Cmp. Biodzem. Physiol. Vol. 91A, No. 2, pp. 317-321, 1988 Printed in Great Britain

~3~9629188 $3.00 f 0.00 CQ 1988 Pergamon Press plc

PATTERNS OF NITROGEN EXCRETION IN THE PHYLUM ECHINODERMATA

WILLIAM B. STICKLE

Department of Zoology and Physiology, Louisiana State University, Baton Rouge, LA 70803, USA. Telephone: 504 388-I i 32

(Recebed 26 fanuury 1988)

Abstract-l. Significant quantities of urea (> 10% of total nitrogen) are excreted by 16 of 22 species of echinoderms, including asteroids (9 of 10 species tested), crinoids (only one tested), holothuroid (three of four tested) and ophiuroids (three of four tested) but not by echinoids (two tested).

2. A higher percentage of total nitrogen excreted as urea occurred during the summer in Pisaster ochracus and during the winter in Ophiuru liizkeni.

3. Factors which may determine the form of nitrogen excreted by echinoderms include the existence of phosphoarginine as the phosphagen in some species, species specific differences in the aerobic perfusion of deep tissues and the production of urea as a byproduct of polyamine biosynthesis.

INTRODUCTION

The classical paradigm of ammonotelism in aquatic animals, ureotelism in amphibious animals with re- stricted water supply and uricotelism in terrestrial animals and in cleidoic eggs (Needham, 1931; Bald- win, 1967) has been challenged by observations of nitrogen excretion patterns from three phylogeneti- tally distinct groups of deuterostomic animals includ- ing echinoderms (Stickle et al., 1982; Stickle, 1985), several species of ascidians (Markus and Lambert, 1983) and the embryonic soft shell turtle Trionyx spinzferus (Packard and Packard, 1983). Because these findings cast doubt on the generality of the Baldwin-Needham hypothesis, the present study was conducted in order to survey the Phylum Echino- dermata to determine the relationship between urea excretion and systematic status.

The objectives of this study are: (1) to determine the rate of ammonia and urea excretion by species of echin~e~s from all extant classes; (2) to determine the percentage of total nitrogen excreted as am- monium and urea by al1 species studied; (3) to determine the effects of aerial exposure on perivis- ceral fluid ammonium, lactic acid and urea levels and the pattern of nitrogen excretion and (4) to relate patterns of nitrogen excretion to phylogeny, season and nutritional status.

MATERIALS AND METHODS

Twenty-two species of echinoderms, belonging to five classes, were studied with respect to their excretory pattern of ammonia and urea. Details of collection location, water temperature, season of the year and nutritive condition are summarized in Table 1. Excretion rates of several of these species included data on the net flux of primary amines (free amino acids). These data were recalculated to exclude primary amine loss since net flux of that class of nitrogenous compounds is determined by the balance between uptake due to active transport by specific membrane carriers (Stew- art, 1979) and diffusive efflux. Percent of total nitrogen

excreted as ammonium and urea was calculated by dividing the pg nitrogen excreted in each form per hour by the sum of nitrogen excreted in both forms. All excretion rates are calculated on an organic tissue basis according to methods given in Stickle et al. (1982) and Stickle (1985). Although-the skeletal contribution to wet weight ‘vat& significantly among classes of echinoderms (Stickle and Diehl, 1987), surprisingly little variation exists in the percentage of wet weight att~butable to organic tissues (Table 1).

Nitrogen excretion rates were determined by placing individual echinoderms into XI-7000 ml of Millipore-Ntered (0.45pm pore size), oxygenated sea water adjusted to 30% S. Blank containers, filled with an equal volume of millipore filtered, oxygenated seawater only, were included as controls. Echinoderms were removed from the excretion containers after 2-6 hr depending on their size and the volume of incubation water. Assay temperature varied with collection location and is given in Table 1.

Duplicate water samples were taken for ammonia and urea analysis and are reported as PM/g organic tissue wt/hr. Ammonia concentration was measured by the phenol- hyp~hlo~te method of Solorzano (I969). All urea in the sample was converted to ammonia by the modified urease technique (Sigma Technical Bulletin No. 640) and then total ammonia and net ammonia determined. Total nitrogen excreted was calculated as pg/g organic tissue/hr by multi- plying the rates of ammonia and urea excreted by the atomic weight of nitrogen excreted in each form, 14.01 and 28.02, respectively. The percentage of nitrogen excreted in each form was calculated after converting all rates to pug N/g organic tissue/hr.

