 2 8-23-57 S1. Teresa 1 8-27-57 S1.Teresa 5 8-29-57 St. Teresa 1 8-10-57 S1. Teresa 2 9- 3-57 S1.Teresa 2 9-14-57 S1. Teresa 2 2-21-58 Alligator Harbor 10 2-28-58 Alligator Harbor 14 4-11-58 Alligator Harbor 7 6- 2-58 Alligator Harbor 2 2 0 4 6- 3-58 Alligator Harbor 1 1 1 10 6-16-58 Alligator Harbor 1 1 7 8 6-17-58 Alligator Harbor 2 2 14 2 6-29-58 Alligator Harbor 1 7-15-58 Alligator Harbor 1 11-13-58 Alligator Harbor 1 11-27-58 Alligator Harbor 1 11-29-58 Alligator Harbor 2 2 8 0 2- 1-59 St. Teresa 1 6-27-59 S1.Teresa 1 7 Totals 76 8 44 34 September 22-November 20 over a period of 10 years in Santa Rosa Sound, Florida. The peak (50 per cent of the annual) spatfal! of individual years occupied 2 to 6 weeks and occurred in any month from May to September (Butler's Fig. 8). Size-frequency distributions (below) indicate a tendency for spring and fall peaks in O. permollis. GROWTH RATE OF OSTREA PERMOLLlS: Growth curves were determined from age-groups followed in serially collected oyster-sponges and from a series of measurements of oysters in one sponge. Age-groups were identi- fied as peaks in each size-frequency distribution, assuming a general uni- formity of growth and well defined periods of spatfall. Seven age-groups were identified. The series of size-frequency distributions was based on five collections from St. Teresa and Alligator Harbor in 1957-1958 (Fig. 12). Age-groups 294 Bulletin of Marine Science [16(2) CD,.., 2 ai '" ~ '" ~ "10~'" C\J ""11> !!! E- ",. Y <:0 Q .;t N ,..,a @ Q 0 N'" ":' "' S1:l31S;"0 .dO 1:l38VVn N 1966] Forbes: Life Cycle of Ostrea permollis 295 were assigned numerals beginning with the oldest apparent group (6). Spat and very small oysters (Age-group 4) and larger oysters (Age-group 5) constituted the bulk of the population on June 28, 1957. Two or three oysters represented a still older Age-group 6. Age-groups 4, 5, and 6 also appeared in the August 10 collection. On August 29, Age-group 5 was represented by the right tail, and the spring, 1957, spatfall (Age-group 4) constituted most of the sample. Age-group 3 appeared by February 21, 1958, and was clearly the spatfall of autumn, 1957. Age-group 4 was still apparent, but Age-group 5 was almost or entirely extinct. Age-group 3 persisted as the oldest group in the November 29 collection, when a new age-group (1) appeared. An intermediate age-group (2) was made up of an early and a late component that may represent spatfall maxima of spring and summer, 1958. Age-group 6 probably settled spring, 1956; Age-group 5, in autumn, 1956; Age-group 4, in June, 1957; Age-group 3, in autumn, 1957; Age-group 2, spring, 1958; and Age-group 1, in November, 1958. Figure 12 shows that age-groups were still distinguishable at a size of 34-42 mm somewhat more than a year following setting. Growth was measured in O. permollis in an oyster-sponge collected at St. Teresa on February 1, 1959, and kept at the end of the Marine Labora- tory pier (Fig. 13). Measurements are of lengths since height could not be measured in situ. Age-groups 1 and 2 comprised the bulk of the popula- tion on February 1 and July 6. Small oysters (14-22 mm) present on July 6 probably represented the spring, 1959, spatfall (Age-group 0). Age-group I was prominent on November 27, while Age-group 2 had nearly disappeared. Growth curves (Fig. 14) were drawn by plotting size (modes of age-groups in Figs. 12, 13) against time and showed that O. permollis attained length and height of about 35 mm in the year following setting. A single oyster measured in S. grubii and followed from February 25- December 1, 1958, grew from 7 mm to 46 mm. Hence, growth estimates from size-frequency determinations may well have been biased downward as a result of mortality of larger oysters or other factors. The largest O. permollis observed were 45-50 mm and probably attained that size in no more than 2 years after setting. DISCUSSION AND CONCLUSIONS The relationship between O. permollis and S. grubii appears to be commensalism. A commensal neither