CONTRIBUTIONS TO THE BIOLOGY OF THE SEA URCHIN EvelDARlS TRlBULOlDES (LAMARCK)l
B. F. McPHERSON Institute of Marine Sciences, University of Miami
ABSTRACT The sea urchin Eucidaris tribuloides (Lamarck) is found in several different habitats off southeastern Florida. This species appears to be particularly adapted for living in rocky and coral reef areas, using its primary spines to anchor itself under rocks or in crevices. It may move from these protective sites at night. On reefs it ingests hard particles of limestone, probably deriving nourishment from boring and encrusting organisims associated with this material. On the patch reefs of Margot Fish Shoal, growth of the gonads in the population took place during spring and summer ] 965-66. There was some gametogenetic activity throughout the year. Sex cells accumulated, forming ripe gonads in late summer and early fall of .I 965 and .I 966. Relative growth rate of the test decreased with increasing size of the urchin, and it also decreased during the slimmer. Urchins probably live at least four to five years. A.lthough the greatest population density was observed on shallow-water, outer reefs, the growth rate there was less than in areas closer to shore. Urchins from different habitats varied in size and in dimensions of the test. Eucidaris was reared from the egg through metamorphosis in 25 days.
INTRODUCTION Recently a number of investigators have been working on the ecology and physiology of various sea urchins (Boolootian, ] 966). The work has centered around relatively few species and practically nothing has been done on any member of the cidaroids. Perhaps this is because areas where most of the work has been carried out (California, New England, and northern Europe) do not have cidaroids in shallow water. Even in places where there are shallow-water representatives, such as Barbados, BWI, the urchins may not be abundant or easily obtained (John Lewis, personal communication). In the West Indian region there are 12 species of cidaroids, one of which is littoral, the rest being found at moderate depth. The littoral species, Eucidaris tribuloides (Lamarck), is relatively abundant in some habitats off southeastern Florida, and this afforded an ideal opportunity to study a cidaroid urchin and to compare its biology with that of other tropical urchins. According to Mortensen (1928), Eucidaris tribuloides occurs from South Carolina and Bermuda to Bahia, Brazil, in the western Atlantic, being very common throughout the West Indian waters. Bernasconi
1 Contribution No. 904 from the Institute of Marine Sciences, University of Miami. This study was carried out under Grant No. WP-00573 from the National Institutes of Health. 1968] McPherson: Biology of Eucidaris 40 I
(1955) reported that E. tribuloides was collected off Rio de Janeiro and the island of Trindade in the South Atlantic. In the northwestern Atlantic it has been collected as far north as Cape Hatteras, North Carolina (Cerame- Vivas & Gray, 1966), and in the Gulf of Mexico it has been reported on Alacran Reef, Campeche Bank (Kornicker et al., 1959), and from St. Petersburg, Florida, at 55 meters (Donald K. Serafy, Florida State Board of Conservation, personal communication). Mortensen also reported that it occurred off the African coast (variety africana), at the Cape Verde Islands and in the Gulf of Guinea, and in the mid-Atlantic at the Azores and Ascension Islands. Over much of its range E. tribuloides occurs from very shallow to relatively deep water; Mortensen (1928) reported that it occurs to 450 m. However, along the edge of its distribution, at least in the North Atlantic, it is absent from the shallow water (North Carolina; St. Petersburg, Florida) . Kier & Grant (1965) studied the distribution of a number of echinoids off Key Largo, Florida, in the Pennekamp Coral Reef State Park. They reported that Eucidaris tribuloides was solitary and widely but sparsely distributed in rocky "niches" and turtle grass (Thalassia testudinum) beds. In the area of study it was collected from the back reef channel, over a mile from shore, seaward to depths of at least 25 meters.
STATIONS Sites were selected to represent several environmentally different habitats (Fig. 1). Long Reef is an outer "dead" reef, composed mainly of rubble and old coral rock. Margot Fish Shoal is located about 1/2 mile inshore from Long Reef and consists of a large expanse of "patch" reefs, each patch separated from others by areas of sand or "sea grass" and deeper water. On this site there was both living and dead coral. A third site was the seaward side of Virginia Key, off the sewage treatment plant. This area has been referred to as "Sewage Beach" (Moore et al., 1963a and b). It is a shallow area of "sea grass" (mostly Thalassia testudinum), sand, and rocks. Eucidaris was found throughout the area, but in small numbers. Regular monthly sampling was begun in May, 1965. Collections on Long Reef were discontinued in September, 1965, because urchins became very scarce, partly because of Hurricane Betsy. Sampling on Margot Fish Shoal was continued through 1 December, 1966, and on Virginia Key through 31 October, 1966. Before the regular monthly sampling was initiated on these areas, intermittent collections had been made. In addition to these three sites, sampling and observations were also conducted at irregular intervals at a number of other locations (Fig. I): Lake Worth jetties (northern inlet); French Reef (an outer reef of coral rubble and rock, mostly "dead"); seaward of Alligator Reef (45-55 402 Bulletin of Marine Science [18(2)
LAKE WORTH _____~ JETTIES
NI
..____SEWAGEBEACH, q VIRGINIA KEY
< d f ..--LONG REEF ri/~F:~;~~~"5" 0 .~/ -----MOLASSIS HUMP 25 - P .~.ALLIGATOR REEF 06 c:/ ,e? ...-1 •• 0 •• G c.--'
800 I FIGURE 1. Chart of southern Florida, showing collection stations. meters, flat mud-sand bottom); and Molasses Hump, Molasses Reef, Key Largo (coral rubble, mostly "dead"). Urchins collected in all the above areas, except number six, were taken in shallow water (1-3 meters), in most cases by diving with mask and flippers. Collections were made off Alligator Reef with the use of a 10-foot otter trawl. In addition to the urchins collected from the above areas, some were obtained from other locations in the Atlantic in order to study variation in shape of the test (see Table 1). 1968] McPherson: Biology of Eucidaris 403 TABLE 1 COLLECTIONSITES FOR Eucidaris trihuloides USED IN STUDIES ONVARIATIONIN DIMENSIONOF THETEST1
1. Margot Fish Shoal. 2. Long Reef. 3. Alligator Reef. 4. Sewage Beach, Virginia Key. 5. Llama Piedrecitas, Cartagena, Colombia; "living" and "dead" coral reef, 1-2 meters (urchins supplied by Dr. R. Pfaff, University of Cartagena, Colombia). 6. Ascension Island; shallow, rocky area (urchins supplied by Mr. R. Kes- singer) . 7. West Africa (E. trihuloides, variety africana); collected from R/V PILLS- BURY;P-275 (1° 24'N, 5° 38'W, 5-38 fms); P-22 (5° 25'N, 0° 01'W, 28 fms).