The effects of 3.08 hr aerial exposure on the perivisceral fluid ammonium, lactic acid and urea concentrations and the pre- and post-emersion ammonium and urea excretion rates were determined for five Pisusrer ochraceus which had been fed prior to the experiment. Water temperaure was 8°C and air temperature was 11°C. Lactic acid concentrations were determined by the lactic acid method given in Sigma kit No. 826-UV.

Ammonia and urea excretion rates of starved and fed echinoderms at different seasons and P. ochraceus exposed to the air were compared by use of Student’s t-test. Ammo- nium, urea and lactic acid concentrations in the perivisceral fluid were also compared with Student’s r-test.

317

318 WILLIAM B. STICKLE

Table 1. Collection and experimental data for the soecies of echinoderms studied

Soecies Literature

citation Collection

location Water temp.

l”C1 Per cent

tissue

Class Crinoidea

Class Ophiuroidea

Class Asteroidea

Florometra serratissima Bamfield, BC

Amphipholix squamata Gorgonocephalus euchnemis Micropholis atra Ophioderma brevispinum Ophiura liitkeni

Friday Harbor, WA 8 Friday Harbor, WA 9

3 Terrebone, LA 23 I Pensacola, FL 23

Friday Harbor, WA 8-13

Luidia foliolata Euasterias troschelii Leptasterias hexactis Orthasterias koehleri Pisaster ochraceus Crossaster papposus Dermasterias imbricota Henricia leuiuscula Pteraster tesselatus Solaster stimpsoni Class Echinoidea Strongylocentrotus

droebachiensis StrongJ4mvltrotus

Friday Harbor, WA 2 Juneau, AK I Juneau, AK 2 Friday Harbor, WA 2 Friday Harbor, WA 2 Friday Harbor, WA 2 Friday Harbor, WA 2 Friday Harbor, WA 2 Friday Harbor, WA

Friday Harbor, WA

1 Juneau, AK

9

8 I3 I3 13

8-13 8-13

I3 I3

8-13 8

I3

IO

5 9

IO II I6

II II 16 8 8

IO 9

I5 7

14

6

purpuratus Class Holothuroidea

Curtmaria mineata Eupentacta quinquesemita Parastichopus calt@rnicus Psolus chitonoides

tnday tiarbor, WA II 8

Friday Harbor. WA I3 9 I Juneau, AK I3 II

Friday Harbor, WA 8 6 Friday Harbor, WA 13 11

Season condition

W, f

W, s W, s S, f S, s SW, s

W, s S, f F, f S, s s-w, f-s s-w, s S, s S, f s-w, s W, s

S, s

W, s

S, s S, s W, s S, s

(I) Stickle et al. (1982), (2) Stickle (1985), (3) Gillham (1985). S, F, W = summer, winter f, s = fed or starved

RESULTS

Total nitrogen excretion rates of all species of echinoderms studied exhibited considerable varia- tion; however, the ambient temperature of the ex- periments ranged from 8 to 23°C (Table 2). Total nitrogen excretion rates were determined during summer and winter for one species of ophiuroid and three species of asteroids (Table 3), in order that Q,, values could be calculated. Total nitrogen Q,, values were 1.92 for Crossaster papposus, 2.86 for Pteraster tesselatus, 3.21 for Ophiura liitkeni and 10.91 for fed and 52.38 for starved Pisaster ochraceus between 8 and 13°C. The highest total nitrogen excretion rates occurred in the active carnivores, P. ochraceus and Evasterias troschelii, and in the crinoid, Floro- metra serratissima, from the north Pacific coast of North America. Relatively high rates of total nitro- gen excretion also occurred in the carnivorous aster- oids, Leptasterias hexactis and Orthasterias koehleri. However, high rates of total nitrogen excretion were also obtained for Micropholis atra and Ophio- derma brevispinum at an ambient temperature of 23C.

