Hydrobiologia 156: 161-173 (1988) P. Sundberg, R. Gibson & O. Berg (eds) Recent Advances in Nemertean Biology ? Dr W. Junk Publishers, Dordrecht - Printed in the Netherlands 161 Water and solute regulation in Procephalothrix spiralis Coe and Clitellio arenarius (Miiller) III. Long-term acclimation to diluted seawaters and effect of putative neuroendocrine structures Joan D. Ferraris & Jon L. Norenburg Mt. Desert Island Biological Laboratory, Salsbury Cove, Me 04672, USA Key words: volume regulation, Nemertina, Oligochaeta, inorganic ion regulation Abstract When acutely transferred to diluted seawater (SW), Procephalothrix spiralis and Clitellio arenarius regulate water content (g H20/g solute free dry wt = s.f.d.w.) via loss of Na and Cl (^moles/g s.f.d.w.). The present study extends these observations to a greater range of salinities and determines the effects of long-term, stepwise acclimation to diluted seawaters. Final exposure to a given experimental seawater (70, 50, 30, 15%) was 48 hours. Osmolalky (mOsm/kg H:0) and Na, K, and Cl ion concentrations (mEq/1) were determined in total tissue water and in the extracellular fluid of C. arenarius. Extracellular volume was determined as the l4C- polyethylene glycol space. Both species behaved as hyperosmotic con formers in diluted seawaters. However, reduction of the osmotic gradient between worm and medium occurred in P. spiralis, but not C. arenarius, in 30 and 15% SW. In both species, osmolality and Na, Cl, and K concentrations in total tissue water decreased with increased dilution of the SW. Water content increased with dilution of the medium but was lower than that which would be predicted based on approximation of the van *t Hoff relation. This indicated the occurrence of regulatory volume decrease (RVD). In P spiralis, in 70 or 50% SW, RVD was accompanied by loss of Na and Cl contents. However, in 30 or 15% SW, Na and Cl contents increased and in worms in 15% SW K content decreased. The latter movements of Na, Cl and K are indicative of cellular hysteresis and were associated with decreased viability, indicating the lower limits of regulatory ability in this species. In comparison, RVD in C. arenarius occurred in all diluted seawaters and was accompanied by loss of Na and Cl contents. InC arenarius, evidence for reduced viability was absent. Removal of the supra- and subesophageal ganglia of C. arenarius resulted in retention of water, Na and Cl (g H:0 or ^moies/g s.f.d.w.) in worms acclimated to 70% SW. Removal of the cerebral ganglia and cephalic glands of P. spiralis did not significantly influence regulation of water content. Introduction P spiralis (Archinemertina) and C. arenarius (Oligochaeta) inhabit rocky intertidal areas on the northeast coast of North America. In this habitat, changes in the salinity of the surrounding seawaters can be of short duration, as with rainfall during low tide, or of long duration, as with the melting of inter- tidai ice sheets in protected bays. Both species are known to regulate water content (g H20/g s.f.d.w.) during short-term acute exposure to decreased salin- ity (Ferraris & Schmidt-Nielsen, 1982a, 1982b) as well as under fluctuating salinity conditions (Fer- raris, 1984). The present study extends the existent observations on the responses of P. spiralis and C. arenarius to include long-term exposure and greater dillution of seawaters. Ability to regulate extracellular, but not intraceilu- 162 lar, water content during short-term hypoosmotic exposure as well as during exposure to fluctuating sa- linity conditions is correlated with the presence of the supra- and subesophageal ganglia in C. arenarius and the cerebral ganglia and cerebral organs in Paranemertesperegrina (Hoplonemertina) (Ferraris & Schmidt-Nielsen, 1982a; Ferraris, 1984, 1985a). Under the same osmotic conditions, the ability of P. spiralis to regulate intracellular water content is similarly independent of the presence of the cerebral ganglia and the cephalic glands (Ferraris & Schmidt- Nielsen, 1982b; Ferraris, 1984). (Regulation of total body water content in