benefits nor harms its host nor feeds on host tissues but may be a "guest at the same table" (van Beneden, 1894). The oyster depends on the sponge for substrate and protection. S. grubii sometimes lacks the oyster (Forbes 1964) and is not known to be harmed or benefitted by its presence. The relationship would tend 296 Bulletin of Marine Science [16(2) 20 en February I, July 6, 1959 November 27, 0::: /959 /959 W I- 2 en 15 >- 0 2 l.L 10 0 0::: 0 W 5 0 en ~ ::J Z 0 6 10 14 18222630 14 18 22 26 30 34 3B 26303438 4246 LENGTH IN MM FIGURE 13. Length-frequency distributions of Ostrea permollis growing in situ in Stelletta grubii from February 1 to November 27, 1959. Age-groups are identified by numerals as in text. toward mutualism if the oyster's pumping of water aided growth of the host or if significant numbers of sponges developed from fragments torn off by crabs when attacking oysters. The only modifications recognized in O. permollis are its yellow color, a somewhat weakened shell, and specific responses to its host at setting. The yellow color indicates conchiolin modification of unknown adaptive significance. O. permollis undoubtedly evolved from an ostreid that set on solid substrates. Several factors aided in development of the commensal habit. To begin with, S. grubii probably sufficiently resembled films of micro- organisms on substrates so that larvae from the ancestral oyster population occasionally set on this sponge. Relative absence of fine, sharp spicules contributed to the suitability of S. grubii as a substrate. The tilted position of ostreid spat favored access to the sea despite siltation and similarly enabled spat on S. grubii to function when partly covered by the sponge. The inhalant region, through which food and oxygen were admitted and larvae and pseudofeces expelled (Fig. 15), was unlikely to become overgrown by the sponge because of its elevation. Contact of the dorsal margin with the substrate favored prompt growth of the sponge over the upper valve. Setting of O. permollis is unmodified from the ancestral condition. Embedment and consequent protection constituted a threshold in adaptation of the oyster to its new adaptive zone. Protection of embedded oysters resulted in selection for ability of pediveligers to respond to S. grubii. Protection relaxed selection for strong dissoconchs in sponge- 1966] Forbes: Life Cycle of Ostrea permollis 297 40 2 35 3 ~ ~ 4 30 z ~25 w 20 N I I I CJ) , I I I , 0:: 15 I I I 0 I I , I I I I I I I I I- I I , 10 I I I <.9 I I I I I Z I I I I W I I .-J I I I I I , 5 I I , I I I I I I I I I I I I I I I I I 0 1957 1958 1959 FIGURE14. Growth rate of Ostrea permollis, based on age-groups identified in Figures 12-13. Dotted lines connect curves to probable age-group spatfan dates. dwelling oysters, and therefore adaptation of their progeny to non-host substrates decreased. Such phenotypes that are intermediate in adaptation to separate adaptive zones are subject to strong selection and thus tend to leave a genetic discontinuity between well adapted populations (Simpson, 1953). Differentiation of O. permollis was accelerated by concomitant effects of its occupancy of S. grubii. First, commensal oysters were partially isolated from corresponding populations on solid substrates since the sponge commonly grows on sandy or sea grass bottom where solid 298 Bulletin of Marine Science [16(2) wa stes CO2 sperm pseudofeces I a rva e Ostrea permollis Stelletta grubii FIGURE 15. Relationships of Ostrea perm ollis to its host sponge and the sea. substrates are widely separated. In fact even if other oysters were abundant nearby, the aggregation of oysters crowded on each sponge constituted a relatively distinct breeding unit. Secondly, aggregation increased the opportunity for each oyster to breed, in contrast to conditions in diffusely scattered populations, and hence increased the selective value of host specificity. Adaptation of O. permollis to its adaptive zone (S. grubii) is perhaps not complete since it can