1 Ecological information on the first four collection sites is given in the text.
METHODS AND MATERIALS For studies on growth of the gonads and reproduction, approximately 15 urchins, ranging in size from 28 to 35 mm in test diameter (t.d.), were collected monthly on Margot Fish Shoal to estimate changes in gonadal size and sexual condition in the population during the year. In addition, urchins ranging from 8 to 39 mm t.d. were collected at this same station in October, 1965, and in August, 1966, to estimate changes in gonadal size with increasing test diameter, and the size at which the first ripe sex cells develop. Varying numbers of urchins were also collected at irregular intervals at Long Reef, Virginia Key, and Lake Worth, to compare with those collected on Margot Fish Shoal. For most of the urchins collected, the volume of the gonads was measured, and a gonadal smear was examined under a microscope to determine if there were ripe sex cells. In order to verify ripeness of ova or spermatozoa in doubtful cases, fertilization experiments were carried out by mixing ova and sperm. Fertilization was considered suc- cessful if any cleavage was observed. Volume of the gonads was expressed 100 gonad volume relatively as 1 . This is the gonad index~. Gonad volume test vo ume was measured by water displacement in a graduated cylinder. The test volume (including spines, which accounted for about 20 to 30 per cent of the test volume) was determined for 42 individuals from Margot Fish
'10 gonad volume ~t~es-t -vo-Il-,m-e-has been used by some authors (Moore et aI., 1963 a and b). 404 Bulletin of Marine Science [18(2) Shoal by water displacement. The cubed diameter of these individuals was plotted against test volume and a straight line regression was cal- culated. In subsequent work the test diameter was measured and the test volume was calculated from this regression line. Histological sections were made from the gonads of most of the urchins collected in the regular monthly samples at Margot Fish Shoal and from some of the urchins collected in other areas. Gonads were fixed in Bouin's solution. Sections were made perpendicular to the axis of greatest length of a gonad at a thickness of 8,..., and were stained with hematoxylin and eosin or with Masson's trichrome stain, the latter being used in most cases. On a few urchins, sections were made through different parts of the same gonad to determine if there were differences in development of sex cells in different parts of the gonad. Microscopic examinations of smears were made on more than one gonad of some urchins to determine if there were differences between gonads in the same individual. There were no evident differences between sections in different parts of the gonad or between smears of different gonads of the same urchin, so in all subsequent work sections or smears were made from any of the five gonads. Growth of the test was recorded as increase in the maximum diameter. Test diameter was measured with vernier calipers to the nearest tenth of a mm. Three methods were used to estimate the growth of the test: ( I) measuring urchins kept in aquaria, (2) tagging urchins, (3) using size- frequency analysis of the population. The last method was not useful in establishing growth rates, but did give estimates of the mean and maximum sizes of individuals in different areas. Urchins collected on Margot Fish Shoal were held in aquaria and measurements were made monthly to determine their test growth. Water flow through the aquaria was maintained at about two liters per minute. Urchins were offered an excess of food (rock infested with the boring sponge Cliona lampa). A few urchins were sacrificed during the year to determine gonad size and sexual condition. The effect of controlled temperature on growth of the test was measured on 21 individuals of Eucidaris held in aquaria. Urchins were measured, tagged, and divided among three aquaria which had running sea water at 180, 22°, and 26°C (flow was about 0.5 liter per minute). Urchins in these tanks were maintained for 54 days (8 December, 1966, to 31 January, 1967) with an excess of food (Cliona lampa). At the end of this time the urchins were remeasured, and the gonad volumes were determined. Several different types of tube tags were originally used for tagging Eucidaris. Tags obtained from the Floy Tag and Manufacturing Company, Seattle, Washington, proved best, and these were used in all tagging experi- ments after August, 1965. Tube tags were slipped on the upper, mature, 1968] McPherson: Biology of Eucidaris 405 primary spines with care not to damage the spine musculature. Mature cidaroid spines lack the epidermal tissue coat found over the spines of other urchins, hence cidaroids will tolerate "fouling" on these spines. Urchins were tagged and released at irregular intervals on Margot Fish Shoal and Long Reef in 1964. In 1965 and 1966, urchins were tagged and released off Virginia Key and on Margot Fish Shoal during most months of the year. On Margot Fish Shoal and Long Reef, urchins generally were collected, tagged, and released on the same day. In a few instances, however, they were brought into the laboratory, tagged and released on a later date. Most of the urchins released off Virginia Key were collected on Margot Fish Shoal. When tagged urchins were re- captured they were measured and returned immediately to the sea. Size-frequency analyses were made at various times on data from Margot Fish Shoal, Long Reef, Virginia Key, and French Reef. These data were obtained from urchins which were collected, measured, and then returned to the sea, and did not include transplanted or tagged urchins. Statistical comparisons were made on the growth of urchins held in aquaria and tagged urchins which were recovered. Initial test diameter was plotted against growth increment, and in some cases regression lines were calculated and drawn. If there was too much scatter for the regres- sions to be significant, a mean growth was calculated. A comparison be- tween growth in different areas was made by regression analysis or comparison of means. The relation between growth, size, and season was tested by correlation analysis. The relationship was determined between dry weight and volume of the gonads for 31 urchins, and between dry weight of the somatic tissue and volume of the test for 26 urchins. For the latter, urchins were decalcified with 20 per cent formic acid and WIN-3000 ionating resin. Comparisons of test shape were made on material given in Table 1. The following measurements (Fig. 2) were made: (1) test diameter (t.d.), (2) test height (t.h.), (3) peristome diameter (measured across Loven's axis); (4) diameter of the apical system (measured across Loven's axis), (5) interporiferous width. Measurements of the test were made with vernier calipers, or with an ocular micrometer (interporiferous width), to the nearest tenth of a mm. A number of methods were used in attempting to rear Eucidaris from the egg through metamorphosis. The larvae were held in finger bowls and glass jars of various sizes. Sea water was usually taken from aquaria at the Institute of Marine Sciences in Miami, but in several cases it was taken from well offshore in the Florida Straits. Both filtered (# 1 What- man) and unfiltered sea water was used in different experiments. The larvae were held in numbers varying from about 5 to 20 individuals per liter of sea water. In most cases the water was changed every other 406 Bulletin of Marine Science [18(2)
INTERAMBULACRUM ' LOVEN'S "'- AXIS / TO. '~, / ~
/, ....•. ....•. , ' "'- , , \ ) PO '-t.0 . \v / ABORAL ORAL / SIDE VIEW VIEW I VIEW
FIGURE 2. Schematic diagram of the test of Eucidaris tribuloides, showing measurements used in this study. T.D. = test diameter; T.R. = test height; A.D. = apical diameter; P.D. = peristome diameter; A. = ambitus; ip. = interporiferous width. day. This was accomplished by carefully removing the larvae with a glass pipet and placing them in fresh sea water. A number of cultures of phytoplankton were tested as food for the larvae, including the flagel- lates, Amphidinium klebsi, Platymonas sp., and Chloromonas sp., and the diatom, Asterionella japonica. Sketches and notes were made as the larvae developed, but since only a few individuals were reared past the early stages and only one individual was reared to the young urchin, the material available was limited. Observations on feeding, behavior, and predators were made at various times at the different stations. Notes on abundance were maintained for all stations, and sampling for density was carried out on French Reef. Estimates of density were made by dropping a one-meter-square iron frame at random over the area to be sampled, and counting the urchins within each meter. GROWTH AND REPRODUCTION Growth of the Gonads.-When the gonads of Eucidaris from Margot Fish Shoal were examined during a period of maximum size, there was an increase in absolute volume from less than 0.1 ml to about 0.7 ml over a test-diameter range of 20 to 30 mm (Fig. 3). Gonad index also tended to increase over this size range, roughly from 1 to 4, indicating that relative size of the gonad increased between 20 and 30 mm t.d. Urchins larger than 30 mm t.d. had widely varying gonad volumes and indices, and there appeared to be no trend in the data indicating changes in relative gonad size with test sizes over the range 30 to 40 mm. 1968] McPherson: Biology of Eucidaris 407
9 E » w 9 9 ~ 2.0 N=45 rJ :J 9 rJ ....J r! 0 > 0 <::( rJ r! z if 9 0 if t:> 9 if 9 1.0 ff rJ \' »
\' rJ \' 9 \' if rJ 0"1>90- \' if if if rJ t- ~ \' \' , 5 10 15 20 25 30 35 40 TEST VOLUME (ml) 20 30 35 39 TEST DIAMETER (mm) FIGURE 3. Gonad volume of Eucidaris tribuloides during periods of maximum ripeness on Margot Fish Shoal.