this species is primarily an in- tracellular phenomenon since the volume of the ex- tracellular compartment is only about 5% of the to- tal.) The putative neuroendocrine nature of cerebral ganglia (primary location of neurosecretory cells), cerebral organs, and cephalic glands as well as re- sponse of these structures, on a cytological level, to osmotic variation has been treated in detail elswhere (Drawert, 1968; Ferraris, 1979a, 1979b, 1979c, 1985b; Ferraris & Schmidt-Nielsen, 1982a). Additional questions addressed in the present study are (1) whether these neurosecretory or neuroglanduiar structures affect water content regulation in P. spira- lis under more extreme conditions and (2) whether the observed influence of the cerebral ganglia on regulation of water content in C. arenarius persists following long-term stepwise acclimation. Materials and methods under intertidial rocks at Hulls Cove and Salsbury Cove, Maine. Animals were maintained in aquaria with recirculating natural, Frenchman Bay seawater (100% SW, Table 1) at 7 ?C for at least one week be- fore use. Surgical procedures Detailed descriptions of the ablation techniques used are provided in Ferraris & Schmidt-Nielsen (1982a, 1982b). A summary follows. Tissues of P. spiralis were ablated by transection of the animals with a microscalpel immediately posterior to the cerebral ganglia. This procedure effectively removes both the cerebral ganglia and the cephalic gland which lies immediately anterior to the ganglia. Tis- sues of C arenarius were ablated by transection im- mediately behind the subesophageal ganglia which also removes the supraesophageal ganglia and the mouth. In other groups of worms of both species (sham-operated) wound healing, without removal of organs, was induced by incising the dorsal body wall in the cephalic region. Ablated and sham-operated worms were returned to aquaria for a 5-day recovery period prior to use in experiments. This is sufficient time for complete wound healing to occur following both types of surgery and for reestablishment of the mouth in ablated C. arenarius; it is insufficient for regeneration of the ganglia (Ferraris & Schmidt- Nielsen, 1982a, 1982b). Control worms were treated in an identical manner but were not operated upon. Collection and maintenance Adult specimens of both species were collected from Experimental procedures Control, ablated and sham-operated individuals of Table I, Seawater composition (meaniS.E.). Seawater Osmolalitv Na K Cl TO (mOsm) (mEq/1) (mEq/1) (mEq/l) 100 948 ? 1.25 461.3 ?1.49 10.2 ?0.05 522.6? 1.45 70 670*1.32 334.3 ?2.93 7.1 ?0.07 370.2 + 0,95 50 478 = 0.85 235.5 ?3.48 5.O + O.07 272.5 ?0.20 30 282 ? 0,65 134.3 ?1.38 3.0 ?0.08 I55.0?0.85 15 143 = 0.48 70.9 ? 0.52 1.5 ?0.03 79.5+0.45 &? either species were simultaneously immersed in diluted seawaters in a stepwise manner so that ex- posure to 70,50,30, or 15% seawater (Table 1) lasted 48 hours. Following a 48 hour exposure, worms were either prepared for analysis or transferred to the next lower seawater concentration. Seawaters were pre- pared by dilution of 100% seawater with tap water. The water in aquaria was kept recirculating. Animals were prepared for analysis by the recon- stitution method as described in detail by Ferraris & Schmidt-Nielsen (1982a). In summary, entire worms (5-JO mg) were weighed to the nearest 0.001 mg (Cahn Automatic Electrobalance, Model 21), dried at 60?C to constant weight, submerged in a small volume (50 n\) of deionized-distilled water and heat- ed in a water bath at 98 ?C for 3 minutes. Specimens were left undisturbed in small plastic tubes at 4?C for about 48 hours to allow diffusion of ions and other osmotically active substances. Tube contents were then mixed and centrifuged and analyses per- formed on the supernatant. Values obtained were corrected for dilution (Ferraris & Schmidt-Nielsen, 1982a). Analyses Osmolality (mOsm/kg H20; Wescor 5100 B Vapor Pressure Osmometer) and Na and K ion concentra- tions (mEq/L; Instrumentation Laboratories, Mod- el 343) were determined on duplicate 5 ftl samples. Cl ion