set on solid substrates, at least under laboratory conditions. That O. permollis has not been recognized on solid substrates in the Gulf of Mexico attests to effectiveness of substrate specificity under ecological conditions, though probability of finding it on solid substrates would also depend on population density. Shortening of life expectancy when not protected by the sponge would also contribute to the apparent absence of O. permollis on solid substrates. ACKNOWLEDGMENTS I thank Dr. Winston Menzel, Oceanographic Institute, Florida State University, for encouragement and aid throughout the investigation; 1966J Forbes: Life Cycle of Ostrea permollis 299 Dr. Luther Franklin for assistance; Dr. Victor Loosanoff, at the time Director of the Biological Laboratory, U. S. Bureau of Commercial Fisheries, Milford, Connecticut, for a most instructive visit to his labora- tory; Mr. Harry Davis for information on rearing of oyster larvae; and Dr. Ravenna Ukeles for inocula and suggestions for culturing algae. SUMARIO CICLO DE VIDA DE Ostrea permollis Y SU RELACI6N CON LA ESPONJA HUESPED, Stelletta grubii Los individuos de la especie O. permollis viven amontonados en la superficie de la esponja S. grubii. La ostra obtiene plankton y oxigeno mediante comunicaci6n directa de su margen inhalante con el mar. O. permollis sufre poca modificaci6n en su adaptaci6n al huesped. La concha, sin embargo, es fnlgil. El significado que para la adaptaci6n pueda tener la "conchilina" amarilla permanece obscuro. Se not6 evidencia de danos ocasionados a las conchas por Polydora y de predaci6n por parte de cangrejos y caracoles. S. grubii protege la concha de Polydora y de los predatores. Se criaron larvas de O. permollis hasta su metamorfosis. Las larvas pediveligeras, el asiento y la metamorfosis fueron iguales que en otras Ostrea. Se observ6 el asiento en S. grubii y, en menor numero, en conchas y en cristal. No parece que las larvas sean atraidas hacia S. grubii desde lejos. EI escoger un substrato especifico depende de la respuesta de las larvas a los substratos durante las distintas etapas del asiento. Para el asiento en cristal 0 en concha se requiere la presencia de una pelicula 0 capa de microorganismos. La respuesta del huesped se relaciona proba- blemente con la preferencia de las larvas de las ostras por determinada peHcula 0 capa de microorganismos. El crecimiento de la esponja huesped trae consigo el empotramiento de la ostra. La postura tipica de las ostras al asentarse favorece el empotra- miento de toda la ostra con excepci6n de su region inhalante. Estudios de frecuencias de tamanos e incidencia estacional de las huevas indican una puesta de huevos maxima en los meses de Junio y Noviembre de los aiios estudiados. Las ostras crecieron hasta aIcanzar por 10 menos 35 mm el primer ano y hasta aIcanzar un largo final de alrededor de 50 mm. Se discuten los factores que Bevan a la ostra a babitos de comensalismo. LITERATURE CITED BUTLER, P. A. 1965. Reaction of some estuarine mollusks to environmental factors. in Biological problems in water pollution. Third seminar, 1962. Dept. Health, Educ., Welfare, U. S. Pub. Health Servo Pub. No. 999- WP-25, pp. 92-104. COLE, H. A. 1937. Metamorphosis of the larva of Ostrea edulis. Nature, 139: 413-414. 300 Bulletin of Marine Science [16(2) 1938. The fate of the larval organs in the metamorphosis of Ostrea edulis. J. Mar. bioI. Ass. U. K., 22: 469-484. COLE, H. A. AND E. W. KNIGHT-JONES 1939. Some observations and experiments on the setting behavior of larvae of Ostrea edu/is. J. Cons. int. Explor. Mer, 14: 86-105. 1949. The setting behavior of larvae of the European flat oyster Ostrea edulis L., and its influence on methods of cultivation and spat collec- tion. Fish. Invest., London, Ser. 2, 17: 1-39. DAVIS, H. C. AND R. R. GUILLARD 1958. Relative value of ten genera of micro-organisms as food for oyster and clam larvae. Fish. Bull., U. S., 58: 293-304. ERDMANN, W. ] 934. Untersuchungun liber die Lebensgeschichte der Auster. 