The monthly mean gonad index for urchins collected on Margot Fish Shoal (in the size range 28 to 35 mm) is given in Figure 4. These data indicate that gonad growth on Margot Fish Shoal occurred between January and October in ]965, and again between February and July in ] 966. In the first period, the gonad index increased about 2.0 units, which would represent an actual increase of 0.4 ml for a 30-mm urchin. During the second period, the gonad index increased about 2.8 units for the females (or 0.9 ml for a 30-mm urchin) and 4.4 units for the males (or 1.6 ml for a 30-mm urchin). Significant decreases in gonad index occurred between the summer of 1965 and the following winter, with minimum values occurring in January for the females (1.4) and in Feb- ruary for the males (1.9). Urchins collected off Virginia Key were larger in test diameter than those collected on Margot Fish Shoal and, correspondingly, the gonads were larger absolutely at the former location than at the latter. In addi- 408 Bulletin of Marine Science [18(2)
6 x w o ~4 o M AM JJASON OJ FMAMJ JA SOND 1965 1966 FIGURE 4. Mean gonad index of Eucidaris tribu/oides on Margot Fish Shoal. Vertical lines give the 95 per cent confidence intervals. tion, the gonad index (relative gonad size) was significantly greater at Virginia Key than at Margot Fish Shoal. Gonad index at the former site ranged from a maximum of 12.0 in March, 1964, to a minimum of 5.9 in September, 1966 (Table 2). Gonad index at Long Reef during the summer of 1965 was not significantly different from that at Margot Fish Shoal during the same period. The gonad indices of nine urchins held in aquaria were all larger than the mean monthly indices (including the 95 per cent confidence intervals) of urchins from Margot Fish Shoal. Reproduction.-The ova of Eucidaris, when viewed in unstained smears, are round, having a diameter of 70p.. The oocytes are the same size as the ova or smaller, and are distinguished from the latter by their large germinal vesicles. The spermatozoa are identified by their extremely rapid motion when a drop of sea water is added to a small piece of testis. The ova of Eucidaris when viewed in stained sections range in shape from circular (about 65/l in diameter) to oval (the largest measured being 60 x 80p.). The nucleus has a diameter of about 7p.. Oocytes are easily identified by their conspicuous germinal vesicle. The largest oocytes are about the same size as the ova, and have germinal vesicles of about 33p. in diameter. It was not possible to distinguish smaller oocytes from primary or secondary ones, or to distinguish either of these from the small oogonia. The spermatozoa of Eucidaris are conical, with a body of about 3p. in length. In the centers of the acini, they tend to form masses in which individuals are not distinguishable. Possibly some conical sex cells seen in the testes were mature spermatids, since active sperm from these testes were not observed in gonad smears. Younger spermatids, spermatocytes, and spermatogonia were not differentiated. 1968] McPherson: Biology of Eucidaris 409 TABLE 2 THE SIZE OF THE GONAD FROM SPECIMENS OF Eucidaris tribuloides COLLECTED OFF VIRGINIA KEY Test Test Gonad Mean diameter volume volume Gonad gonad Date (mm) Sex (ml) (ml) index index 11 Mar., '64 42 is 49.0 7.0 14.9 46 ? 63.4 7.0 11.0 45 is 59.6 7.0 11.7 12.0 38 ? 37.1 4.0 10.8 2 Sept., '64 48 c;> 71.7 11.0 15.3 49 c;> 76.0 7.0 9.2 46 c;> 63.4 2.5 3.9 9.0 42 c;> 53.4 5.0 9.8 31 ? 21.7 1.5 6.9 6 Dec., '65 47 ? 67.5 3.5 5.2 41 c;> 49.0 3.2 6.5 6.5 46 c;> 63.4 5.0 7.9 4 May, '66 43 c;> 55.9 8.0 14.3 49 c;> 76.0 6.0 7.9 11.1 29 Aug., '66 44 c;> 55.9 4.0 7.2 49 is 76.0 11.5 15.1 11.2 27 Sept., '66 36 ? 32.1 2.6 8.1 30 ? 19.8 1.0 5.1 25 c;> 14.0 0.6 4.3 33 c;> 25.4 1.3 5.1 5.9 32 ? 23.4 2.0 8.5 25 Q 12.8 0.5 3.9 27 Q 15.3 1.0 6.5 31 Oct., '67 37 Q 34.5 3.6 10.4 34 ? 27.5 1.4 5.1 37 ? 34.5 3.8 11.0 8.2 34 ? 27.5 3.4 12.4 27 ? 15.3 1.0 6.5 44 ? 55.9 2.0 3.9 The non-germinal cells in the gonads of Eucidaris are called nutritive phagocytes (after Holland & Giese, 1965). These exist in two phases: globulated and deglobulated, Globulated nutritive phagocytes were clearly demonstrable in the gonads of Eucidaris by gently squashing a small piece of gonad in a drop of sea water beneath a cover slip. Each nutritive phagocyte is typically a spherical-to-oval cell (slightly smaller than the ova) containing globules of various sizes. Cell boundaries were not distinguishable in stained sections, but large vacuoles and globules, which are characteristic of this phase, were conspicuous. With Masson's tri- chrome stain these phagocytic cells appeared as a green mass, sometimes completely filling the acini. At certain times the phagocytic cells contained 410 Bulletin of Marine Science [18(2) FIGURE 5. Histological sections through the gonads of Eucidaris tribuloides. A, ovary, stage 1; B, ovary, stage 2; C, ovary, stage 3; D, ovary, stage 4. Scale lines are 100,u. particles which stained gray to black and which appeared to be derived from germinal-cell debris. The gray appeared to be cytoplasm in different degrees of disintegration, and the black appeared to be nuclei or nuclear debris. Deglobulated nutritive phagocytes were not individually distinguishable, but appeared in section as net-like, non-germinal material in the acini 1968] McPherson: Biology of Eucidaris 411 TABLE 3 CLASSIFICATION OF THE GONADAL CONDITION IN Eucidaris tribuloides Stage Description 1. Acini relatively empty, except for "net" (deglobulated nutritive phago- cytes); gonads small. 2. Acini relatively empty, except for "net" (deglobulated nutntlve phago- cytes); sex cells evident, particularly around the base of the germinal layer. 3. Nutritive material filling the acini; few sex cells. 4. Nutritive material filling the acini except for sex cells which form continu- ous bands around the base of the germinal layer. Females have mostly young oocytes. Males lack spermatozoa, but have earlier stages of sex cells. 5. Acini filled mainly with developing sex cells; nutritive material reduced. In females, mature and younger oocytes (and usually some ova) in acini. In males, spermatozoa and earlier stages of sex cells in acini. 6. Ripe sex cells filling acini; nutritive material mostly absent. 7. Acini mostly empty, particularly in lumen. Nutritive "debris" may be evi- dent, but this does not appear to be associated with discrete cells. Relict ova or spermatozoa may be present. Earlier stages of sex cells mostly absent or inconspicuous. when stained green with Masson's trichrome stain (Fig. 5,A). This phagocytic material tended to cover the oocytes or ova with a thin layer. Spermatozoa tended to associate with this phagocytic net, and were prob- ably also covered with a thin layer, although this was not demonstrated. There was no obvious difference between phagocytic cells of ovaries and testes in Eucidaris, except for their inclusions. After examining all histological sections, urchins from Margot Fish Shoal were classified into seven arbitrary stages of gonad development. A description of each stage is given in Table 3, and Figures 5, 6, 7, and 8 illustrate these stages. In order to study the annual reproductive changes in the population, tables, based on examination of sections, were prepared, giving the number of individuals in each stage for each monthly sample. Various stages could then be rated as typical for different periods during the year. Typical gonads for each period were then described, based on microscopic examina- tions of sections and smears, and on gonad volumes. Typical gonads during the winter of 1965-66 were characterized by deglobulated nutritive phagocytes which formed conspicuous "nets" in the acini (7 December, 1965,4 January and 2 February, 1966). Most germi- nal cells in the ovary were immature (oocytes or earlier stages), but there were a few relict ova which appeared to be in various stages of disintegra- 412 Bulletin of Marine Science [18(2) FIGURE 6. Histological sections through the gonads of Eucidaris tribuloides. A, ovary, stage 5; B, ovary, stage 6; C, ovary, stage 7; D, ovary, stage ], showing relict ova. Scale lines are lOa!'