concentration (mEq/l) was measured by coulometric titration (Buchler-Cotlove Chloridom- eter) on single 10 fil samples. Extracellular (PEG) space determination 163 assay for 14C-PEG. The in vivo and in vitro metho- dologies and associated calculation of fractional ex- tracellular space are described in detail in Ferraris & Schmidt-Nielsen (1982a and 1982b, respectively) and are summarized as follows: In vivo Animals were subjected to the same 48 hour stepwise acclimation to various seawaters. Individual C arenarius received an intracoelomic and intravascu- lar micropuncture injection of 14C-PEG (0.01 fid) in the appropriate seawater solution. The injected substance was allowed to equilibrate with the ex- tracellular compartment for 2 hours. Worms were then weighed and a volume of mixed coelomic fluid and blood (extracellular fluid) was collected. Worms were then dried and reconstituted as above. )4C- PEG concentration in extracellular fluids and in tis- sues were determined and the fractional extracellular space calculated. The fraction of the extracellular fluid was determined in a total of 40 worms stepwise acclimated to 100, 70, 50, 30 or 15% seawater. In vitro P. splralis was subjected to experimental seawaters as above. Worms were sliced into 2 mm pieces and the tissue incubated in a 14C-PEG solution for 2 hours. The incubation medium contained 100, 70, 50, 30, or 15% SW plus 14C-PEG (0.25 (id). Following equilibration, tissues were prepared for analysis by the reconstitution method. Tissue samples and in- cubation media were analyzed for l4C-PEG concen- tration and the fractional extracellular space calcu- lated. The fraction of the extracellular fluid was determined in a total of 79 worms stepwise acclimat- ed to 100, 70, 50, 30 or 15% seawater. ,4C-polyethylene glycol M.W. 4000 (l4C-PEG) was used as a marker for extracellular fluid in both spe- cies; however, the techniques employed differed. PEG space was determined for C. arenarius in vivo, whereas, the same was determined for P. spiralis us- ing an in vitro method. The very small size of P. spiralis, in combination with an acoelomate body plan, precluded obtaining sufficient extracellular fluid even by micropuncture methods to accurately Calculations Total body water content [gram H20/gram solute free dry weight (g H:0/g s.f.d.w.)j was calculated af- ter the method of Schmidt-Nielsen etat. (1983). This method of determination of water content is more accurate than that based solely on dry weight. Since regulatory volume decrease (RVD) is associated both with a change in water content and with a decrease t 164 in the amount of solutes, RVD results in a change in the specific weight of the cells. Calculation of g H2OZg s.f.d.w. corrects the dry weight for the change in specific weight. The method, as used in the present study, has been described in detail (Ferraris & Schmidt-Nielsen, 1982a). Solute contents (^moles/g s.f.d.w.) were calculated by multiplying the solute concentration in tissue water (^Eq/ml) by the total tissue water content in g/g s.f.d.w. Fractional extracellular fluid was calculated as fractional PEG space after the method of Ferraris & Schmidt-Nielsen (1982a, 1982b). Intracellular water was obtained by difference. Since the PEG space was not determined on all individuals in the present study we recognize a possible limitation in this meth- od. Data were compared using a one way analysis of variance followed by Student-Newman-Keuis Mul- tiple Range test for separation of significant means. Data referred to as significantly different have a statistically significant difference at least at P < 0.05. Extracellular and intracellular solute determination Solute concentrations in the extracellular fluid of C. arenarius were determined by removal and analysis of a mixture of coelomic fluid and blood as described in Ferraris & Schmidt-Nielsen (1982a). Samples were analysed for Na, K and Cl ion concen- trations (mEq/1). Extracellular solute concentra- tions were used in conjunction with the fractional PEG space to calculate extracellular ion content (fimoles/g