Ammonium excretion rates varied directly with temperature for both fed (Q10 = 10.80; P < 0.02) and starved (Q,, = 10.42; P c 0.01) Pisuster ochraceus while urea excretion rates varied directly with tem- perature for sea stars in both nutritional conditions (fed Q,, = 42.45; P > 0.05; starved Q,, = 462.25; P < 0.01). Interestingly, starvation resulted in a significant increase in the rate of urea excretion in this species during the summer (P c 0.05) but not during the winter (P > 0.05). Ammonium excretion rate varied directly and urea indirectly with temperature in Ophiura liitkeni, ammonium excretion rate varied directly with temperature in Crossaster pupposus,

while the NH: excretion varied inversely and urea excretion directly with temperature in Pteraster tesse- latus.

Urea was synthesized endogenously in the ophi- uroids Amphipholis squamata, Ophioderma brevi- spinum and Ophiura liitkeni during the winter; in the asteroids Luidia foliolata, Pisaster ochraceus, Derma- sterias imbricata, Pteraster tesselatus and Solaster stimpsoni; and in the holothuroids Cucumaria mineata, Parastichopus californicus and Psolus chito- noides. All of these species had been starved prior to the determination of the pattern of nitrogen ex- cretion.

Perivisceral fluid ammonium and urea concen- trations of Pisaster ochruceus increased significantly but not substantially during 3.08 hr aerial exposure and returned to pre-emersion levels during 4.78 hr reimmersion in 30’?&1 S seawater. Lactic acid concen- tration did not change significantly during or after aerial exposure (Table 4). Ammonium concentration increased by only 54% and urea concentration by 32% during 3.08 hr emersion.

No significant difference exists between the am- monium or urea excretion rates of the sea stars before and after aerial exposure. Aerial exposure of P. ochraceus for a period of time typical for its position in the lower intertidal zone, did not cause a substan- tial accumulation of ammonium ions, lactic acid or urea at 8-11°C.

Eight of the species of echinoderms studied are ureotelic ( > 50% total N excreted as urea), including three species of ophiuroids, four species of asteroids and one species of holothuroid (Fig. 1). In total, 16 of the 22 species are ureogenic (> 10% of total N excreted as urea) with respect to protein catabolism. Neither species of echinoid studied excreted an appre- ciable quantity of urea.

Patterns of nitrogen excretion in echinoderms

Table 2. Excretion rates of 22 species of echinoderms. NH: and urea rates are given as PM/g organic tissue/b and total nitrogen is given as pgN/g organic tissue/hr

Total Grow Soecies N NH: Urea nitrogen

319

Class Crinoidea FIorometra serratissima

Class Ophiuroidea Amphipholix squamata Gorgonocephalus euchnemis Micropholis atra Ophioderma brenispinum Ophiura b’itkeni

summer winter

Class Astetoidea Luidia folioluta Evasterias troschelii Leptasterias hexnctis Orthasterias koehleri Pisaster ochraceus

summer, f summer. s winter, f winter, s

Crossaster papposus summer, s winter, s

Dermasterias imbricota Henricia leviuscula Ptemster tesfelatus

summer, s winter, s

Solaster stimpsoni Class Fkhinoidea

S. droebachiensis s. purpuratus

Class Holothuroidea Curumaria mheota Eupenracta quj~quesemita Par~tichDpus caiifornicus Psolus ehitomides

s = starved, f = fed. S = Strongyiocentrotus.

