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Notice: © 1996 Marine Biological Association of the United Kingdom. This manuscript is an author version with the final publication available and may be cited as: Young, C.M., Tyler, P. A., & Gage, J. D. (1996). Vertical distribution correlates with pressure tolerances of early embryos in the deep-sea asteroid Plutonaster bifrons. Journal of the Marine Biological Association of the United Kingdom, 76(3), 749-757. http://dx.doi.org/10.1017/S002531540003143X

~ <\":J \Q J. mar. bioi. Ass. UX (1996), 76,749-757 749 Printed in Great Britain

VERTICAL DISTRIBUTION CORRELATES WITH PRESSURE TOLERANCES OF EARLY EMBRYOS IN THE DEEP-SEA ASTEROID PLUTONASTER BIFRONS

CRAIG M. YOUNG*, PAUL A. TYLERt AND JOHN D. GAGE+

"Division of Marine Science, Harbor Branch Oceanographic Institution, 5600 US Highway 1 N, Fort Pierce, Florida 34946, USA. 'Department of Oceanography, The University, Southampton, S09 5NH. lDunstaffnage Marine Research Laboratory, Scottish Association for Marine Science, PO Box 3, Oban, Argyll, PA34 4AD

The astropectinid asteroid Pluionaster bifrons (Wyville Thomson) occurs on the conti­ nental slope of the north-east Atlantic between 1000 and 2500 m depths. As in most deep­ sea , the factors limiting bathymetric distribution of this are unknown. Eggs were fertilized in vitro and incubated through the early embryonic cleavage stages at pressures that correspond to depths from 0 to 3000 m. The highest percentage of normal development occurred near the peak of the species distribution (2000 m), and virtually no normal development occurred at a pressure corresponding to 3000 m depth. Develop­ mental rate was retarded at pressures higher and lower than those found near 2000 m. These experiments indicate that embryonic pressure tolerances could determine both the upper and lower bathymetric limits of distribution for this species.

INTRODUCTION

Distributional limits of deep-sea animals have been attributed to various physical and biological factors such as temperature (LeDanois, 1948; Carney et al., 1983), pres­ sure (Somero et al., 1983), substratum characteristics (Haedrich et al., 1975), or water masses (Gage, 1986; Rowe & Menzies, 1969), but virtually all evidence to date has been correlative and there have been few attempts to test any of the proposed hypotheses experimentally. Many authors have assumed that control of distribution occurs in the larval stages (Rowe & Menzies, 1969: Cutler, 1975). In a thorough but necessarily speculative review of the factors controlling bathymetric distributions, Carney et al. (1983) issued an appeal for experimentation and predicted that further understanding of deep-sea distributions will likely come only as we are able to test the causes. One of the most consistently documented bathymetric patterns in the world ocean is an abrupt increase in the number of species appearing on the slope at between 1000 and 1400 m depth (Gage, 1986; Haedrich et al., 1975, 1980; Mills, 1972; Menzies et al., 1973). Some workers have suggested that this faunal change lies at a natural boundary that marks the beginning of the bathyal zone (Menzies et al., 1973). Secondary increases in appearance and disappearance of species occur at various depths, depending on the taxon underconsideration. For example, Vinogadova (1962) showed that many inverte­ brate phyla undergo a major faunal transition at -3000 m. 750 CRAIG M. YOUNG, PAUL A. TYLER AND JOHN D. GAGE