s.f.d.w,). Total tissue ion content was measured and intracellular solutes (^moles/g s.f.d.w.) were obtained by difference. In P. spiralis, sufficient extracellular fluid was not obtainable, even by micropuncture methods, to accurately assay for ion concentrations Experimental protocol The relation of the change in water content (V-J/VJ) to the simultaneously occurring change in tissue os- molality (T|/x2 was used to discern the occurrence of RVD during exposure to diluted seawaters. The Vi and V2 values are the water contents (g H20/g s.f.d.w.) of tissues (in this case entire worms) at the beginning and end of a given seawater exposure, respectively; n-j and ir2 are the tissue water osmolali- ties (mOsm/kg H20) of the same tissue for the be- ginning and end, respectively, of the same period (Ferraris, 1984). In a hypoosmotic medium, RVD may occur (1) while cells are swelling, as a swelling- limitation phase, and (2) subsequent to swelling, as a net volume loss. Both phases are accompanied by a decrease in solute content. Following acclimation to a hypoosmotic medium, if V^/V^ were less than ir,/7T2 water content would have changed less than would be expected given the change in tissue osmo- lality and the occurrence of RVD would be indicat- ed. Results There were few consistent, significant differences among control, ablated and sham-operated animals. Hence, for clarity, all groups of a given species are referred to collectively as C. arenarius or as P. spira- lis. Those differences among groups that occurred primarily involved the responses of C. arenarius in 70% seawater and P. spiralis in 30% seawater. Ail differences that were statistically significant (P < 0.05) are either reported in the text or appear in appropriate figures. Osmolaliiy and ion concentrations in total tissue water C. arenarius was isoosmotic to the medium in 100% seawater but was significantly hyperosmotic to all other media (Fig. 1). The osmotic difference be- tween worms and medium was approximately 45 mOsm in 70% SW whereas that in 15% SW was 85 mOsm. P. spiralis was slightly hyperosmotic to 100% SW (Fig. 1). In experimental seawaters P. spiralis v/as sig- nificantly hyperosmotic to the medium maintaining an average difference of 75 mOsm in 70 and 50% o 95.0 i- U 80.0 ? 75.0 h 70.0 E + UJ Z O1000 r x o> \ 500 - E o E r 150 r 50 0 250 50 h 0 250 r- UJ L 10? I" V 50 165 %l ODQ ooa 1? Coo P.SPIRALIS C. ARENARIUS O CONTROL ? O ABLATED * D SHAM ? n=8? ISOOSMOTIC _%L Otf Ceo *o?rj -?- $Sg %B %B ^OD OCD \ .001 .002 .0035 l/OSMOLALITY .007 Fig. I. Percent water, osmolality, andK, Na, and Ct concentrations [mean, S.E. (S.EIs smaller than size of symbols)] in the total tissue water of control, ablated and sham-operated P. spiralisand C. arenarius after exposure to diluted seawaters. an = 5 at 0.007 and n = 6 at 0.0015 for P. spiralisonly. * Ablated animals significantly different (P < 0.05) from either control or sham-operated worms; "ablated worms significantly different from both control and sham-operated animals. 166 SW. When worms were in 30 or 15%SW, the gradient between worm and medium was only about 20 mOsm. Survival of this species in 15% SW was low (62.5%, n = 8); reduction in mobility and re- sponse to touch was obvious in worms in both 30 and 15% SW. Percent water in C. arenarius increased signifi- cantly with each progressive dilution of the medium. There was an average overall increase of 13.4% (Fig. 1). Ablated C. arenarius were significantly higher in percent water than either control or sham- operated animals in 70% SW but differences among groups were not significant at any other sea water di- lution. P. spiralis also increased in percent water with decreasing seawater concentration (Fig. 1). Howev- er, in the case of this species, the overall increase in percent water from worms in 100% SW to those in 15% SW was 21.5%. Ablated worms were lower in percent water than other groups when worms were acclimated to 30% SW. In C. arenarius the concentrations of all ions measured in total tissue water (Na, K and Cl mEq/1) decreased with the concentration of