10

2 3

12 59

4 1

2 10 18 1

5 6 5 5

1 I 2 7

1 2 I

I2 3

2 16 2 7

2.04 + 0.25 0.40 rf 0.07 39.80 + 4.43

0.29 + 0.29 0.82 f 0.07 I .34 f 0.34

0.77

0.01 rf 0.01 0.01 k 0.01 1.00 + 0.29

0.42

14.93 * 0.70 II.91 tt 1.24 46.80 + 9.60

27.30

1 .oo + 0.42 0.06

0.01 * 0.01 0.11

6.97 F 1.67 3.89

0.41 f 0.05 0.21 + 0.02 2.94 f 0.62 0.14+0.03

1.00 0.27 0.60 0.60

1 I .59 f 0.08 45.24 k 9.37

23.04 25.21

2.07 2 0.44 1.13*0.19 0.63 * 0.07 0.35 i 0.02

0.39 + 0.22 0.86 i. 0.22 0.06 * 0.01 0.04 & 0.01

40.65 F_ 11.65 40.02 it: 8.76 12.31 F 2.8

5.53 r 0.39

0.78 0.56

0.58 + 0.21 0.97 + 0.71

0.00 0.00

0.39 rt 0.31 0.04 & 0.01

10.93 7.87

17.99 i: Il.67 14.56 i: 9.76

0.24 0.33 15.53 0.62 FO.18 0.04 9.18 2 2.73

0.53 0.05 9.70

1.79 0.62 k 0.24

0.06 0.00 + 0.00

27.01 9.08 i: 3.21

0.51 + 0.45 2.09

0.48 + 0.42 0.04

0.04 2 0.03 0.09 & 0.01

20.56i 18.15 31.1

5.93 ri: 0.81 12.45 t 0.07

0.35~0.11 0.71 * 0.07

Table 3. Seasonal and nutritional differences in the nercentaae of nitrogen excreted bv echinoderms

Group Species Season N~t~tjonal

status

Percentage excreted

NH; Urea

Class Ophiuroidea Ophiura liitkeni

Class Asteroidea Pisaster achraceus

Crossaster popposus

Pteraster tesselatus

Abbreviations as for Table I

S S 96 4 W S 22 78

S f 80 20 W f 85 15 S s 46 54 W s 80 20 S s 100 0

W s 100 0 S s 27 73 W s 89 II

DISCUSSION

Surprisingly, the majority of species of echino- derms studied to date are ureogenic and eight species are ureotelic. Urea is probably synthesized from the metabolism of metabolically labile protein rather than from dietary sources. Three of the four species of ophiuroids which were ureotelic had been starved prior to the determination of excretion rates, as had six of the seven species of asteroids, and all four species of holothuroids. To date, the urea excretion rate of only Leptasterias hexactis has been shown to

be related to the ingestion rate of dietary arginine (Shirley and Stickle, 1982; Stickle et al., 1982).

Conflicting selective forces of water availability for the dilution of the end products of protein catabolism versus the energetic cost of synthesizing those end products are usually invoked to explain the Baldwin- Needham hypothesis of the patterns of excretory products observed throughout the animal kingdom (Hoar, 1984). Indeed, deep tissues of echinoderms are poorly perfused, resulting in the accumulation of anaerobic metabolites (Shick, 1983; Bookbinder and Shick, 1986). Perivisceral fluid of both P&aster

320 WILLIAM B. STICKLE

Table 4. Perivisceral fluid ammothm, lactic acid and urea concentrations of five fisa~ter ochraceus are given as Z f SE in PM/I. Excretion rates of ammonium ions and urea are also given for the same sea stars as tiMin

organic tissuejhr prior to and after re-immersion -

Solute Pre-emersion P Emersion P Re-immersion

Periuisceral jluid Ammonium 6.28 f. 0.52 CO.01 9.71 f 0.82 <O.Ol 6.30 k 0.54

NS ~~

Lactis acid 79* 13 NS 131 k42 NS 119+44 NS

Urea 7.3 * 0.5 NS 9.7 f 0.8 NS 6.3 i 0.5 NS

EXU62 ttd

Urea 0.06 f 0.01 NS 0.05 * 0.01 Ammonium 0.63 + 0.07 NS 0.82 + 0.06

Probability values are based on Student’s I-test comparisons as P values. NS = P > 0.05.

tless trlnolder Floroma tra sbrratlsrlma

Class ophluroldea nmphiphOiiS squamate 6orgonocephafus oucnamls Mlcrophelis atra Ophloderma braL?ispiaam

Ophtara tUt&ant

Class Istereldee Luldls lotfotata

Euastarias troschalll Lsptasterles henactts Orthastartas koahkri

P/raster ochreceus

Crossastar papposus

Oarmesteriar fmbrlrata Honrftle Ieuisuscuta Ptoraster tesselatus Solaster sttmpsonl

Claar Echlnoldea StronggIorentrotus droabachlansfs S. pufpura tus

Chsa llolathuroldea Cucumorta mlneata Eupantarta qUiItqusS8IIIlta

Parastlchopus roflfornltus

Psolus chltanoldes

Fig. I. Percentage of total nitrogen excreted as urea is given for 22 species of echinoderms studied. Urea excretion in excess of 10% of total nitrogen excreted indicates urea is produced from protein metabolism. Where multiple determinations of the percent of total nitrogen excreted as urea were made, the highest

percent urea excreted is given to indicate the species potential for excreting urea.

ochraceus and Strongylocentrotus droebachiensis con- tains an appreciable concentration of lactic acid, 79.1 k 12.7 ( f SEM; N = 5) and 149.5 f 36.8 (N = 6) PM/I, respectively (unpublished data). How- ever, S. droebachiensis does not excrete urea while P. ochraceus does; therefore inadequate perfusion of the deep tissues is not the sole selective force respon- sible for ureotelism in the echinoderms.