The hydrostatic pressures found at abyssal depths cause death or adverse sub-lethal effects in both adults and embryos of most shallow-water marine invertebrates (re­ viewed by Hugel, 1972; Vemberg & Vemberg, 1972). Likewise, many adult deep-sea organisms, including bacteria, protozoans, metazoan invertebrates, and fishes, are barophilic, requiring high pressures to undergo normal physiological functions (e.g. Turley et al., 1993; Yayanos, 1978, 1981a; Yayanos et al., 1981b; Jannasch & Wirsen, 1983; Childress & Somero, 1979). The physiological basis for barophilia involves pressure induced volume changes within cells, organelles, and molecules (Somero et al., 1983; Somero, 1992a,b). In a series of detailed studies on cellular effects of high pressure, embryos of the sea urchin Arbacia punctulata developed abnormally at high pressures (Marsland, 1938, 1970; Zimmermann & Marsland, 1964). Such pressure effects are exacerbated by the low temperatures present in the abyssal zone (Marsland, 1950). We have recently demonstrated barophilia in embryos of Echinus affinis, a regular sea urchin living at a depth of -2000 m in the North Atlantic (Young & Tyler, 1993). This finding is significant from an ecological standpoint because the lower pressure limit tolerated by embryos of E. affinis correlates well with the upper limit of bathymetric distribution. However, the potential role of pressure in setting the lower limit of this or any other deep-sea species has not been investigated. We have investigated the poten­ tial role of pressure in regulating the lower distribution of ten littoral and bathyal echinoids from tropical seas (Young et al., 1996), and five additional littoral echinoids from the coasts of France and Britain (Young & Tyler, unpublished data). Every shallow-waterspecies thathas been tested to date (including those reviewed by Marsland, 1970) is able to withstand pressures much greater than those found near the lowerlimits of their vertical ranges. Here we provide preliminary data on the pressure physiology of developing em­ bryos of the lowerbathyal sea star Plutonaster bifrons, which is only the second barophilic invertebrate species whose embryoshavebeen reared in vitro. Plutonasier bifrons spawns small (120 urn) eggs which are of a size that would be expected to produce planktotrophic larvae (Tyler & Pain, 1982). It has a discrete but extended reproductive period ranging from early January to April. This reproductive season would place larvae in the water column just prior to the spring phytoplankton bloom, which varies in timing by as much as six weeks each year (Rice et al., 1986), but which usually begins in early May. The postlarval development has been described by Sibuet & Cherbonnier (1972). Adults feed on a variety of benthic invertebrates, including protobranch bivalves and other asteroids, as well as on carrion (Tyler et al., 1993, 1994). Analysis of gut contents and pyloric caeca weights indicates that feeding activity and nutrient storage vary season­ ally and out of phase with the gametogenic cycle (Tyler et al., 1993, 1994). Plutonaster bifrons occurs on the continental slope from the Faeroe Channel to South Africa (Clark & Downey, 1992). Its bathymetric range in the Rockall Trough region of the north-east Atlantic is from 1000 to 2500 m (Gage, 1986; Gage et al., 1983) but the species has been reported as shallow as 630 m and as deep as 2965 m elsewhere (Clark & Downey, 1992). The data we report here show an approximate correlation between pressure tolerances of the cleaving embryos and adult bathymetric range, suggesting the possibility that both the lower and upper limits of this slope species may be determined by pressure physiology in early life-history stages. ,GE PRESSURE TOLERANCES OF ASTEROID EMBRYOS 751

MATERIALS AND METHODS ~rsesub-lethal ~rtebrates (re- Plutonaster bifrons (Figure lA) were collected by Agassiz Trawl from a depth of 2200 dult deep-sea m on 10 March 1993 at Station M (Gage et al., 1983) on the Hebridean Slope off northern :l.dfishes, are Scotland. Unctions (e.g. Wirsen, 1983; lIves preSSure ro et al., 1983; ugh pressure, Lighpressures Ie effects are md,1950). , a regular sea ~r,1993). This Jressure limit f bathymetric limit of this or ted the poten- t and bathyal )ral echinoids data). Every lby Marsland, .elower limits