. tion (Fig. 6, D). The sex cells of the males were also mostly immature. A few individuals, however, had active sperm (observed in microscopic smears) ; these were probably relict. During this period, the number of germinal cells in the gonads varied. Some gonads contained only a few sex cells which were sparsely distributed throughout the acini (stage I; Figs. 5,A; 7,A). In others, there appeared 1968] McPherson: Biology of Eucidaris 413 FIGURE 7. Histological sections through the gonads of Eucidaris tribuloides. A, testis, stage 1; B, testis, stage 2; C, testis, stage 3; D, testis, stage 4. Scale lines are 100".. to be many sex cells around the base of the germinal layer (stage 2; Figs. 5,B; 7,B). Various gradations existed between these two stages, and division between the two was somewhat subjective. Gonad volume during this period was relatively low. Typical gonads during the spring in 1965 and 1966 were characterized by the globulated nutritive phagocytes which filled the interior of the acini, 414 Bulletin at Marine Science [18(2) FIGURE 8. Histological sections through the gonads of Eucidaris tribuloides. A, testis, stage 5; B, testis, stage 6; C, testis, stage 7; D, ovary, staOge4, showing typical condition during summer of 1965 and 1966. Scale lines are 100M. replacing the "net" formed by the deglobulated phase of the phagocytes. In general, this non-germinal material differed from that observed later during the summer in that it appeared very homogeneous, containing only globules and vacuoles, while later in the summer it appeared more heterogeneous, containing what appeared to be germinal debris. Gametic 1968] McPherson: Biology of Eucidaris 415 development was variable. Some ovaries were practically devoid of oocytes or ova (stage 3; Fig. 5,C), while others contained large numbers of oocytes around the base of the germinal layer (stage 4; Fig. 5,D). Sim- ilarly, some testes contained very few sex cells (stage 3; Fig. 7,C), while others contained large numbers concentrated mainly around the base of the germinal layer (stage 4; Fig. 7,D). Various gradations existed be- tween stages 3 and 4, and the division between the two was subjective in many cases. Very few individuals with ripe sex cells were observed during this period by microscopic examination of gonad smears. Gonad volume during this period tended to be higher than it was earlier in the winter, and this was probably related to the accumulation of nutrients by nutritive pbagocytes. During tbe summer of 1965 (June-September) gonads were character- ized by globulated nutritive phagocytes containing what appeared to be germinal debris (stage 4; Fig. 8,D). Most of the urchins were ripe on 12 October (stage 6; Figs. 6,B; 8,B). Gonad index was maximal on this date (for 1965) and decreased sharply thereafter, indicating that spawning probably took place during the period 12 October to 16 November. During the summer of 1966, urchins ranged from stage 4 (resembling those which were typical for the summer of 1965) through stage 6. This was the period when the greatest number of ripe (stage 6) and nearly ripe (stage 5) urchins were found, and when the gonad volumes were maximal. Apparently spawning took place earlier this year than in 1965, as indicated by the sharp decrease in gonad volume (gonad index) after 31 August. Figure 6,A shows a typical ovary in stage 5 during the summer. Oocytes reached their maximum size and filled most of the acini, although some ova were evident in the lumen. Figure 6,B shows a typical ripe ovary in stage 6 during the summer. Ova filled most of the acini. Nutritive phagocytes were inconspicuous or absent. The connective tissue layer was thin and inconspicuous. Figure 8,A shows a typical testis in stage 5 during summer. Sex cells bad practically filled the acini, replacing much of the nutritive material. Active spermatozoa were evident in live smears from urchins in this stage. Figure 8,B shows a typical testis in stage 6, sampled during summer. Spermatozoa were concentrated in masses which filled most of the acini. Earlier stages of sex cells were still seen at the base of the germinal layer in a few places, but by far the most abundant sex cells were the sper- matozoa. Nutritive phagocytes were inconspicuous or absent. The con- nective tissue layer was thin and inconspicuous. Typical gonads during the fall in 1965 and 1966 were characterized by the reduced germinal layer, and in some cases there was a conspicuous lumen in the acini. The phagocytic tissue was inconspicuous or absent, 416 Bulletin of Marine Science [18(2) TABLE 4 SIZE (TEST DIAMETER) AT WHICH RIPE SEX CELLS FIRST DEVELOP IN Eucidaris tribuloides ON MARGOT FISH SHOALl Test diameter (mm) Unripe Spermatozoa Ova 8 X 9 X 10 X 10 X 11 X 13 X 14 X 14 X 14 X 15 X 16 X 16 X 17 X 18 X 19 X 19 X 20 X 20 X 21 X 22 X 23 X 23 X 24 X 1 Urchins were examined during October 1965 and August 1966. and the germinal layer consisted mainly of what appeared to be germinal debris. ]n some cases, the lumen contained relict ova or spermatozoa. The connective tissue layer tended to be quite conspicuous. As mentioned, gonad volume (gonad index) tended to be low during this period. A typical ovary and testis for this time of year (16 November, 1965, and 14 October, 1966) are shown in Figures 6,C and 8,C (stage 7). Twenty-five urchins, ranging from 8 to 24 mm test diameter (t.d.), were examined during the period August-October (period of maximal ripeness) to estimate the size (t.d.) at which the first ripe sex cells were developed at Margot Fish Shoal. All urchins over 20 mm had ripe sex cells. The smallest male with active sperm had a test diameter of 14 mm, and the smallest female with ripe eggs had a test diameter of 19 mm (Table 4). Since collections were irregular in areas other than on Margot Fish Shoal, the annual reproductive cycle cannot be delineated with certainty in these areas. On Long Reef, urchins were found with ova on 15 July, 1965. lndi- 1968] McPherson: Biology of Eucidaris 417 45 40 E'30 E ~20 0:: w I- W ~ 0 o I- (j) N= 40 46 114 63 49 39 95 56 46 54 65 95 ~40 20 ~ ~ ~ F t~t ~~~ ~ ~ J FMAMJJ ASOJ FMAMJ JA 1965 1966 FIGURE9. Size frequency of Eucidaris tribuloides collected on Margot Fish Shoal (percentage). viduals were found with spermatozoa on this date and also on 12 May and 5 January, 1965 (only one individual out of eight had active sperm on this last date). Sections of gonads from urchins collected monthly between May and August, 1965, revealed that most urchins on Long Reef were in gonad stages similar to those on Margot Fish Shoal at the same time (stages 3 and 4). A few ripe male urchins (stage 6) were collected on 12 May and 15 July, 1965. Ripe sex cells were found in urchins collected off Virginia Key on 11 March, 1964, 4 May, 1966, 29 August, 1966, and 31 October, 1966, and they were not found in samples collected on 2 September, 1964, 6 December, 1965, and 27 September, 1966. The few urchins examined from Lake Worth on 6 July, 1965, and 29 March, 1966, were unripe. Tag Returns.-The primary spines of Eucidaris develop on interambulacral plates around the apical system. During their growth they are covered with an epithelium of tissue, but after they reach their maximum length this epithelium is lost (Mortensen, 1928). Both the spines and the plates on which they develop move down the test as more new spines and plates are formed aborally (Cutress, 1965). The time required for a particular spine to move from an apical to an ambital position on the test depends on the growth rate of the urchin. Primary spines are shed at certain 418 Bulletin of' Marine Science [18(2) N = 222 71 189 229 198 40 . E E ~ 20 0:: W I- ~ ~ ~ W ~ r ~ « 0 1964 0 I- (J) N=94 26 48 51 W 40 I- 20 ~ ~r-~ J F M A M J J A SON o J F 1965 FIGURE 10. Size frequency of Eucidaris tribu/oides collected on Long Reef (percentage) . times, and generally this occurs in the ambital or oral position on the test (Cutress, 1965). Loss of spines did not appear to be an important factor affecting recovery of tagged urchins, since relatively few untagged urchins were recovered at the tagging stations, and since most urchins held in aquaria kept their tags for many months. Only mature, apical spines which had only recently lost their epithelium were used for tagging, and the time required for these to move down the test to an ambital or oral position is on the order of months or even years. Since loss occurred mainly on lower positions on the test, relatively few tagged spines were lost over a period of several months. There was no indication that tags were shed from the spines. There appeared to be no seasonal trend in the recovery of tagged urchins. The total recovery rate in 1965 and ] 966 was 50 per cent of the releases off Virginia Key, and 37 per cent of the releases on Margot Fish Shoal. Two possibilities may be suggested to explain the higher rate of recovery in the former area: (1) conditions for collections were 1968] McPherson: Biology of Eucidaris 419 -E E -0: N=51 52 90 W 49 I- W ~ c:t 040 I- (f) W I- 20 J F M A M J J A s 1963 1965 FIGURE 1]. Size frequency of Eucidaris tribuloides collected on Sewage Beach, Virginia Key (percentage). better off Virginia Key than on Margot Fish Shoal, and thus more re- coveries were made on the former ground than on the latter; (2) there may have been a higher mortality on Margot Fish Shoal. The total recovery rate of urchins on Long Reef in 1964 was 15 per cent. This rate is not comparable with the above rates since: (1) the tags were slightly different; (2) the periods from tagging to recovery were different. None of the urchins released on Long Reef in August, 1965, were recovered, presumably because they were killed by Hurricane Betsy in September of that year. Growth of the Test.