Development of the statocyst in Aplysia californica I. Observations on statoconial development

63

Hearing Research, 49 (1990) 63-78

Elsevier HEARBS 01434

Development of the statocyst in ApZysia californica I. Observations on statoconial development Michael L. Wiederhold 1*2,Jyotsna S. Sharma I, Brian P. Harrison 2

Driscoll

’ and Jeffrey L.

’ ~e~urtmeRt of ~iol~~ngolo~, Uniuers~~ of Texas Health Science Center, San Antonio, Texas and 2 Audie L. Murmur Memorial Veterans Administration Hospital, San Antonio, Texas, U.S.A.

(Received 17 July 1989; accepted 4 January 1990)

The gravity receptor organs of gastropod molluscs, such as Aplysia californica, are bilateral paired statocysts, which contain dense statoconia within a fluid-filled cyst. Gravitational forces on the statoconia are sensed through their interaction with ciliated mechanoreceptor cells in the wall of the cyst. Larval Aplysia contain a single statolith within each statocyst; when the animals grow to a critical size, they begin producing multiple statoconia, a process that continues throughout life. The number of statoconia is highly correlated with animal weight but poorly correlated with age, indicating that stone production is related to total metabolism. The single statolith has an amorphous internal structure whereas the multiple statoconia have calcification deposited on concentric layers of membrane or matrix protein. The statohth appears to be produced within the cyst lumen but the multiple statoconia are produced within supporting cells between the receptor cells. Large adult animals have statoconia larger than those in early ~st-metamo~~c animals which have just started producing multiple stones. The m~mum statocyst diameter at which the receptor-cell cilia can suspend the statolith in the center of the cyst lumen is 45 pm; production of multiple stones begins when the cyst reaches this size. The mechanisms by which statocoma production is initiated and controlled are discussed. Statocyst; Statoconia; Otoconia; Gravity; Mineralization

Introduction Statocysts are gravity receptor organs found in invertebrates. They are generally fluid-filled, saclike organs with the luminal surface of the wall lined with sensory cells. They contain dense stones, either a single statolith or multiple statoconia, that fall under the influence of gravity, stimulating the underlying sensory cells. The term statocyst is generally used for invertebrates, and is analogous to the otocyst in vertebrates. Because of their relatively simple structure, the large size of the sensory receptor cells and viability of in vitro preparations, several invertebrate statocysts have been studied extensively, both physiologically and anatomically (Coggeshall,

Correspondence to: Michael L. Wiederhold, Department of Otoiaryngology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284-7777, U.S.A.

1969; Wiederhold 1974, 1977; Gallin and Wiederhold, 1977; Alkon, 1975; Spangenberg, 1979; Budelmann, 1988; Janse, 1982, 1983; Williamson, 1988). Thus, the physiology of the statocysts is reasonably well-understood in adult animals with simple statocysts containing only 13 sensory receptor cells, as in Aplysia and Hemissenda, and in the cephalopods Octopus, Sepia and squid, in which the statocysts are as complex as the mammalian vestibular end organs. A schematic drawing of a section through an adult Aplysia statocyst is shown in Fig. 1. Six sensory receptor cells are shown, with cilia projecting into the cyst lumen. The statoconia sink, and come into contact with the beating cilia of receptor cells at the bottom of the cyst. This interaction causes a conductance increase, leading to depolarization of the receptor cells (Wiederhold, 1974, Gallin and Wiederhold, 1977). These cells have strongly nonlinear current-voltage characteristics, similar to those of vertebrate hair cells (Wiederhold, 1977;

0378-S955/90/%03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)

64

Corey and Hudspeth, 1979). Action potentials are generated in the receptor-cell axons, which project to the cerebral ganglia, providing information about the direction and magnitude of gravitational forces and acceleration. The cilia on the sensory receptor cells are motile and beat continuously. This can be seen in live preparations. Under favorable optical conditions, the ciliary beating can be visualized directly. This movement is also evident from the fluctuation in light reflected from the statoconia or statolith. The ciliary motility is integral to the sensory function of the receptor cells; if motility is blocked chemically, the movement of the statoconia cease and physiologic responses to a standard tilt are eliminated or greatly reduced (Wiederhold, 1978). Thus this preparation, although quite different in some respects from vertebrate auditory and vestibular organs, gave some of the first indications of active and nonlinear processes in ciliated mechanoreceptor cells. We now wish to investigate the formation of the statoconia which are analogous to vertebrate otoconia. In Ap&sia, the statocyst is easily accessible, being encased in only soft tissue. The limited number of statoconia allows quantitation of the number, size distribution, structure and composition of the statoconia as well as developmental changes in these parameters. These attributes make this a favorable system in which to study the formation of statoconia and possible environmental influences on this process. In A. punctata, statocysts appear as six-celled organs on the side of the foot primordium when the embryo reaches the 300-cell stage (2-3 days after fertilization) (Saunders and Poole, 1910). They are thus the earliest neural structures to develop. Statocysts arise as ectodermal invaginations that close to vesicles and then constrict off from the ectoderm (Raven, 1958). A study of neurogenesis in A. californica showed that an ‘otocyst’ can be distinguished as early as the gastrula-to-segmentation cavity stage embryo (2-3 days after fertilization) (Jacob, 1984). In larvae and adults, the statocysts are associated with the pedal ganglion. After metamorphosis, as the animal undergoes detorsion, the statocysts are positioned posterior to the buccal mass in the circumesophageal ring of ganglia.