the medium (Fig. 1). Primary reduction was seen in Na and Cl concentration. P. spiralis also successively decreased in tissue ion concentration (mEq/1) but primary reduction oc- curred with respect to K rather than Na reflecting an acoelomate body construction (Fig. 1), Ion concentrations in extracellular fluid Na concentrations (mEq/1) in the extracellular fluid of C arenarius were either iso- or hyperionic to that of the medium (Table 2), In worms in 100, 30, and 15% SW, average fluid to medium ratios (F: M) were 1.04, 1,01, and 1.12, respectively. In worms in 70 and 50% SW, F:M ratios increased to 1.26 and 1.31, respectively. Extracellular K (mEq/1), however, was consistently hyperionic to the seawater. Fluid to medium ratios for K progressively increased (i.e., 2.60, 3.28, 4.25, 5.39 and 10.52) with dilution of the medium. In contrast, extracellular Cl concentration was strongly hypoionic to full strength seawater Table 2. Extracellular ion concentrations (mEq/1) in C. arenarius (mean = S.E.) during exposure to diluted scawatcrs. Seawater Control Ablated Sham-operated 100% Na 473.6 K 10.5 Cl 513.9 70% Na 322.0 K 7.4 Cl 372.2 50% Na 239.3 K 5.3 Cl 274.5 30% Na 139.6 K 2.9 Cl 159.9 15% Na 72.4 K 1.6 Cl 79.3 492.9- 8.15 (5) 27.1 * 0.88 364.5* 14.78 (8) 403.2 = 9.84 22.8- 1.96 327.4? 15.95 291.2 ? 16.79 24.1 = 1.63 225.3=11. II 142.9= 5.55 17.1= 2.54 162.5= 9.85 81.3= 4.90 17.8= 3.56 75.9= 7.75 489.9 ? 15.93 (5) 26.3 ? 0.47 374.5+17.68 {8} 406.4+ 5.23 26.8 ? 3.72 314.5? 16.39 324.9 ?18.79 22.4= 2.49 202.0= 2.75 139.2 + 6.99 16.4 ? 1.31 147.3 ? 12.35 83.7= 4.34 16.0= 0.68 107.3+14.90 492,6= 13.08 28,6= 2.4S (3) 372.8 = 14.22 18) 412.1 = 11.66 13) 23.3: 1.29 |3) 300.7= 9.17 306.9 ? 9.96 21.1 = 0.90 233.4 = 25.04 141.9= 7.83 13.4= 0,34 158.5= 6.02 77.2? 4.17 16.7= 2.58 83 8= 9.0! n = 4 except where otherwise indicated in parentheses. 167 (F: M = 0.72). With dilution of the medium Cl con- centration became less hypoionic; F: M ratios equaled 0.84, 0.80 and 0.97 in 70, 50 and 30% SW, respectively. In worms in 15% SW, extracellular Cl concentration in control and sham-operated worms was isoionic to the medium {F:M = 1.01); an un- usually high Cl concentration in ablated worms raised the average fluid to medium ratio to 1.12 in worms in this seawater. In spite of the high Cl con- centration in ablated worms, differences in extracel- lular Cl concentration among the groups were not significant. Comparative information on P. spiralis was not available due to the very small size of this species and the small volume of the extracellular compartment (approximately 5% of total body water content). Water and solute content Water content (g HUO/g s.f.d.w.) in C arenarius in- creased significantly with each dilution of the medi- um (Fig, 2), Overall, total water content increased by an average of 7.7 g HiO/g s.f.d.w. As also reflected by percent water, the amount of water in ablated worms was significantly higher than in in control or sham-operated worms after a 48 hour exposure to 70% SW. P. spiratis similarity increased in water content with increased dilution (Fig. 2). However, while this species was initially significantly lower in water con- tent than C. arenarius, by the end of the experiment P. spiralis had gained an average of 10.7 g H:0/g s.f.d.w. and had surpassed the water content of C. arenarius. Ablated P.spiralis in 30% SW contained significantly less water than did either control or sham-operated, animals. In order to estimate the occurrence of regulatory volume decrease (cf. Ferraris & Schmidt-Nielsen, 1982a), the relative water contents (v\/V,) of worms in various sea waters were plotted against the relative osmolalities (JTI/JTI) of the same animals (Fig. 3). C arenarius, in all experimental seawaters, demonstrated a lower water content than might be expected if these animals had behaved as true os- momeiers. Differences were significant (P < 0.05) in all seawaters except 70% and increased with decreasing osmolality. Similarly, P. spiralis had a lower water content than expected in all experimen- tal seawaters (P < 0.05 except 