-Size frequency analyses indicated that individuals of Eucidaris tended to be smallest on Long Reef, slightly larger on Margot Fish Shoal, and largest off Virginia Key (Figs. 9, 10, 11). In all three areas there was little change during the year in the size of the urchins sampled. The mean size fluctuated between 20 and 25 mm test diameter at Long Reef, 27 and 32 mm at Margot Fish Shoal, and 40 and 44 mm at Virginia Key. Urchins collected seaward of the outer reefs in relatively deep water off Alligator Light (45-55 m) tended to be larger than urchins from Long Reef. The urchins from this deeper water had a mean size of 35 mm. 420 Bulletin of Marine Science [18 (2) .10 • • E .08 E ~ o a: .06 W Cl.. I ~ .04 ~ o a: (!) .02 10 20 30 40 50 60 INITIAL TEST DIAMETER (mm) FIGURE 12. Growth of Eucidaris tribuloides held in aquaria. Data from two-month periods, with all seasons combined. Y = 0.100 + (-0.002X). Growth rates of Eucidaris in aquaria, and at Virginia Key, and Margot Fish Shoal were all significantly different, being greatest in aquaria and least at Margot Fish Shoal. Growth per day, determined from monthly tag and aquarium returns, was plotted against test diameter, and regression lines were calculated; regression analyses were used to compare the different growth rates (Figs. 12, 13, and 14). The data from Long Reef were few, and there was no significant regression. In this case mean growth was determined and·compared with mean growth of samples from Margot Fish Shoal over the same size range. There was no significant difference in the mean growth between these two areas. Growth of Eucidaris is plotted against time in Figure 15. This growth was determined from the above regression lines (aquaria, Virginia Key, and Margot Fish Shoal). The age of the smallest urchin (6 mm) was not known, so this was arbitrarily plotted as six months. An estimate of minimum growth of very small urchins was obtained from a buoy which 1968] McPherson: Biology of Eucidaris 421 E .08 E >- • ~ .04 3: o a: (.!) .02 • 10 20 30 40 50 INITIAL TEST DIAMETER (mm) FIGURE 13. Growth of Eucidaris tribuloides off Virginia Key. Data includes all tag returns on urchins out for more than 100 days. Y = 0.062 + (-O.OOlX). had been anchored in mid-water for seven months off Miami. A small individual of Eucidaris (7 mm) was found on the buoy in March, 1967. This urchin must have settled from the plankton and grown to this size within the previous seven months. Urchins approaching the maximum sizes observed on their respective grounds (from size frequencies) were probably at least four to five years old. Urchins in aquaria reached the maximum size observed (about 50 mm) in approximately the same time. Urchins approaching maximum sizes in these locations showed little or no growth, so actual ages were not determinable. There appeared to be no significant differences in dry weight of somatic tissue for a given test diameter between urchins collected during different seasons or at different locations (Fig. 22). Growth in test diameter, then, may be converted into growth in somatic tissue for various sized urchins (Table 5). Relative growth was estimated by dividing the mg per day by the mean somatic weight. This growth decreased with increasing size of the urchin (Fig. 16). In aquaria, where data covered the greatest size range, urchins 422 Bulletin of Marine Science [18(2) .-.. .08 E E '-" >- o<:J: .06 0:: W 0... I ~ .04 ?> o 0:: (!) • .02 .01 10 20 30 40 50 INITIAL TEST DIAMETER (mm) FIGURE14. Growth of Eucidaris tribu/oides on Margot Fish Shoal. Data includes all tag returns on urchins out more than ]00 days. Y = 0.035 + (-O.OOlX). in the 15-20-mm size group grew about seven times as fast as those in the 45-50-mm size group. Over the same size range of 15 to 30 mm, relative growth rate decreased more rapidly on Margot Fish Shoal than off Virginia Key or in aquaria. Urchins from Margot Fish Shoal in the 25-30 mm group had roughly the same relative growth rate as urchins held in aquaria in the 45-50-mm group (Fig. 16). Urchins at Virginia Key, Margot Fish Shoal, and in aquaria, had signifi- cantly lower mean test-growth rates in summer than during the rest of the year (Fig. 17). This was determined by correcting the growth rate of each individual for size (from the above regressions), and calculating means for two-month periods. Off Virginia Key, growth of the test was roughly twice as great during the period October, 1965-May, 1966, as during May-September, ]965, and June-October, 1966. At Margot Fish Shoal, growth rate was lower than at Virginia Key, but again the mean appeared to be roughly twice as great from October, 1965-Feb- 1968J McPherson: Biology of Eucidaris 423 ___ • A 50 ~. /. ___ ·S E 40 . .~. E a: ./ / w / . f- w 30 M ~ .. / ----.-. 10 •/ / ,,. ,, " 10 20 30 40 50 60 70 MONTHS FIGURE 15. Growth of the test of Eucidaris trihuloides in aquaria (A), at Sewage Beach on Virginia Key (S), and at Margot Fish Shoal (M). Estimated from Figures 10, 11, and 12. Seasonal differences in growth not considered. ruary, 1966 as in either May and July, 1965, or March-November, 1966. In aquaria, growth rate was greater than at either Virginia Key or Margot Fish Shoal, but there appeared to be less seasonal variation, the winter maximum being only about 20 per cent greater than the summer minimum. There was no significant correlation between test growth and temperature in the above areas. Although test growth slowed during summer, there appeared to be little fluctuation during the rest of the year, and hence over much of the temperature range. In addition, the 21 urchins held in aquaria from 8 December to 3 I January at three different temperatures showed no significant differences in growth of the test or in gonad size. The growth-rate means were 0.031, 0.038, and 0.033 mm per day for the temperatures 18 0, 22 0, and 26°C, respectively. These data may indicate that during anyone period or season growth is relatively independent of temperature, at least within the above range. (H. B. Moore suggested that variation in test growth may have obscured a correlation with tem- perature). Variation in Dimensions of the Test.-Variation in shape of the test was 424 Bulletin of Marine Science [18(2) TABLE 5 MEAN GROWTH OF THE TEST (SOMATIC TISSUE) IN MM AND MG PER DAY FOR Eucidaris tribuloides Test Months Mean wt. Mean growth of test diameter in size somatic tissue Relative (mm) group (gm) mm/day mg/day growth Aquaria 15-20 3 0.19 0.063 1.13 5.9 20-25 3 0.34 0.054 1.62 4.8 25-30 4 0.55 0.045 2.28 4.1 30-35 6 0.84 0.036 2.52 3.0 35-40 5 1.20 0.027 2.70 2.3 40-45 11 1.80 0.D18 2.16 1.2 45-50 15 2.55 0.010 2.00 0.8 V irginia Key 15-20 4 0.19 0.046 0.74 3.8 20-25 5 0.34 0.038 1.02 3.0 25-30 6 0.55 0.030 1.45 2.6 30-35 8 0.84 0.021 1.54 1.8 35-40 10 1.20 0.013 1.60 1.3 40-42 6 1.60 0.006 1.30 0.8 Margot Fish Shoal 15-20 10 0.19 0.D18 0.32 1.8 20-25 12 0.34 0.013 0.39 1.1 25-30 22 0.55 0.008 0.40 0.7 30-32 26 0.84 0.004 0.15 0.2 first considered within a limited geographical region. Measurements of urchins collected from different habitats off the southeastern Florida coast are given in Figure 18. For the measurements used, there was no significant difference in the shape of the test between urchins from Margot Fish Shoal and Long Reef or between urchins from Alligator Reef and Virginia Key. The Margot-Long Reef urchins, however, did differ significantly from the Alligator-Virginia Key urchins. Urchins from the former areas with the same test diameter tended to have higher tests and larger peristomes, but smaller apical systems, than those from the latter areas. Since test shape varied significantly between some localities, the nature of the habitats in these localities is of interest. Margot Fish Shoal and Long Reef resemble each other in that both are rocky, offshore areas. The other two areas differed considerably, the site off Virginia Key being an inshore, rocky and "sea grass" area, and that off Alligator being an offshore, deep-water habitat with a flat, mud bottom. Since test shape may vary with habitat, the latter must be considered in comparisons between widely separated (geographically) populations of 1968] McPherson: Biology of Eucidaris 425 o AQUARIA 6 6 VIRGINIA KEY 5 • MARGOT 4 3 2 :r: I- 3: a a:: <..9 w > I- « --.J w a:: 0.2 0.2 0.4 0.6 0.8 1.0 2.0 3.0 SOMATIC TISSUE (gm DRY) FIGURE 16. Relative growth of the test of Eucidaris tribuloides in aquaria, at Sewage Beach on Virginia Key, and at Margot Fish Shoal. urchins. The data available indicate that the habitats at Cartagena and Ascension probably more closely resemble the type at Margot Fish Shoal and Long Reef than those at the two other locations in Florida. For this reason, urchins from Cartagena and Ascension were compared with those from Margot Fish Shoal and Long Reef. There were no significant differences in the measurements used to indicate test shape in these four areas (Fig. 19). The type of habitat off West Africa is not known, since urchins were trawled from water at depths ranging from 9 to 69 meters. 426 Bulletin of Marine Science [18(2) .06 .04 AQUARIA .02 MARGOT ~ .01 o '- E E I I- 3: o 0:: (9.02 VIRGINIA KEY .01 M J ASONDJ FMAMJ A SON 1965 1966 FIGURE 17. Seasonal test growth of Eucidaris tribuloides in aquaria, on Mar- got Fish Shoal, and off Virginia Key. (Corrected to a size of 30 mm test diameter. ) Since the collecting sites were relatively deep, these urchins were compared with the specimens taken off Alligator Reef. African urchins from station P-22 (51 m) showed no significant differences from those off Alligator Reef in diameter of the peristome and apical system, but they tended to be higher (Fig. 20). Urchins from station P-275 (9-69 m) were mostly smaller than those from station P-22 and Alligator Reef. Interporiferous width at the ambitus showed no significant differences between populations. Total Growth.-Growth may be measured as increase in somatic or gonadal tissue. These have been dealt with separately for urchins from Margot Fish Shoal, but it is important to consider them together, in order to determine total growth. There appeared to be no significant seasonal differences in dry weight of the gonads for a given volume of gonads, or in dry weight of the somatic tissue for a given volume of test (Figs. 21, 22). Growth of both test and gonads, then, can be expressed in gm of dry weight, and the sum of these considered as total growth. 