The post-hatching development of A. cafifornica has been subdivided into 13 stages, based on behavioral and morphological characteristics visible in living specimens (Kriegstein, 1977). The stages are summarized here as they relate to statocyst development. Stages:

l-6:

7-8:

9-12:

13:

newly-hatched to fully-developed planktonic veliger larva ready to metamorphose. Statolith prominent in intact animal. Animal length approximately 70-400 pm. onset and completion of metamorphosis. Animal length 400500 pm_ Statocyst relatively unchanged. early to late juvenile stage; statocyst and statoconia obscured by pigmentation, shell and connective tissue surrounding it. Single statolith appears unchanged at light microscope level. Multiple statoconia first appear. Animal length: 0.7-20 mm. beginning of egg laying and thus adulthood of metamorphosed animal. Animal length > 20 mm. Multiple statoconia formation and growth of statocyst continue.

Bidwell et al. (1986) first noted that the larval Aplysia statocyst has a single statolith whereas McKee and Wiederhold (1974) found up to 1000 statoconia in adults. Appearance of the statolith is dependent on the presence of strontium in the sea water during a critical 24-h period at four days after fertilization (Bidwell et al., 1986). In this study we examined the addition of stones through the life of the animal and their site and mode of formation. We examined the number, size and form of statoconia in animals of varying age and weight. The statocysts in the different age groups were also studied by transmission electron microscopy (TEM) to compare the structure of cells and organelles that may be involved in statoconia formation. Materials and Methods Aplysia californica were obtained in various developmental stages from the Aplysia Rearing

65

Facility of the Howard Hughes Medical Institute at Woods Hole, MA. The particular stages obtained were post-metamorphic (stage ll), 10 g, 30 g, and > 50 g. Large animals, 600-700 g, were obtained from Pacific Biomarine, Venice, CA. Animals were maintained in a continuous-flow recirculating marine aquarium (Neptune Lobster Tank Co.) containing artificial sea water (Instant Ocean) maintained at 1519°C. One group of 20 post-metamorphic animals was maintained at room temperature in a 500 cc container of artificial sea water that was changed daily. All animals were fed Grassilaria sp. which was maintained in a separate artificial sea water system at room temperature. Embryonic stages and hatchlings were obtained from egg cases laid by adults that were maintained in our laboratory. The post-metamorphic animals’ statocysts were examined by placing the whole animal on a slide with a drop of sea water; pressure on a cover slip was used to displace the shell. This allowed visualization of the intact statocyst with its contained statoconia, in relation to the adjacent pedal ganglion. For animals larger than 0.5 g, the circumesophageal ring of ganglia was dissected and the right and left statocysts placed separately on slides. The diameter of the statocyst was measured, the statocysts were punctured with a fine, sharp needle and the statoconia allowed to flow out. A coverslip was placed on the slide and the number of stones counted. The size of the statoconia was determined by examining the statoconia from the dissected statocyst under polarized light. Fifty to 65 statoconia within a randomly selected area of the slide were measured. An image analysis system (Cue-2) was used to measure the cross-sectional area of each statoconium and an average diameter derived from this measurement, assuming a circular cross-section. For light and electron microscopy, intact animals (if less than 1 cm long) or dissected statocysts were fixed by immersion in 2.9% glutaraldehyde in 0.15 M cacodylate buffer, prepared with artificial sea water (1,004 mOsm, pH 7.35) for four hours and triple-rinsed in sodium cacodylate buffer. Specimens were post-fixed in 1% osmium tetroxide for one hour, dehydrated in

a graded series of ethanols and embedded in Spurr’s resin. Thick sections (l-4 pm) were cut through the statocyst and stained with 0.5% Toluidine blue in 1.0% sodium borate for examination by light microscopy. For TEM, 90 nm sections were cut and stained with uranyl acetate and Reynolds lead citrate, and examined with a Philips 301 electron microscope. The distributions of statoconia diameters were compared between groups of animals of different sizes using the Kolmogorov-Smirnov test of equality of distribution functions. Results