veloping ern- md barophilic 'ifrons spawns lanktotrophic eriod ranging e in the water timing by as in early May. )972). Adults res and other Figure 1. (A) Adult Plutonaster bifrons collected from the Hebridean Slope. (B) Newly fertilized ovum contents and of Plutonaster bifrons surrounded by spermatozoa: fu, female nucleus and mn, male pronucleus are ,vary season- visible in the cytoplasm. (C) Late 2-cell embryo initiating second cleavage incubated at 200 atm. (D) Irregular 2-cell embryo incubated at 1 atm. (E) Normal4-cell embryo from 200 atm culture. Scale bar applies to B-F. F, typical embryo incubated at 300 atm pressure that has undergone several irregular nnel to South cleavages. ugh region of 1983) but the Ovaries were dissected from two females and suspended in cold (4°C) sea-water. At where (Clark the time of dissection, many oocytes already appeared to be mature (no germinal ltion between vesicles were visible). Testes were removed from a single male and macerated in cold e, suggesting sea-water. Sperm became very active immediately upon dilution and were used to ~cies may be inseminate eggs teased from the ovaries within a few minutes thereafter. In a subse- 752 CRAIG M. YOUNG, PAUL A. TYLER AND JOHN D. GAGE quent trawl, we found a female with a gonad containing numerous primary oocytes. Incubation in a sea-water solution of I-Methyladenine for 12 h resulted in germinal vesicle breakdown of ~20% of the eggs. However, no viable sperm were available by this time, so additional cultures were not obtained and the experiments could not be repeated a second time. We cleaned much of the gonad debris from cultures by passing the embryos through various grades of nitex screen. Nevertheless, because gametes were obtained by dissec­ tion and maceration, only a small fraction of each culture consisted of mature eggs that were fertilized successfully. The total percentage of full-sized eggs undergoing cleav­ age (including both normal and irregular cleavages) ranged from 19·35 to 33·75% (mean: 24·32%; SO ±4·84; N==12) in the various cultures. As cleavage was the only sure indication that any given egg was mature enough to develop, all subsequent analyses involve only eggs that underwent at least one normal or abnormal division. Embryos were incubated in lO-ml plastic scintillation vials. Each vial was given a 3-ml aliquot of zygote suspension from a single large culture containing the mixed eggs from two females (egg numbers averaged 299·7 per vial; SO ±83·5) then filled com­ pletely with cold sea-water and capped. Two replicate vials were assigned to each of six pressure treatments: 1 , 50 , 100 , 200 , 250 and 300 atm (units are reported in atmos­ pheres rather than the S.l. unit Pascal to facilitate conversion to sea-water depths: e.g. 100 atm == 1000 m). The 1 atm treatments were maintained in a bottle of water (to dampen any temperature oscillations) in a 4°C walk-in cold room. The other treatments were placed in small aluminium pressure vessels filled with freshwater, pressurized by hand using an Enerpac hydraulic pump, and maintained in the same4°C cold room. We justify incubation at a single temperature because surface and bottom temperatures during the reproductive season differ by only 5°C and the respective water masses within the mixed layer (which may extend to 750 m) and below the pycnocline are virtually isothermal (Ellett & Martin, 1973). After 24 h, pressure vessels were depressurized and the contents of incubation vials were emptied into fingerbowls. Full-sized eggs in each culture were examined under a compound microscope at 40x magnification and questionable embryos were checked at a magnification of 100x. The cleavage stage of each normal embryo was noted, and all embryos that had undergone irregular, asymmetrical or highly asynchronous cleav­ ages were counted.

RESULTS

Sperm were active and aggregated around eggs at 1 atm pressure (Figure 1B). Some apparently normal early cleavages (Figure 1C & E) occurred at all pressures tested and in all cultures, but the percentage of embryos undergoing normal development varied significantly among the various pressure treatments (Figure 2). The lowest percentage of normal development (2·25%) was found at the highest pressure tested, 300 atm, which represents a depth greater than the maximum at which Plutonaster bifrons has been reported to occur. A Scheffe multiple comparison test following one-wayANOVA indicated that the 300 atm value differed significantly from all other values (Table 1). PRESSURE TOLERANCES OF ASTEROID EMBRYOS 753

Table 1. One-way ANOVA (A) and the results ofa Scheffe a-posteriori multiple comparison test (B) on the percentage ofnormal development values from Figure 2. In (B), treatments that are not significantly different are indicated by asterisks aligned in the same vertical column.

A.ANOVA: Source of Variation dJ. SS MS F p

Between 5 7679.0 1563.00 64.03 0.000 Within 6 143.9 23.98 Total 11 7822.0

B. Multiple comparison: Ranked Pressure treatment Non-Significant Groups

200 atm 250 atm 100 atm 50 atm 1 atm 300 atm

Approximately 20% of embryos developed normally at the three lowest pressures tested (1, 50, 100 atm), all of which are lower pressures than those at which the adults occur. None of these three low pressure treatments were significantly different from each other (Figure 2; Table 1). Higher, but significantly different percentages of normal development were found at the pressures that occur within the normal depth range of P. bifrons (200 and 250 atm). An average of 81·64% cleaved normally at the pressure equivalent of 200 m depth (Figure 2).