1968] McPherson: Biology of Eucidaris 427 0: ....w w ::. , -; S y~ 2.56 0.49X MARGOT(Mlo 20 + "0 Y~2.71 + 0.27X LONGREEFILl ° ..J- Y~3.69 + 0.29X ALLIGATOR (ALI. u" E Y~2.21 + 0.31X SEWAGE BEACH{S)X 0:- " 10 0: ....W W ~__ ~ -I S-AI ::;; 20 y~ 1.51 + 0.43X MARGOT "o_ y~ 1.27 + 0.42X LONG REEF WE ::. E Y' 1.52 + 0.35X ALLIGATOR 0- •... Y' I.70 + 0.34X SEWAGE BEACH V) 10 [( w a. Y' -2.72 + 0.69X MARGOT •... 20 :I: Y, - 2.66 + 0.69X LONG REEF "'- y, -5.06 + 0.6BX ALLIGATOR ....~! V) 10- ....w 10 20 30 40 50 60 70 60 TEST DIAMETER (mm) FIGURE 18. Variations in shape of test for individuals of Eucidaris tribuJoides collected off southeastern Florida. Growth is manifested mainly by increases of somatic tissue in urchins smaller than 20 mm test diameter. The growth rate of these smaller urchins is not known, but at a size of 18 mm an individual has a mean weight of 0.19 gm of dry tissue (Table 5). Growth from 18 to 30 mm in test diameter on Margot Fish Shoal probably required about three and one-half years, and resulted in a mean increase in somatic tissue of 0.49 gm, so that the total somatic growth was assumed to be 0.68 gm at the end of this time. Gonad size over these three and one-half years increased by about 0.6 ml or 0.13 gm (as determined from Fig. 3). This would be a minimal estimate for growth, since it does not take into consideration any spawning and subsequent growth during this period. It seems quite likely that spawning would have taken place during this period, since all urchins larger than 20 mm had ripe sex cells. If spawning occurred in urchins in the size range 20 to 30 mm, growth of the gonads would have been greater than the above estimate. Assuming total spawning occurred twice during the period, a maximum estimate of gonad-tissue growth would be 0.05 plus 0.08, plus 0.13, or a total of 0.26 gm (Table 6). These estimates would give a total growth (to a size of 30 mm) of 0.81 to 0.94 gm, with somatic growth accounting for 71 to 84 per cent of the total. In urchins larger than 30 mm on Margot Fish Shoal, somatic growth 428 Bulletin of Marine Science [18(2) --" M·L y. 2.05 + 0.30X CARTAGENA(Cl' ~CO M-L ~O y. 3.77+ 0.33X CARTAGENA(Cl' ~-_:~ ~ M-L y. -2.56 +0.67X CARTAGENA(C)' 'i: 20- w_C> ./ J: E ASCENSION 0 , N • B I-E MARGOT- LONG REEF REGRESSIONLINE, ~ 10 I- / M-L 10 20 30 TEST DIAMETER (mm) FIGURE 19. Variations in shape of test for individuals of Eucidaris tribuloides from Cartagena, Ascension, Margot Fish Shoal, and Long Reef. rate decreased markedly (Table 5). In the size range 30 to 32 mm, the mean somatic growth per day was 0.15 mg or 54 mg per year. Growth of the gonads must be estimated from changes in mean gonad volume of the population. If individuals in the population are synchronized in gonad development, changes in mean volume of the gonads will reflect growth, since all individuals will ripen and grow together. How- ever, if urchins grow and mature at different times, increases in gonad size in the population will give low estimates of individual gonad growth. Therefore, an estimate of gonad growth in individuals based on gonad TABLE 6 ESTIMATED GONADAL GROWTH IN Eucidaris tribuloides ON MARGOT FISH SHOAL Size Mean gonad volume Mean gonad weight (dry) Number of group (ml) (gm) spawnings 15-20 negligible negligible 0 20-25 0.2 0.05 1 (?) 25-30 0.4 0.08 1 (?) 1968] McPherson: Biology of Eucidaris 429 lr W f- W ~ 20 y. 3.69 + 0.29X 0_ • AI ...J E u- _-- '0 il: 10 '.-0 Y. 1.52 + 0.35X o ~~ __ Al EUCIDARIS TRI8ULOIDES VAR AFRICANA • AI 8 ~/ WEST AFRICA Y. -5.08 + 0.68X • .8:../,,;' P _ 2750 .•...: p: 22· o ~,'t .a ,/ EUCIDARIS TRIBULOIDES o o~" ALLIGATOR REEF(AIJ I!. o I t I 10 20 30 40 TEST DIAMETER (mm) FIGURE 20. Variations in shape of test for individuals of Eucidaris tribuloides from Alligator Reef, and E. tribuloides, variety africana from West Africa. growth in the population is a minimal value. It may equal or exceed the population growth by an unknown amount, but will not be smaller than the population growth. Estimates of gonad growth from the population on Margot Fish Shoal were 0.4 ml or 90 mg in 1965, and 1.2 ml or 260 mg in 1966. Total growth then may be estimated as 0.14 to 0.31 gm per year, with somatic growth accounting for 16 to 36 per cent of the total. Somatic growth and gonadal growth were both considerably greater off Virginia Key than on Margot Fish Shoal. Urchins that grew from 15 to 30 mm (0.49 gm) on Margot Fish Shoal in three and one-half years would have reached a size of 43 mm (1.65 gm) off Virginia Key in the same time. Mean gonad volumes off Virginia Key reached a maximum value of about 6.0 ml (1.3 gm) for urchins this size (43 mm), which was nearly nine times as great as the gonad size on Margot Fish Shoal at the end of the three and one-half years. Larval and Postlarval Development.-Attempts to rear Eucidaris from the egg through metamorphosis were made many times during 1964-1966, but were successful only once, in October, 1964. The phytoplanktonic 430 Bulletin of Marine Science [18(2) E E f- :r: (!) w 3: 2.0 >- a:: o o <[ z o (!) o WINTER 1.0 o • SUMMER N = 31 0.5 o 2 3 4 GONAD VOLUME (mil FIGURE 21. Dry weight of the gonads of Eucidaris tribu/oides plotted against gonad volume (Margot Fish Shoal). Y = 0.04 + 0.18X. species offered as food seemed to have no effect on the development of the larvae even though cells of some were seen in the larval gut. Changing water, using water from different sources, and maintaining different con- centrations of larvae also seemed to have little effect on development. Generally, larvae developed to a 2-armed stage, and then failed to pro- gress even though they might live for more than a month. The early development of E. tribuloides to the 2-armed stage (about two weeks old) has been described by Tennent (1922). Essentially, the results observed in this study agree with his descriptions. Individuals of Eucidaris fertilized on 5 October, 1964, reached a 4-armed stage on 16 October, a 6-armed stage on 18 October, and an 8-armed stage on 20 October. On 23 October, pedicellariae were seen on some individuals, with one at the base of the larval body appearing best developed and most conspicuous (Fig. 23). The larvae at this time were characterized by large ciliated lobes (epaulettes) which formed continuous rings around their middle. These lobes were not as conspicuous as those described for E. metularia by Mortensen (1937). On 29 October, several larvae had tube feet and spines, and the pedicellariae appeared 1968] McPherson: Biology of Eucidaris 431 [J 3.0 0 ..-.. MARGOT E 0 WINTER 0'1 2.0 A SUMMER - I W 0 SEWAGE BEACH ::> VIRGINIA BEACH (f) (f) I- 1.0 U I-- o 5 10 20 30 40 50 90 TEST VOLUME (m I) FIGURE 22. Dry weight of the somatic tissue of Eucidaris trihu/oides plotted against test volume at different season and stations. functional. On 31 October (25 days after fertilization), one individual metamorphosed into a young urchin (Fig. 24). There were several others partly through metamorphosis, but these never completed the process; they remained in this intermediate state for five to eight days before dying. (The mean temperature during the developmental period was 25°C.) The newly metamorphosed urchin lived for 15 days, but failed to show any growth during this period. It was kept in a 200-ml finger bowl, and the water was changed daily. It moved around the bowl, and would "right" itself in about 20 seconds when turned over. The mouth appeared to be unopened. 432 Bulletin of Marine Science [18(2) FIGURE 23. Larva of Eucidaris tribuloides, 20 days old. Scale line is 100,". All the plates on the small urchin were fenestrated. There were at least two types of spines evident on the plates: small spines, about lOOp- in length, with triforked ends; and larger spines, about 250-300p- in length, which tapered gradually to a point. The small spines appeared to be formed from three fenestrated ridges. They were more common on the apical plates. The larger spines were located mainly on the plates of the cornea. They also were fenestrated, and appeared to be formed from three or more serrated ridges which ran throughout most of the length of the spine. There was a third type of structure on the plates of the cornea, which consisted of a small, rounded protuberance mounted on a short, fenestrated rod (Fig. 25). Three of these structures were seen on the newly metamorphosed urchin. These were located in three separate ambulacral regions. Each structure was about 60p- in length, and resembled a sphaeridium. This was extremely interesting, since cidaroids supposedly lack sphaeridia. The exact nature of the small, rounded structures seen 1968] McPherson: Biology of Eucidaris 433 FIGURE 24. Eucidaris Iribuloides, 15 days after metamorphosis, oral vIew. Scale line is 1001'. on this individual will remain uncertain until more material becomes available. Mortensen (1937) does not mention any protuberances, similar to those described above, for Eucidaris melularia. However, he did not rear this species through metamorphosis, but found the young, at which time the smallest were 0.5 mm. These urchins may have been more advanced than the single individual of E. tribuloides reared from the egg. POPULATION DENSITY The abundance of Eucidaris varied considerably in the areas where it was found. In the "sea grass" flats off Virginia Key, only a few individuals were seen during most field trips. This scarcity was also indicated by the fact that only a few were washed up on the beach after Hurricane Betsy, while there were hundreds of individuals of Lylechinus variegatus and