Fig. 1 is a schematic drawing of a section through the statocyst of a young adult Aplysia. Six sensory receptor cells can be seen as well as numerous statoconia. The statoconia range from 2-20 pm in diameter. In embryonic through early post-metamorphic specimens, the statocyst contains only a single statolith, as illustrated in Fig. 2. Besides a difference in average size, there are structural differences between the statolith and the statoconia. As illustrated below, the statoconia have a prominent lamellar structure whereas the statolith has an amorphous appearance in crosssection. At stage 1, when larval Aply,sia have just hatched, the statocysts are fully formed, with 13 sensory receptor cells separated by supporting cells. Each statocyst contains a single statolith at this stage. In one late-stage embryonic animal, 70 pm in diameter (estimated to weigh 185 ng), the statocysts were 16 pm in diameter; one statolith was sectioned and was 6.7 pm in diameter. The single statolith is maintained through metamorphosis until the animal reaches approximately 1 mm in length. Then production of multiple statoconia begins. In adult animals between 170 and 700 g, individual statocysts contained from 569 to 935 statoconia, with the largest number counted in a 198 g specimen. The number of statoconia was determined in animals of varying weight and age. Fig. 3 shows the increase in number of statoconia per statocyst with increasing animal weight. The solid curve is a

66

Fig. 1. Schematic drawing of a section through the statocyst of an adult Aplysiu calijornica. Six receptor cells (RC) are shown with cilia (C) projecting into the cyst lumen. These are separated by several supporting cells (SC), whose luminal surface bear microvilli (Mv). Numerous statoconia (St) are seen within the cyst lumen and two are in supporting cells. Receptor cells have prominent endoplasmic reticulum (ER), multivesicular bodies (MVB) and lamellar bodies (L). Both supporting and receptor cells have large nuclei (N). Many mitochondria (Mi) are seen in receptor cells.

best-fit fourth-order polynomial, used to draw attention to the trend in the data; no theoretical model is implied. All post-metamorphic animals which were estimated by volume calculation to weigh less than 1 mg had a single statolith in each statocyst. The multiple statoconia were first seen in animals estimated to weigh 1 to 3 mg (slightly over 1 mm in length, Stage 11 and 12.) The dramatic increase in statoconia number as weight increases from 10 to 100 mg appears to occur during stage 12. Beyond this stage, statoconia appear to be added throughout life, at least until animals attain a weight of approximately 1 kg. In the 198 g Aplysia in which one statocyst had 935 statoconia, the other side had 696. When accurate counts could be obtained from both statocysts, the

average of the two was used in the plot of Fig. 3; no trend was noted for either the right or left statocyst to have more statoconia and this may thus represent intragenic variability. When plotted against animal age, the number of statoconia is seen to generally increase with increasing age (Fig. 4) but this relationship is less consistent than that with weight illustrated in Fig. 3. Aplysia less than seventy-five days old always have a single statolith and all animals over 110 days have multiple statoconia but some animals up to 108 days also had a single statolith while one animal had more than 100 statoconia at 80 days post-hatching. The statocyst and single statolith are fully formed in embryonic animals before hatching from

Fig. 2. L&t micrograpb of a 4-&m section through the statocyst of an early postmetamorphic animal, 60 days post hatching, weigtihingless than 1 mg. Note relationship between receptor-eel1 cilia and the single statolith. Three cilia are indicated by arrowheads pointing to approximately the midpoint of their ieugth.

the egg case. In statocysts of late-stage embryos within the egg case and of viable veliger larvae, the beating of sensory-cell cilia causes conspicuous movement of the statolith while maintaining it in the center of the cyst lumen. As noted above, the statolith in late-stage embryos are approximately 6 pm in diameter. Early postmetamo~~c animals, appro~mately 60 days post-hatc~ng and near 1 pm in length, all had statoliths between 9 and 12 pm in diameter, with a mean of 10.3 ym. Animals just over I mm in length, estimated to weigh l-2 mg, retained the large single statolith, but also had up to 35 smaller statoconia, from 3 to 8 pm in diameter. Fig. 5 shows a 2 pm section through the statocyst in an animal 1.2 mm in length (101 days old), which illustrates the single statolith and 3 small statoconia (this animal was maintained at room temperature).

The statolith diameters from ten post-metamorphic animals with single statoliths, five l-2 mg animals with 3 to 16 statoconia, seven juvenile and eight adult animals were measured and are plotted in Fig. 6. All animals included in Fig. 6 were maintained at 15-19°C. The multiple statoconia in the l-2 mg animals are clearly smaller than those in large adults; in the I-2 mg animals, the statoconia diameters range from 3-8 pm (mean = 5.07, SD = 1.32). Animals from 0.1 to 0.5 g (animal length, l-2 cm) had statoconia ranging in size from 2 to 17 pm diameter with a mean of 9.28 pm (SD = 3.28). Larger animals, from 170-700g had statoconia ranging from 3-20 pm in diameter with a mean of 11.10 pm (SD = 3.35). Fig. 7a demonstrates that the distribution of statoconia diameters in 0.1-0.5 gram animals is shifted to smaller values, compared with large adults from

68 INCREASE PER

IN NUMBER

STATOCYST

WITH

OF STATOCONIA

INCREASING

ANIMAL

WEIGHT

INCREASE

1

).O( 11

0.01

0.1

ANIMAL

PER

1 WEIGHT

10

100

IN NUMBER

STATOCYST

WITH

OF STATOCONIA INCREASING

AGE

1r

(gm)

Fig. 3. Scatter plot of increase in number of statoconia PG~ statocyst with increasing weight of specimens. All animals estimated to weigh below 1 mg (from volume calculations) had a single statolith and are entered at the 1 mg abscissa value. In this and other figures, the curve is a fourth-order, simple power-series fit to the data. The r values are the nonlinear regression coefficients.