70% SW). Differ- ences between expected and demonstrated water contents were more pronounced in C. arenarius than in P. spiralis. K content (^moles/g s.f.d.w.) in C. arenarius did not decrease with acclimation to diluted seawaters (Fig. 2). In P. spiralis, there was also no significant loss of K (pmoles/g s.f.d.w.) after stepwise acclimation to 70, 50 or 30% SW, however, K content dropped markedly after acclimation to 15% seawater (Fig. 2). Na content (^moles/g s.f.d.w.) (Fig. 2) in C. arenarius decreased with each dilution of the medi- um, Na content (^moles/g s.f.d.w.) in ablated worms was higher (/J<0.05) than in control and sham- operated animmals in 70% seawater with no signifi- cant difference occurring in any other seawater con- centration. In P. spiralis there was a decrease {P < 0.05) in the Na content (jimoles/g s.f.d.w.)of all groups after ex- posure to 70% seawater; further reduction occurred in 50% SW (Fig. 2). Loss of Na, however, was fol- lowed by a significant Na gain in worms in 30% sea- water and again in 15% seawater. Differences in Na content among the three groups were significant only in 30% SW. For both species, a pattern similar to that which occurred in Na content was demonstrable with re- spect to Cl content (jtmoles/g s.f.d.w.) (Fig, 2). In C. arenarius significant Cl (pmoles/g s.f.d.w.) was lost with each dilution of the medium except the last (15% SW). Ablated worms contained more Cl (jtmoles/g s.f.d.w.) than sham-operated worms (P < 0.05) in 70% SW. In 30% seawater Cl content in ablated animals was only lower than that in con- trol worms {P < 0.05). In P. spiralis, Cl (^moles/g s.f.d.w.) was lost in 70% SW and in 50% SW (P < 0.05) (Fig. 2). The amount of Cl in worms then progressively increased with acclimation to 30 and 15% seawater. Cl content was lower (P < 0.05) in ablated animals than in the control groups in 30% seawater with no significant differences occurring among groups in other sea- waters. 168 14.00 r e E 8.00 o x 4.00 2.00 L ? E a. t 200 Ooa Ceo oco I" CtP^Ooa O^ % P. SPIRAL IS C. ARENARIUS O CONTROL ? O ABLATED * a SHAM ? ffl/gm s. f. d. w. 1000 i- ? o E + D 400 Z 200 1- ?. \ oO o? o.. 1000 r ? i_ 400 h 200 L- \ \OQ o I?II?II?I i?i .001 .002 .0035 1 / OSMO L ALITY ?S _l=j .007 F'g. 2. Water (gram H:0/gram solute free dry weight), K, Na, and Cl contents (jimolcs/g solute free dry weight) (mean, S,E) in control. ablated and sham-ope rated P. spiralisand C, acenar/wv after exposure to diluted seawater. an = 5 at 0.007 and n = fiat 0.0015 for P. spiraiis only. ?Ablated animals significantly different (P < 0.05) from either control or sham-operated worms: "ablated worms significantly different from both control and sham-operated animals. 169 60 5.0 4.0 - 'Ol "* P. SPIRALIS C. ARENARIUS O CONTROL ? o ABLATED * a SHAM ? n = 8? ?- V2A, = nl/TT2 0.0 4.0 5.0 6.0 Fig. 3. Relative water content (Vi/V,) (mean, S.E.) in control, ablated and 5 ham-ope rated P. spiralis and C. arenarius'm 100% and dilut- ed seawaters compared with the relative osmolalities (JT/IT;) (mean, S,E.) of the same animals, J n : T[/?; about 6.0 for P. spiralis only. 6 at T,/T, about 1.3 and n = 5 at Extracellular (PEG) space and the water content of the extra- and intracellular compartments In C. arenarius, the extracellular space, /, e. the frac- tion of the total tissue water in which l4C- polythylene glycol was distributed, did not differ (P < 0.05) between control and ablated worms in any seawater concentration {Table 3). The mean val- ue obtained in a given seawater was thus used in all calculations involving PEG space. Extracellular TableS. I4C-PEG space as percent of total water content during exposure to diluted seawaters (mean ? S.E.). Seawater I'M C. arenarius P. spiralis (n=l6> Control Ablated Control + ablated (n = 4) (n = 4) (n = 8) 100 ?i46.58?3.4l :!47.41?3,83 !|47.01 ?2.38 J4.09 + 0.390 70 J40.33 ?2.89 ;;4l.79?3.53 \\ 41.06?2.13 j 3.50 ?0.259 50 135.91 ?4,95 j33.87?4.53 I 34.89 + 3.13 :4.26?0.471 30 j 46.08+. 1.11 j 50.24 + 2.86 !;48.16+1.62 ;5.07 ?0.461 IS 150.89 + 7.12 ; 54,17 + 4.15 ;52.53?3.86 8.74 ?0.696