Tripneusles ventricosus washed up by the hurricane. During 1963-64, Eucidaris was quite abundant on Long Reef. As many as 222 urchins were collected in an hour by two people, most being returned after measurements were made. During 1965, they be- 434 Bulletin of Marine Science [18(2) f-----< O.Olmm FIGURE 25. Appendage on newly metamorphosed individual of Eucidaris trihu/oides. came noticeably less abundant, so that by the summer of that year only about 30 to 40 urchins could be collected within an hour. In the fall of 1965, after Hurricane Betsy, only one or two urchins could be found on the reef. A number of trips were made to Long Reef during the following year, but there appeared to be no recolonization in this area. Several species of urchins were quite abundant on French Reef in the summer of 1965. Most of these urchins were removed by Hurricane Betsy in September of that year. There appeared to be some recoloniza- tion of the reef by the following summer, but numbers of urchins were still considerably less than those recorded in 1965 (Table 7). Size-frequency analyses made on French Reef indicated that the mean size of Eucidaris decreased from 28 mm on 9 August, 1965, to 26 mm on 28 September, then to 21 mm on 17 March, 1966, and finally to 17 mm on 30 July (Fig. 26). Another sample collected on 30 July, 1966, on an area about a mile south of French Reef (Molasses Hump) was com- posed primarily of very small urchins (mean size, 8 mm) (Fig. 26). These data indicate that small urchins probably were recruited to the population TABLE 7 NUMBER OF URCHINS COLLECTED ON FRENCH REEF Area Number of individuals sampled ------(square Eucidaris Ec/rinometra Echinometra Dit/dema Tripneustes Date meters) tribliloide.l' Illcunter viridis antillarum ventriCO.l'll.l' 9 Aug., '65 50 93 70 43 42 28 Sept., '65 25 8 2 17 May, '66 50 18 4 3 30 30 July, '66 50 21 3 1 4 4 1968] McPherson: Biology of Eucidaris 435 N= 127 78 55 54 41 23 40 ~ E E- O:: w 30 f- .1 W ~ r J J A S 0 N 0 J F M A M J J A 1965 FIGURE 26. Size frequency of Eucidaris tribuloides on French Reef and Molasses Reef. M = Molasses Reef. on French Reef some time before or during the summer of 1966, but that their numbers were small and they had little effect on density. Species other than Eucidaris were represented on French Reef mostly by small, immature individuals. Even during the period of highest density (nearly two urchins per square meter on French Reef in August, 1965) the number of individuals of Eucidaris never approached that reported elsewhere for other species. Moore et al. (1963a) reported that Lytechinus variegatus may have a density of up to 15 adults or 250 young per square meter in "sea grass" flats off Miami. Kitching & Ebling (] 961) reported that Paracentrotus lividus reached densities of over 10 per square meter. Echinometra lucunter had a mean density of three urchins per square meter at Pigeon Key, Florida, in August, 1965 (McPherson, unpublished). PREDATORS AND PARASITES Several invertebrates were observed attacking Eucidaris. The gastropod, Charonia variegatus, was found feeding on it twice on Margot Fish Shoal (15 April, 1964, and 23 June, 1965). C. variegatus fed on a number of these urchins in an aquarium. The gastropod, Cypraecassis testiculus, attacked Eucidaris in aquaria by boring through the test, but it removed little or no soft, internal tissue. When Echinometra lucunter was placed in aquaria, however, it was attacked and eaten by Cypraecassis. The stone crab, Menippe nodifrons, was observed feeding on Eucidaris in aquaria. 436 Bulletin of Marine Science [18(2) TABLE 8 INCIDENCE OF GALLS ON Eucidaris tribuloides, CAUSED BY THE SNAIL, Mucronalia nidorum Date and location With galls Without galls Margot Fish Shoal: 26 February, '64 1 53 ] 5 April, '64 0 21 1 June, '64 0 24 3 September, '64 1 44 15 March, '65 2 21 12 May, '65 3 111 23 June, '65 1 62 27 July, '65 0 49 12 October, '65 2 87 Total ]0 472 Long Reef: 15 April, '64 2 70 1 June, '64 1 180 3 September, '64 10 219 31 October, '64 0 198 29 December, '64 I 33 23 June, '65 0 48 27 July, '65 2 49 -- Total 16 797 French Reef: 11 July, '65 2 125 9 August, '65 5 73 - -- Total 7 198 The Spanish hagfish, Bodianus rufus, was seen feeding on Eucidaris at Margot Fish Shoal after the urchins had been dislodged from beneath rocks, and were lying free on the bottom. J. E. Randall (personal com- munications) has found the spines or tests of Eucidaris in the following fishes (mainly from the Virgin Islands and Puerto Rico): Batistes vetula, Diodon hystrix, D. holocanthus, Anisotremus surinamensis, Haemulon plumieri, Halichoeres radiatus, and Lachnolaimus maximus. He feels that Eucidaris was probably not a major dietary item in any of these fishes. A number of individuals of Eucidaris were collected which were infected with the small gastropod, Mucronalia nidorum (Table 8). This snail bores into the primary spine, causing a small gall four to six mm in diameter (Pilsbry, 1956). Galls may contain one to several snails, and 1968] McPherson: Biology of Eucidaris 437 sometimes a number of egg capsules. Galls were seen on urchins from all field stations, with roughly two to three per cent of the urchins ex- amined being infected. BEHAVIOR AND FEEDING Eucidaris is a sluggish echinoid. During much of the time, it remains relatively stationary. It does not "cover" or burrow into sand. It may bore into rocks, or at least enlarge crevices in rocks, but this habit is not as well developed as in some other echinoids, such as Echinomefra lucunter. During the day, individuals found on the reef are invariably beneath rocks or in crevices. They use their large primary spines to anchor themselves firmly to the rocks. Some become so firmly entrenched that they cannot be removed without breaking their spines or their tests. At night, however, Eucidaris may move from its protective hole or crevice. This was observed during two night dives on Margot Fish Shoal, on 13 July, and 2 August, 1966. Urchins were located before sunset, and observations were made about an hour after sunset. On these nights, 16 urchins were seen on the sides and tops of the rocks, and three were counted which were still beneath rocks or in crevices. Diurnal movement has been reported in other echinoids which live in crevices during the day and move out into the open at night: Eucidaris metuiaria (Mortensen, 1928); Centrostephanus rogersii (Sinclair, 1959); Diadema sefosum (Thornton, 1956); D. anti/larum, Echinometra iucunter (Dr. R. H. Chesher, personal communication). Moore (in Boolootian, 1966) feels that at least some of these urchins move out at night to feed. Although Eucidaris may move from its crevice at night, it apparently moves only short distances as an adult. Tagged urchins were always recovered in the field within a few meters of the point of their release. Many other echinoids appear to be more mobile, and various species have been reported to migrate and aggregate (Elmhirst, 1922; Lewis, 1958; Moore et ai., 1963a; Reese, in Boolootian, 1966; Tennent, 1910). The gut of specimens of Eucidaris collected on Margot Fish Shoal and Long Reef contained primarily particles of CaCOa. This material con- sisted of fragments of coral, gastropod shells, Foraminifera, echinoid spines, but mostly of unidentified particles. Algal and sponge remains, though not as common as the fragments of CaCOa, were often important constituents of the gut content. Individuals from the grass flats off Virginia Key contained mostly bottom sediment, but algae and Thaiassia were minor constitutents. Some urchins taken in the rocky areas off Virginia Key had the rock-boring sponge, Cliona lampa, in their guts. The material in the gut and the feces of Eucidaris did not appear to be 438 Bulletin of Marine Science [18(2) in discrete pellets. This is unlike some other urchins which form rounded pellets covered with mucus-like secretions (Fuji, 1962; Lewis, 1964). The guts of more than 100 urchins from Margot Fish Shoal were ex- amined; 76 per cent contained some food. A delay of a few hours from the time of collection to examination makes the above a minimal estimate for the number of urchins with food in their gut, since some individuals recorded as empty might have contained food at the time of their collection. There did not appear to be a seasonal trend in these data. DISCUSSION OF GROWTH AND REPRODUCTiON Growth and size of the gonads of Eucidaris appeared to be somewhat independent of gametogenesis. On Margot Fish Shoal, growth resulted from the accumulation of nutrients (in nutritive phagocytes), which took place mainly during spring and early summer, and then from the accumula- tion of ripe sex cells, which took place mainly in late summer (] 966) and early fa]) (1965). Gonad size did not reflect a particular gonad stage, but was probably related to the nutrients which had been available to the urchin. Decreases in gonad size indicated spawning or resorption. Boolootian (1966) stated that echinoids in general accumulate nu- trients, and that this is followed by activation of gametogenesis. For Eucidaris, however, gametogenetic activity appeared to be relatively widespread throughout the year (as indicated by bands of cells at the base of the germinal layer which appeared to be in the process of develop- ment), but the accumulation of ripe sex cells appeared to be more limited. This suggests that some mechanism may be operating independently of gametogenesis in controlling the accumulation of ripe sex cells. Holland & Giese (1965) have suggested that nutritive phagocytes may play an important part in regulating the rate of accumulation of spermatozoa in the lumen of Strongylocentrotus purpuratus. Apparently phagocytosis in this species ceases in the gonad at approximately the same time that spermatozoa begin to accumulate in the lumen. The sex cells of Eucidaris appear to be ingested by phagocytic cells during much of the year, but at certain times sex cells built up in the lumen, and phagocytic cells became reduced. Phagocytosis, then, is a possible mechanism for controlling the accumulation of ripe sex cells. Urchins apparently spawned on Margot Fish Shoal in the fall of ]965 (after 12 October) and in late summer of ]966 (after 5 July), as indicated by decreases in the gonad indices and ripe sex cells. It is interesting to note that spawning was observed in the laboratory only in periods of maximum ripeness, on 12 October, 1965, and 31 August, 1966. Out of 15 urchins brought in from Margot Fish Shoal on each of these dates, four and five urchins, respectively, spawned within a few hours after being placed in aquaria. 