0

25

50

75

ANIMAL

100 AGE

125 (days

150 since

175

200

225

250

hatching)

Fig. 4. Scatter plot of the increase in number of statoconia per statocyst with increasing animal age, in days post hatching Since the data do not indicate a single-valued relationship, no regression anaiysis was performed.

Fig. 5. Photomicrograph of a 2-pm section through the statocyst of a post-metamorphic animal, 1.2 mm long showing the single statolith and three statoconia. Tbis animal was 101 days post hatching and was maintained after metamorphosis at room temperature

69 DISTRIBUTION

OF STATOCONIA

IN APLYSIA

OF DIFFERENT

DIAMETERS SIZES

60 m 50 =

!

2

40

:

30

0

0

2

4

6

6

Statoconio

10 Diameter

12

Postmetamorphic

m

1 -

2 mg

Aplysia

6X3

0.1

-

9 Aplysia

I

170

14

16

0.5 -

310

16

9 Aplysio

20

(rm)

Fig. 6. Plot of distributions of statoconia and statolith diameter for Ap.$iu in four size ranges. Cross-hatched plot is statolith diameter in early post-metamorphic animals with only the statolith (Number of statoliths measured =16). Wide rightpointing diagonal bars are statoconia from post-metamorphic animals in which both the statolith and statoconia could be identified (Number of statoconia measured = 13). Left-pointing diagonal bars are statoconia from 0.1-0.5 g animals (N = 317). Solid bars are from adults from 170-310 g (N = 311).

animals to 10.99 pm in 700-g adults, an increase of 116%. As the animals grow, the diameter of the statocyst also increases. As noted above, embryonic statocysts are approximately 16 pm in diameter just before hatching. These animals are nearly spherical in shape, with a diameter of 70 pm; they are estimated to weigh 185 ng. Early post-metamorphic animals raised at 15-19OC all had statocysts between 35 and 45 pm in diameter. Fig. 8 demonstrates the growth of the statocyst with increasing animal weight. In the largest animal examined (698 grams) the statocysts were 246 pm in diameter. Fig. 8 indicates that this growth continues throughout the animal’s life with diameter increasing proportional to the logarithm of animal weight beyond 0.1 g. The relationship between increasing size of the statocyst and increasing number of statoconia is DISTRIBUTION

15

170-700 g. These distributions are significantly different (P < 0.0005) using the KolmogorovSmirnov test. However, Fig. 7b shows that the distribution for 170- to 310-g animals is not distinguishable from that for 668- to 698-g animals. Using the Kolmogorov-Smirnov test, the probability that these two distributions are equal is 0.997. Thus, the distribution of statoconia sizes reaches its final form by the time the animal reaches 170-200 gram weight, even though the number of statoconia continues to increase beyond this size (Fig. 3). The diameters of multiple statoconia show greater variability than that of the single statolith. In all post-metamorphic animals, the statolith was between 9 and 12 pm. In contrast, statoconia range from 2 to 20 pm. Both the statolith and the average statoconia size increase as the animals grow. Statoliths increased from 6.7 pm in a latestage embryonic animal to 12 pm in post-metamorphic animals, a 79% increase. Some earlier embryos contained statoliths estimated to be approximately 4 pm from observations made through the egg case. The statoconia increase from an average of 5.09 pm in l-2 mg post-metamorphic

=

10

2 :! t

5

E b

OF STATOCONIA

IN APLYSIA

TA

OF DIFFERENT

DIAMETERS SIZES

I m

0.1 170 -0.5 - 7009 Aplysic 9 Aplysio

0 0

2

4

6

8

10

12

14

16

16

20

l5 T6

0

0

IBI

2

4

6

6

Statoconia

10

12

Diameter

14

16

668 170

16

-

696 310

9 Aplysia

20

(rm)

Fig. 7. Comparison of distributions of statoconia diameters for animals of different size, as in Fig. 6. (A) Comparison of statoconia from 0.1-0.5 g animals (N = 317) with those from 170-700 g animals (N = 544). Distributions are significantly different, P < 0.0005. (B) Comparison of 170-310 g adults (N = 311) with 668 to 698 g animals (N = 233). Distributions are equivalent, P = 0.997.

70 INCREASE

fN NUM6ER

WITH INCREASING

OF SlATOCONlA

STATOCYST DIAMETER

INCREASE IN STATOCYST DIAMETER WITH INCREASING ANIMAL WEIGHT 250

P 6 t

200 -.

150-e

2 h c 0 g vi

lOO-50.50 oc -7

: -6

: -5

: -4

: -3

Log [ANIMAL

: -2

: -1

: 0

: 1

: 2

wmkiT (gm)]

Fig. 8. Scatter plot of increase in statocyst diameter with increasing animal weight. Smallest animal was embryonic, 70 pm in diameter, estimated to weigh 185 ng. Largest animal was 698 g.