5. Ober die Entwicklung und die Anatomie der "ansatzreifen" Larvae von Ostrea edulis. Wiss. Meeresuntersuchungen N. F. Abt. Helgoland, 19 (6): ]-25, 6 pIs. FORBES, M. L. 1964. Distribution of the commensal oyster, Ostrea permollis, and its host sponge. Bull. Mar. Sci. Gulf & Carib., 14 (3): 453-464. GALTSOFF, P. S. 1964. The American oyster, Crassostrea virginica Gmelin. Fish. Bull., U. S., 64: 1-480. HOPKINS, S. H. 1958. The planktonic larvae of Polydora websteri Hartman (Annelida, Polychaeta) and their settling on oysters. Bull. Mar. Sci. Gulf & Carib., 8 (3): 268-277. HORI, J. 1933. On the development of the Olympia oyster, Ostrea llirida Carpenter, transplanted from the United States to Japan. Bull. Jap. Soc. sci. Fish., 1: 269-276. HORST, R. 1883. A contribution to our knowledge of the development of the oyster (Ostrea edlilis L.). Rep. U. S. Comm. Fish., Part 10, Report for 1882: 159-167. 1886. The development of the oyster (Ostrea edulis L.). Rep. U. S. Comm. Fish., Part 12, Report for 1884: 891-911. JACKSON, R. T. 1888. The development of the oyster with remarks on allied genera. Proc. Bast. Soc. nat. Hist., 23: 531-550, 4 pIs. KNIGHT-JONES, E. W. 1951. Gregariousness and some other aspects of the setting behavior of Spirorbis. J. Mar. bioI. Ass. U. K., 30: 201-222. KORRINGA, P. 1941. Experiments and observations on swarming, pelagic life and setting in the European flat oyster, Ostrea edulis L. Arch. Neerland. ZooL, 5: 1-249. 1951a. On the nature and function of the "chalky" deposits in the shell of Ostrea edulis Linn. Proc. Calif. Acad. Sci., Ser. 4, 27: 133-158. 1951b. The shell of Ostrea edulis as a habitat. Arch. Neerland. ZooL, 10: 31-152. 1952. Recent advances in oyster biology. Quart. Rev. BioI., 27: 266-365. 1966] Forbes: Life Cycle of Ostrea permollis 301 LAGLER, K. F. 1952. Freshwater fishery biology. Brown, Dubuque, Iowa, x + 360 pp. LOOSANOFF, V. L. 1954. New advances in the study of bivalve larvae. Amer. Scient., 42: 607-624. LOOSANOFF, V. L. AND H. C. DAVIS 1963. Rearing of bivalve mollusks. in R. S. Russell, ed. Advances in marine biology. Vol. 1, pp. 1-136. Academic Press, New York, xiv + 410 pp. MENZEL, R. W. 1955. Some phases of the biology of Ostrea equestris Say and a comparison with Crassostrea virginica (Gmelin). PubI. Inst. Mar. Sci. Univ. Texas, 4: 69-153. NELSON, T. C. 1924. The attachment of oyster larvae. BioI. Bull., 46: 143-151. 1957. Some scientific aids to the oyster industry. Amer. Scient., 45: 301-332. PRYTHERCH, H. F. 1934. The role of copper in the setting, metamorphosis and distribution of the American oyster, Ostrea virginica. Ecol. Monogr., 4: 49-107. RANSON, G. 1960. Les prodissoconques (coquilles larvaires) des Ostreides vivants. Bull. l'Inst. Oceanogr. Monaco, (1183): 1-41, 136 figs. SCHEER. B. T. 1945. The development of marine fouling communities. BioI. BulL, 89: 103-121. SCHELTEMA, R. S. 1961. Metamorphosis of the veliger larvae of Nassarius obsoletus (Gastro- poda) in response to bottom sediment. BioI. Bull., 120: 92-109. SENO, H. 1929. A contribution to the knowledge of the development of Ostrea denselamellosa. J. imp. fish. Inst., 24: 129-135, pIs. 3-4. SIMPSON, G. G. 1953. The major features of evolution. Columbia University Press, New York, xx + 434 pp. THOMSON, J. M. 1954. The genera of oysters and the Australian species. Aust. J. Mar. Freshw. Res., 5: 132-168. VAN BENEDEN, P. J. 1894. Animal parasites and messmates. Appleton, New York, 274 pp. WILSON, D. P. 1954. The attractive factor in the settlement of Ophelia bicornis Savigny. J. Mar. bioI. Ass. U. K., 33: 361-380. 1955. The role of micro-organisms in the settlement of Ophelia bicornis Savigny. J. Mar. bioI. Ass. U. K., 34: 531-543. WOODS HOLE OCEANOGRAPillC INSTITUTION . 1952. Marine fouling and its prevention. U. S. Naval Institute, Annapolis, 388 pp. YONGE, C. M. 1960. Oysters. Collins, London, xiv + 209 pp., 17 pIs. ZOBELL, C. E. AND E. C. ALLEN 1935. The significance of marine bacteria in the fouling of submerged surfaces. J. Bact., 29: 239-251.