1968] McPherson: Biology of Eucidaris 439 The difference in spawning time during the two years indicates that this event was not directly controlled by light, day length, or temperature. Spawning occurred near the maximum mean temperature in 1966, while it occurred after mean temperatures had decreased several degrees in 1965. In 1966, spawning occurred during periods of greater length of day and light intensity than in 1965. Although the above external factors did not serve as triggers for the release of sex cells, anyone of them might have controlled or initiated gonad development prior to spawning. Tem- perature was higher during the pres pawning periods in 1965 than in 1966 (data supplied by Miami Marine Research and Test Station, Inc., Miami). The mean gonad index of Eucidaris was greater and the spawning was completed earlier during the cooler year of 1966. A larger mean gonad index for a population may indicate either a greater individual growth, or a more synchronized development of the gonads by the individuals in the population. Other authors have reported a correlation in sea urchins between gonad size in the population and temperature, with gonads being larger at lower temperatures (Moore et al., 1963a and b; Boolootian, 1966). The fact that adult urchins transplanted from Margot Fish Shoal to Virginia Key grew faster on the latter grounds suggests an environmental effect which was more advantageous for growth at the Virginia Key site. Similarly, the greater growth in aquaria suggests more advantageous con- ditions for growth of the adults than those found either on Margot Fish Shoal or off Virginia Key. Low abundance of Eucidaris in areas of high growth, such as the site off Virginia Key, may indicate that conditions are not advantageous for younger stages, or that younger stages fail to reach these areas. Wave exposure may be an important factor in the growth of Eucidaris. Outer reefs, such as Long Reef, are exposed to greater wave action than inner reefs, such as Margot Fish Shoal, and the water becomes progres- sively calmer on inshore areas such as Virginia Key. In aquaria, of course, there is no wave action. Growth of Eucidaris was greatest in the calmest areas and decreased with increasing wave action. Size of Eucidaris (and presumably growth) increased seaward of the outer reefs in deeper water where wave action was again reduced. Wave action might affect either the food available to the urchin or the time available for feeding. Some of the larger individuals of Eucidaris from both aquaria and the field showed little or no growth over periods of many months, indicating that they could be considerably older than four to five years. It has been reported that individuals of this species have been maintained in aquaria for periods up to seven years (Moore, in Boolootian, 1966). The decrease in relative growth rate of the somatic tissue of Eucidaris with increasing size may be explained by either a decreasing total growth 440 Bulletin of Marine Science (18(2) rate or an increasing growth of gonads. Evidence indicates that the latter may at least partly explain the decreasing growth of the test since gonad growth becomes increasingly important as the urchin approaches its maxi- mum test size. Moore & Lopez (1966) found that Moira atropos had an increasing relative spawn production with size, and that at its maximum size all available energy was diverted to gonad production. Even though gonad growth may become increasingly important as urchins increase in size, the total growth rate, at some point, will decrease as urchins become older. The decreased rate of test growth of Eucidaris during summer is prob- ably the result of gonad development during this period. Lewis (1958) reported that Tripneustes esculentus (= ventricosus) had a similar de- crease in rate of test growth during spring and summer and that this coincided with a period of gonad growth. Echinus esculentus also has an alternation between gonad growth and test growth (Moore, 1937). Eucidaris is a relatively slow-growing species when compared with some other tropical sea urchins. It required at least four to five years to reach the maximum sizes observed in this study. Over much of this time it grew at only one to two mm per month, or even less. Moore et al. (l963a) reported that off Miami Lytechinus variegatus grew quite rapidly during its first year, reaching a test diameter of about 50 mm. After this, growth was considerably slower, and possibly the surviving urchins reached a size of 70 to 78 mm by the third year. They stated that about two years is the normal life span for this species off Miami. In the same area, Mc- Pherson (1965) reported that Tripneustes ventricosus grew rapidly during its first year, reaching a size of about 75 mm (maximum size in the area studied was about 109 mm). This corresponded roughly with the mean size for the population, indicating that most of the test growth was com- pleted during the first year. Randall et al. (1964) reported that juveniles of Diadema antillarum grew at a rate of 3.5 to 6.7 mm per month. Variation in the shape of the test has been described in a number of echinoid species, and at least in some cases this variation is correlated with environmental differences (see Swann, in Boolootian, 1966). This variation may be genetic, or it may be environmentally induced or selected. The cause could only be determined with certainty through rearing ex- periments in the laboratory. The shape of the test of Eucidaris is more variable between different habitats than between widely separated but ecologically similar habitats. This suggests the possibility that test shape may be environmentally modi- fied, either through induced changes or selection of certain forms. The observed variation does not appear to be correlated with depth of water, since urchins living in the shallows off Virginia Key had measurements similar to those from deep water. These last two habitats are similar, in 1968J McPherson: Biology of Eucidaris 441 that both are relatively calm. In the wave-washed areas, such as Margot Fish Shoal and Long Reef, test shape was quite similar. It has been sug- gested that regular sea urchins might tend to be flatter in areas of heavy wave action, due to a continual pull by the tube feet (Thompson, 1917; Moore, 1935; McPherson, 1965). This would not explain the above variation, however, since Eucidaris tends to be flatter in calmer water. Swann (1958), for Strongylocentrotus droebachiensis, and Moore et al. (1963a), for Lytechinus variegatus, reported that successive year groups represented in a single population showed significant differences in shape of the test. The latter authors attributed these differences in shape of the test to abnormally low temperatures. SUMARIO CONTRIBUCION AL ESTUDIO DE LA BIOLOGIA DEL ERIZO DE MAR Eucidaris tribuloides (LAMARCK) El erizo de mar Eucidaris tribuloides (Lamarck) se encuentra en varios habitats diferentes frente al sudeste de la Florida. Esta especie parece estar particularmente adaptada para la vida en areas rocosas y de arrecifes coralinos, usando sus espinas primarias para fijarse bajo las rocas 0 en las grietas de las mismas. Durante la noche puede moverse de estos sitios protegidos. En los arrecifes ingiere particulas calizas duras, probablemente nutriendose de los organism os que viven incrustados 0 enterrados en estos materiales. En los arrecifes de Margot Fish Shoal el crecimiento de las gonadas en la poblaci6n tuvo lugar durante la primavera y el verano de 1965-66. Hubo alguna actividad gametogenetica durante todo el ano. Las celulas sexuales se acumularon, formando gonadas maduras, en los finales del verano y principios del otono de 1965 y 1966. La proporci6n del crecimiento relativo de la concha disminuy6 con el aumento de tamano del erizo y tam bien disminuy6 durante el verano. Los erizos probablemente viven por 10 men os de cuatro a cinco anos. Aunque la mayor densidad de poblaci6n fue observada en aguas someras de arrecifes exteriores, la proporci6n del crecimiento alli fue menor que en areas cercanas mas pr6ximas a la costa. Los erizos de diferentes habitats varian en tamano y dimensiones de la concha. Eucidaris fue criado desde el huevo hasta su metamorfosis, en 25 dias. LITERATURE CITED BERNASCONI, I. 1955. 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