100

150

200

L 0

STATOCYST DIAMETER (pm)

Fig. 9. Scatter plot of increase in number of statoconia with increasing statocyst diameter. All animals included were raised in circulating artificial sea water aquarium maintained at 1519°C. Note that afl animals with statocyst diameter greater than 45 gm have multiple statoconia and that all statocysts 44 pm in diameter or smaller had only a single statoiith.

Fig. 10. TEM of two statoconia within a supporting cell and a portion of one in the cyst lumen. Note microvilli which identify the cell as a supporting cell. Note lamellar structure of statoconia. This animal was 125 days post-hatching and weighed 10.7 g. AT21

Fig. 11. TEM of one statoconium within the lumen. This animal weighed 0.51 g. AT23

in Fig. 9. All Aplysia reared in artificial sea water between 15 and 19°C with statocysts less than 44 pm in diameter have single statoliths. Multiple statoconia are seen in all animals with statocysts greater than 45 pm diameter. The age at which multiple statoconia first appear varied from 64 to 85 days. The statocyst illustrated in Fig. 5 has multiple statoconia but the statocyst is only 36 pm in diameter. This animal was maintained at room temperature (23’C) and many of these animals were found to have multiple statoconia with smaller statocysts than were observed in animals reared at lower temperatures. In embryonic animals, the statolith is much larger than the thickness of any cells in the statocyst, so it is likely that the statolith is formed in the cyst lumen. However, the first statoconia to be formed are small enough to have been formed illustrated

intracellularly. Fig. 10 illustrates portions of two fully formed statoconia, approximately 3 pm in diameter, enclosed within the luminal membrane of a supporting cell. The cell is identified as a supporting cell by the presence of numerous microvilli. The supporting cells bear microvilli, whereas the receptor cells are characterized by numerous large cilia (McKee and Wiederhold, 1974; Wiederhold et al., 1986; Wiederhold et al., 1989). For comparison, an electron micrograph of a statoconium within the cyst lumen from another animal prepared identically is shown in Fig. 11. Similar lamellar arrangements, electron density and characteristic small (approximately 0.1 pm) vacuoles, where calcified material has apparently been lost in preparation, are seen in both the intracellular and the intraluminal statoconium. In contrast, the TEM in Fig. 12 shows the amorphous

Fig. 12. TEM of a statolith

from a 1.2 mm early post-metamorphic

character of the interior of a statolith. rings have been seen in statoliths.

No lamellar

Discussion The change from a single statolith to multiple statoconia occurs at the stage when the locomotor and feeding behavior is changing dramatically in the life cycle of Aplysia. The planktonic larva is a suspension feeder while the slow, crawling adult is a grazing herbivore (Carefoot, 1987). The veliger larvae, which have functional statocysts, swim upwards after hatching. This upward swimming is suggested to be an innate negative geotaxis (Hadfield and Switzer-Dunlap, 1984). The immature form of the statocyst, with the single statolith, would signal both linear and angular acceleration quite differently from the mature statocyst with

animal,

69 days post-hatching.

AT62.

multiple statoconia. In the early form, with the statolith in contact with the cilia of all 13 receptor cells continuously, any acceleration would be expected to affect the receptor potential and rate of action potential generation in all of the receptor cells and their axons. In the adult statocyst, with multiple statoconia all in the bottom one-third of the statocyst lumen, only those receptor cells on the bottom of the cyst are activated by accelerations and those cells whose cilia are not in contact with statoconia are silent (Wiederhold, 1974, Gallin and Wiederhold, 1977). Integration over all 13 sensory neurons in the immature statocyst would appear to give more information about the linear accelerations a pelagic veliger would experience in turbulent water. The adult form would be better suited to signal the direction of the gravity vector and the roll experienced by an adult as it falls

13

through sea water when it becomes dislodged from an algae mass. This would allow it to settle with the foot downward. The pelagic sea butterfly, Clione limacina, has a similar statocyst with a clump of statoconia which Tsirulis (1974) illustrates as aggregated and maintained in the center of the cyst lumen. The crawling land snail Helix (Laverack, 1968) and pond snails Pomacea paludosa (McClary, 1968) and Viuiparus uiviparus (Zaitseva et al., 1980) all have detached statoconia at the bottom of the cyst. Thus, in all of these species, pelagic forms appear to have centrally suspended statoliths or statoconia, while crawling and benthic species have independent statoconia which settle to the bottom of the cyst. Cragg and Nott (1977) have attempted to distinguish phylogenetic groups on the basis of statoconial number in bivalve pediveligers. They report that the pediveligers of Paleotaxodonta (nut shell), Pteriomorpha Pterioda (scallops) and Heterodonta (hard-shell clams) all have a single statolith in each statocyst while the Pteriomorpha Mytiloida (mussels) and some Pterioida (silvershells and oysters) have several statoconia in each statocyst. Since no information on the adult statocyst is given, it is not possible to say if multiple statoconia are added in the adult. In comparing statocyst diameter and statoconia number and size in various groups of molluscs, we find that Aplysia has among the largest statocysts. Only the prosobranch gastropods, Pomacea paludosa (Stahlschmidt and Wolff, 1972) and Paludina (Bullock and Horridge, 1965) have larger statocysts, being 400-500 pm and 450 pm, respectively. Aplysia does have the largest number of statoconia reported. McClary (1968) reports the number increasing from 3 to 670 during development in Pomacea paludosa. Arkett (1988) first recorded spontaneous, small-amplitude, postsynaptic potentials from prototrochal ciliated cells at about 45 h after fertilization in the veliger larva of the prosobranch, Calliostoma ligatum. Thus, the observation that the statocyst is one of the first organs to develop in Aplysia, first appearing 2-3 days after fertilization (Jacob, 1984), suggests that the statocyst could play a geotaxic role very early in the animal’s development. Cash and Carew (1989)