Another possible explanation for the excretion of urea by many species of echinoderms is that urea is a by-product of polyamine synthesis during gameto- genesis. Polyamines are involved in DNA, RNA and protein synthesis (Heby, 1981) but their specific function in the division and growth of mammalian cells remains unclear (Pegg, 1986). Watts et al. (1987) determined spermine, spermidine and putrescine con- centrations in the pyloric caeca and gonads of adult Aster&s vulgaris. They hypothesize that polyamines are important in the regulation of cellular prolifera- tion activity in these tissues during the annual repro- ductive cycle. Urea is a by-product of the synthesis of

spermidine from arginine (Tabor and Tabor, Bishop et al., 1983).

1976;

Dramatic changes occur in cellular proliferation and differentiation during the reproductive cycle of echinoderms including Pisaster ochraceus (Mauzey, 196X; Nimitz, 1976). Likewise, tremendous seasonal changes occur in the biochemical composition and rate of synthesis of lipids in P. ochraceus (Greenfield et al., 1958; Allen and Giese, 1966). The extremely high seasonal Q,, values for nitrogen excretion and shifts in the pattern of NH: and urea excretion in P. ochraceus, and probably Ophiura liitkeni and Pteraster tesselatus, are likely to be coupled with reproductive cycle variation in cellular proliferation and biochemical substrates metabolized.

Interspecies differences in urea excretion are prob- ably related to the distribution of the type of phos- phagens both within individuals and among species. Both arginine kinase and creatinine kinase have been recorded in species of holothuroids, asteroids, ophi- uroids and echinoids, but only arginine kinase has

Patterns of nitrogen excretion in echinoderms 321

been reported for the crinoids (Watts, 1975). The conversion of arginine to ornithine and urea by arginase can result in the excretion of urea.

Aerial exposure of Pisaster ochraceus during the winter did not result in a significant shift of the perivisceral fluid towards an anaerobic condition as determined by no significant change in lactic acid concentration. Likewise, although perivisceral fluid ammonium and urea concentrations increased significantly during aerial exposure the relative in- crease was not substantial. It appears unlikely that aerial exposure is much of a selective force, as a result of the accumulation of toxic quantities of these solutes, for the excretion of urea by P. ochraceus or other echinoderms in the lower intertidal zone.

Although the selective forces of water availability and the metabolic cost of synthesis have been used to construct the Baldwin-Needham hypothesis, other selective forces, which are likely to influence variation in the excretion of urea by echinoderms, include the distribution of phosphoarginine versus phosphocreatinine, species specific differences in the aerobic perfusion of deep tissues and seasonal differences in the production of urea as a by-product of polyamine synthesis during the annual reproduc- tive cycle.

Acknowledgemenfs-Much of the research reported on in this study was carried out at the University of Washington’s Marine Laboratory at Friday Harbor. I am extremely grateful to its Director, Dr Dennis Willows, for allowing me access to this superb facility. A number of other species were collected by Dr Stanley Rice of the National Marine Fisheries Service Laboratory in Auke Bay, Alaska and kindly sent to me to allow an extension of the comparative study. Finally, thanks go to the personnel at the Bamfield Laboratory for collecting Florometra serretissima and allow- ing me access to their facilities for a short stay while nitrogen excretion patterns were determined. Financial support for this study came from a summer fellowship and sabbatical leave provided by Louisiana State University and from funds provided by the Petroleum Refiners Environmental Council of Louisiana. Investigators who assisted in various aspects of this study include Dr Thomas Sabourin, Dr Thomas Shirley and Mr Bradley Gilham. I am extremely grateful to these individuals for their companionship and input into this comparative study.

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