100

....c 80

0 1 50 100 200 250 300 Pressure (atm)

Figure 2. Mean percentages (±l SO) of Plutonaster bifrons embryos developing normally at various pressures. 754 CRAIG M. YOUNG, PAUL A. TYLER AND JOHN D. GAGE

Similar kinds of abnormalities were observed at all pressures. The most common abnormality we observed was drastically unequal blastomere sizes in what should have been equal holoblastic cleavage (Figure ID & F). Asteroids generally have a loose organization of blastomeres wherein the spatial relationships of cells are not fixed in the early cleavage stages (Chia & Walker, 1991). This was observed in P. bifrons, and such embryos were counted as normal. The developmental rate of normally cleaVing em­ bryos also varied with pressure (Figure 3). Only those developing at 200 atrn reached the 32-cell stage after 24 h. A small number of embryos at 50 atm and 250 atrn attained the 16-cell stage and embryos in all other treatments attained maximally the 8-cell stage (Figure 3). The presence of large numbers of 2- and 4-cell embryos even at 200 atrn suggests a large amount of variation in the time of fertilization, which probably indicates that eggs from these cultures attained maturity and were fertilized over an extended time period.

50 50 1atm 50 atm 40 40 30 30 20 20 10 10 0 0 en 8 0 2 4 16 32 2 4 8 16 32 2:- .0 50 50 E 100 atm 200 atm W 40 40 0> c 30 30 '5. 0 20 20 Q) > 10 10 Q) 0 0 0 \+0- 0 2 4 8 16 32 2 4 8 16 32 ~ 0 50 50 250 atm 300 atm 40 40 30 30 20 20 - 10 10 -

0 0 I I III 2 4 8 16 32 -2 4 8 16 32 Number of Blastomeres

Figure 3. The percentage (±1 SO ) of embryos of Plutonasterbifrons attaining various cleavage stages after 24 h incubation at six different pressures. PRESSUI~E TOLERANCES OF ASTEI~OID EMBRYOS 755

DISCUSSION

Previous studies with echinoid embryos have suggested that lower limits of bathymetric distribution are probably not set by pressure tolerances of embryos. For example, the early embryos of the echinoids Arbacia lixula and Paraceniroius lividus from the Mediterranean Sea can develop normally to hatching and beyond at 150 atm, which represents a depth more than 1000 m deeper than either species naturally occurs (Young & Tyler, unpublished data). The same has been observed for numerous other species of shallow-water echinoids in the Bahamas, Hawaii and UK waters (Young et a1.,1996; Young & Tyler, unpublished data). Several bathyal from Baha­ mian and British waters also show very broad pressure tolerances that should allow their occupation at greater depths unless other factors restrict their distributions. Plutonaster bifrons represents the first example of a species whose early development is significantly inhibited by pressures that correspond closely to the lower bathymetric limit and as such is the only documented case in which both upper and lower limits could be set by pressure tolerances. Our data are entirely correlative and do not allow us to invoke pressure as a solid cause of the observed distributions. It is possible that even though pressure tolerances are relatively narrow, bathymetric range may be controlled in a proximate way by recruitment, predation, food supply, or physiological responses to abiotic factors other than pressure. It is also possible that pressure tolerances become broader in later larval, juvenile, or adult stages. We can say, however, that in the absence of other controls, the early cleaving embryos of P. bifrons would be restricted in their development to a narrow range of pressures found between about 1500 and 2500 m on the continental slope. Eggs of this species are negatively buoyant, so early development would be expected to occur near the adults. Embryogenesis under high pressure has been studied in only two species from lower bathyal or abyssal depths (Young & Tyler, 1993; this study). Itis not possible therefore to make generalizations about the role of barophysiology in embryonic and larval life. Circumstantial evidence however, indicates that the situation may be com­ plex. For example, some species of gastropods (reviewed by Bouchet & Waren, 1994) and possibly some echinoids with floating eggs (Young & Cameron, 1987) undertake long vertical migrations during the larval stages, traversing as much as 400 atrn of pressure change as well as substantial changes in temperature. We do not know if these species simply have very wide tolerances to pressure, or if physiological characteristics change during migration to permit different ranges of pressure tolerance in different life history stages. In order to understand the role of barophysiology in the adult, embryonic and larval ecology of deep-sea animals, many more species must be exam­ ined through their entire life history.

We thank Captain JeffLong and the crew of RRS 'Challenger' for providing excellent support for this project at sea. The research was supported by a NERC grant to J.D.G. and by NSF grant OCE-9116560 to CM.Y. Travel funds were provided by a NATO collaborative research grant (CRG-900628). This is contribution number 1095 from Harbor BranchOceanographic Institution. 756 CRAIG M. YOUNG, PAUL A. TYLER AND JOHN D. GAGE

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Submitted 25 April 1995. Accepted 11 September 1995.