found that the greatest increase in the number of neurons occurs during stage 12 in Aplysia. This neuronal proliferation occurred in each of the central ganglia simultaneously and the authors, therefore, suggested the action of a general developmental signal or trigger, perhaps a hormone. This corresponds to the developmental stage where we find the first production of multiple statoconia. The better correlation between statoconia number and Apbsia weight (r = 0.978) (Fig. 3) than between number and age (Fig. 4) is likely due to the dependance of weight on maintenance conditions such as food and environmental conditions, including water temperature, pH and oxygen tension. Kriegstein et al. (1974) have shown that the growth rate of Aplysia is temperature dependant. While Aplysia may eat up to one-third of their body weight in algae each day, feeding rate is thought to be influenced by temperature, water salinity, body size, type of food, time of day, season, density of animals, reproductive status and past feeding history (Carefoot, 1987). Hirsch and Peretz (1984) report that Aplysia grow continuously in laboratory culture without approaching a limiting size. Kuslansky et al. (1978) did note satiation in Aplysia. In our observations, the animals will eat almost continuously, if food is available, and the growth rate is strongly dependent on the amount of algae presented. The data of Figs. 3 and 4 indicate that the rate of production of statoconia is related to the total metabolism of the animals, rather than a fixed time schedule of development. McClary (1968) found in the fresh-water snail Pomacea paludosa that the ‘statolith’ (as he terms what we are calling statoconia) size and number were related to snail size rather than age. This study also found two types of statoliths: a lamellate and a non-lamellate type with the latter being found almost exclusively in young snails of O-30 days. He found statocysts of very young snails to carry only small ‘statoliths’ with a wide variation in size in any one adult statocyst. Peretz and Adkins (1982) studied weight gain and allometric measurements of organs to find a reliable index of age for Aplysia. The size of the internalized shell was found to be the most reliable index. The continued growth of the statocyst and the increase in statoconia number appear to

74

follow the continued growth of the animals. In addition, statoconial number is a me&tic (countable) characteristic that warrants consideration as a taxonomic character. Mayr (1969) pointed out that meristic characters permit greater accuracy than continuous measurements because they are less subject to observational errors. We have compared statoconial number and size in animals from several sources: wild-caught adults from Santa Monica Bay, CA; post-metamorphic animals raised in Atlantic Ocean water at Woods Hole, MA and embryonic material from breeding adults in our aquarium. Bidwell et al. (1986) found normal statolith formation required the Sr content of artificial sea water to exceed 4 parts per million (ppm). Although all animals used here were derived from Santa Monica Bay stock, one might question whether the Sr content differs in the various media, contributing to the variation in data seen in Fig. 6 through 9. Culkin (1975) states that ‘average’ sea water contains 8.0 ppm Sr whereas Nicol(l967) gives 8.5 ppm in ‘ typical’ sea water. Kinsman and Holland (1969) measured 7.2 ppm Sr in Atlantic Ocean sea water off of New Jersey. Bernstein et al (1987) measured 7.7 ppm Sr in the North East Pacific Ocean sea water. Gallagher (personal communication) has measured Sr content of 7.5 ppm in Atlantic Ocean sea water at Woods Hole, MA. Instant Ocean artificial sea waters is specified to contain 10 ppm Sr. Thus, all of the natural and artificial sea waters in which our animals were reared and maintained (as well as other non-estuarial sea waters) have ample Sr for normal statolith development. We have not investigated whether Sr is required for production of multiple statoconia. As shown in Fig. 9, multiple statoconia are produced when the statocyst reaches 45 pm in diameter. In statocysts smaller than this, the sensory receptor-cell cilia are always seen to project to the single statolith (e.g., Fig. 2). In adult animals, the maximum ciliary length is 12 pm. Thus, if cilia grow to this length in the early post-metamorphic animals, the maximum diameter of the statolith is 12 pm and the cells of the statocyst wall are 3 to 4 pm thick, a statocyst of 42 to 44 pm diameter would be the largest in which the cilia could support the statolith in the center of the cyst lumen. As the cyst grows beyond

this size, the statolith would fall, thus interacting with just a few receptor cells, rather than with all 13 simultaneously. It could be that this shift to activating a few, rather than all receptor cells, is what triggers the production of multiple statoconia; one would certainly expect different central integration of the input from the array of receptor cells. As discussed above, with one central statolith, linear, or possibly even angular acceleration would affect all 13 receptor cells simultaneously whereas in adult animals, only those cells at the bottom are depolarized by acceleration (Gallin and Wiederhold, 1977). Some efferent influence from the cerebral ganglia, associated with such changes in peripheral input might trigger multiple statoconia formation. Another trigger could be direct contact of the statolith with the supporting cells or their microvilli when the statolith ‘falls’ to the cyst wall. A mechanosensitive response in these cells could lead to increased calcium conductance and, thus, increased intracellular calcium activity which, in turn, could initiate mineralization of the statoconia. Coggeshall (1969) noted that the statocyst diameter was approximately 200 pm and ‘approximately constant irrespective of the size of the animal.’ He examined Aplysia from 1 to 300 g. From the data of Fig. 8, the statocyst should double over this weight range, an increase that would appear small compared to the ten-fold concomitant increase in body length. As our animals increased from 70 pm (newly hatched) to over 30 cm in length, a factor of over 4,000, the statocyst diameter only increases by a factor of 15.6, from 16 to 250 pm. The continuous addition of statoconia is significant because it indicates that the conditions for mineralization are present throughout the life of the animal. The distributions of statoconia diameters shown in Fig. 6 suggests that the range and mean is greater in larger animals. Two possibilities can be distinguished: 1) statoconia produced in large animals could be larger than those produced in smaller animals or, 2) once a stone is ejected into the cyst lumen, it may continue to grow. The lamellar whorls seen in the receptor cells (Wiederhold et al., 1986; Wiederhold et al., 1990) may be exocytosed and carry membrane complex to the growing statoconia in the lumen.

However, the fact that the largest statoconia in the largest animals (700 g) are only 6 percent larger than those in 0.1 to 0.5 g animals suggests that the statoconia do not grow in the cyst lumen and favors the first hypothesis. Clearly the early postrnet~o~~c animals with few statoconia (1-2 mg animals in Fig. 6) produce smaller statoconia than the larger animals. On the other hand, the single statolith does grow in the cyst lumen, from approximately 4 pm, the smallest diameter we have measured in embryonic animals, to 12 pm in post-metamo~~c specimens. In adults with many statoconia, it has not been possible to identify the statolith, so we do not know how large it can become. Several investigators have questioned the role of the statocysts in enabling different molluscan species to orient to gravity. Willows (1985) found few deficits in locomotion after statocyst extirpation in the nudibranch Phestille sibogue. Removal of both statocysts in the prosobranch, Pomacea paludosu had little or no effect on the rate and direction of movement, activity level, position at rest, righting ability, ciliary feeding and ability to crawl to the surface for inspiration (McClary, 1966). In both of these studies, observations were made 4 to 5 days after the surgical manipulations. Moffett and Snyder (1985) found that the statocyst nerve regenerated, which allowed the pulmonate snail Melamp~ bide~tat~ to right itself several days after the statocyst nerve was sectioned. Janse (1981) showed that the statocyst works in conjunction with an oxygen-sensing mechanism to allow the snail Lymnea stagnalis to display either positive or negative geotaxis to seek Oz-rich conditions. Wolff (1975) demonstrated convincingly that, if observations are made before the statocyst or its nerve is allowed to regenerate, Pomacea with both statocysts removed move in random directions on a turning platform, whereas normal animals always move to the center of rotation. This experiment avoided the confounding chemical and tactile cues which experimental animals were likely using in those studies that concluded that the statocysts are not important for orientation. Thus, there can be little doubt that these organs do function to sense natural or artificially created ‘gravity’. In a study of the veliger larvae of the

nudibr~ch, ~ostanga p~chru, Chia et al. (1981), note that the weight distribution of the swimming larva automatically orients it with the velum directed upward and the heavier shell directed downward. Thus, the orientation of the larva during swimming should be monitored chiefly by the four polarized hair cells that encircle the base of the statocyst. The non-polarized hair cells act to monitor sinking in the pediveliger larva’s life; during a fall the statolith is forced to the roof of the statocyst cavity where the non-polarized hair cells are located. There is disagreement in the literature as to the site of generation of the statoconia in molluscan statocysts. Laverack (1968) concluded that in the snail Helix, the stones are generated in the large cells. Although we now know that these are the sensory receptor cells, Laverack assumed that the small cells were the receptor cells and that the large cells must be supporting cells. In contrast, several reports illustrate small statoconia within invaginations of the luminal surface of supporting cells. Tsirulis (1974) illustrates 5 statoconia in one supporting cell and another app~ently emerging from a supporting cell in the statocyst of the sea butterfly, Clione Zimacina. Kuzirian et al. (1981) show what is described as a forming statoconium within a supporting cell in Hermissenda. Cragg and Nott (1977) describe one non-ciliated cell in each statocyst of the scallop Pecren maximw with inclusions resembling the luminal statoconia they describe and suggest that stones are exocytosed from this supporting cell into the cyst lumen. Geuze (1968) illustrates statoconia at the apical surface of supporting cells in the pond snail Lymnaea stagnalis. He punctured the statocysts in some animals, withdrawing both the statolymph and the statoconia. Within 48 hours, the statocysts regenerated and at 12 hours after the puncture, statoconia of low electron density were seen within vacuoles at the luminal surface of poorly differentiated cells. In normal juvenile animals, statoconia are illustrated within broken vacuoles adjacent to the luminal surface of supporting cells. Geuze interprets these observations to indicate that the statoconia are formed within the vacuoles in the supporting cells and this entire vacuole is expelled into the cyst lumen, where the vacuole disintegrates.

76

In the Aplysia statocyst, the electron micrograph of Fig. 10 gives particularly clear evidence that a fully formed statoconium is enclosed by the intact luminal membrane of a supporting cell. The numerous microvilli on the lurninal surface distinguish this cell from a sensory receptor cell. This would suggest that when the statocyst reaches sufficient size that its cilia can no longer suspend the single statolith within the center of the cyst lumen, some signal ‘turns on’ the supporting cells to begin producing, or calcifying, intracellular statoconia. As suggested above, this could be some efferent neural signal from the ganglia or could be due to mechanical stimulation of the supporting cells by the falling statolith. Under the latter mechanism, mineralization would continue due to mechano-stimulation by the statoconia. Such mechanisms would not likely be involved in production of the statolith, since in embryonic material the statolith is much larger than the supporting cells (Wiederhold and Sharma, 1990). Presumably the statolith is formed by precipitation within the cyst lumen. The ring structure so prominent in the statoconia is not seen in the statolith. The rings appear to be layers of membrane or matrix protein laid down on the developing stone within the supporting cell. Determining the identity of this material would allow us to explore further the mechanisms by which mineralization of the statoconia is initiated and controlled. Salamat et al. (1980) studied otoconial development in fetal rats and conclude that they are formed in the endolymph. They suggest that the calcification is laid down on a filamentous network derived from secretory vesicles at the apical surface of sensory epithelial cells. Ann&o (1980) and Anniko et al. (1987), using X-ray micro probe elemental analysis of the fetal mouse labyrinth, concluded that vesicles at the apical surface of supporting cells have a high calcium concentration, which is released into endolymph beneath the otolithic membrane to calcify the otoconia. Several types of otoconia have also been observed in the macular regions of young rats by Ross and Peacor (1975). These authors suggest that multifaceted, rounded and transitional forms may arise from the addition of calcite on the terminal faces of the multifaceted ‘seedling’. Lim (1973, 1984) however, suggests that these rounded forms may

be due to the pressure exerted by the gel these otoconia are embedded in. In embryonic chick, Fermin et al. (1987) show fibrillar material arising from the microvilli of supporting cells, which is suggested to form the primitive otolithic membrane. As calcification begins, this membrane segments to form the otoconia (Fermin and Igarashi, 1985). Thus, in vertebrate systems, the otoconia are mineralized in endolymph, a very low-calcium fluid. Bosher and Warren (1978) measured a calcium concentration of 23 PM in cochlear endolymph of rats. In Aplysia, statolymph appears to communicate freely with extracellular fluid, since no resistance or potential difference could be measured between the statocyst lumen and the sea water bathing an in vitro preparation (Gallin and Wiederhold, 1977). Thus, statolymph probably has a calcium concentration near that of sea water (10 mM). The supporting cells would be expected to have very low intracellular calcium, as in most cells, so for the statoconia to become calcified within these cells, some mechanism must lead to a calcium influx. This could be achieved by a mechanosensitive calcium conductance at the luminal surface of the supporting cells or their microvilli. Acknowledgement This work was supported by the National Aeronautics and Space Administration Space Biology Program and Veterans Administration Medical Research funds. References Alkon, D.L. (1975) Responses of hair cells to statocyst rotation. J. Gen. Physiol. 66, 507-530. Anniko, M. (1980) Development of otoconia. Am. J. Otolaryngol. 1, 400-410. Anniko, M., Wikstrom, S.O. and Wroblewski, R. (1987) X-ray microanalytic studies on developing otoconia. Acta Otolaryngol. (Stockh.) 104, 285-289. Arkett, S.A. (1988) Development and senescence of control of ciliary locomotion in a gastropod veliger. J. Neurobiol. 19, 612-623. Bidwell, J.P., Paige, J.A. and Kuzirian, A.M. (1986) Effects of strontium on the embryonic development of Aplysia californica. Biol. Bull. 170, 75-90. Bernstein, R.E., Betzer, P.R., Feely, R.A., Byrne, R.H., Lamb, M.F. and Michaels, A.F. (1987) Acantharian fluxes and

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