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Authors (or delegates for foreign authors) will receive page proofs of articles shortly before publication. They will be charged the current cost of printers' time for corrections to these (other than corrections of printers' or editors' errors). Other than these charges for authors' alterations. The Biologi- cal Bulletin does not have page charges. Reference: liiol Hull 178: 1-9. (February, 1990) The Role of Arachidonic Acid and Eicosatrienoic Acids in the Activation of Spermatozoa in Arenicola marina L. (Annelida: Polychaeta) M. G. BENTLEY1*, S. CLARK2, AND A. A. PACEY1 ^Gatty Marine Laboratory, University of St. Andrews, St Andrews, Fife, KY16 8LB. Scotland, U.K., and 2Dove Marine Laboratory and Department of Biology, University of Newcastle upon Tyne, Newcastle upon Tyne, NE1 7RU, U.K. Abstract. Partial purification of a sperm maturation factor (SMF) in the intertidal polychaete Arenicola ma- rina has implicated arachidonic acid, an arachidonic me- tabolite, or a similar substance as the active factor from the prostomium. The effects of a number of 20-carbon fatty acids on inactive spermatozoa are investigated, and this reveals that only arachidonic acid and 8,1 1,14-eico- satrienoic acid cause sperm activation. The use of argen- tation thin-layer chromatography to separate fatty acids with varying degrees of unsaturation reveals a compo- nent in prostomial lipid extract, which co-migrates with eicosatrienoic acids. Investigations using cyclooxygenase and lipoxygenase result in a loss of sperm-activating properties of both prostomial extract and fatty acids. The use of cyclooxygenase and lipoxygenase inhibitors has no effect. Bovine serum albumin (BSA) reduces the sperm activating properties of both fatty acids and prostomial extract in a dose-dependant way. Additional purification procedures using: (a) organic solvents and aqueous buff- ers and (b) ODS silica cartridges, demonstrate that the active fraction of prostomial extract co-elutes at every step with the 8,1 1,14-eicosatrienoic acid standard. Gas chromatography of methyl esters of prostomial lipid ex- tract reveals the presence of a peak with an identical re- tention time to the methyl ester of authentic 8,1 1,14-ei- cosatrienoic acid standard. The results described here provide strong evidence that the active SMF in prosto- mial homogenate is not a fatty acid metabolite but the parent acid 8,1 1,14-eicosatrienoic acid. These results could only be made unequivocal by full structural analy- Received 28 August 1989; accepted 30 November 1989. * To whom all correspondence should be addressed. sis using mass spectrometry and NMR following capil- lary gas-liquid chromatography. Introduction Arenicola marina is a common intertidal polychaete. Its reproductive cycle is annual, with most populations found around the coasts of the British Isles spawning in the autumn or early winter (Howie, 1959). The repro- ductive biology of this species has been reviewed by Howie (1984). In both sexes, gamete proliferation occurs in the gonads, and early germ cells are released into the coelomic fluid where gametogenesis proceeds (Ash- worth, 1904; Newell, 1948). Females approaching matu- rity are characterized by many oocytes that have com- pleted vitellogenesis but are arrested in prophase of mei- osis I; maturing males have many sperm morulae (Howie, 1959). Sperm morulae are plates of several hun- dred fully differentiated immotile spermatozoa, which are bound together at both the head and distal ends of the flagella (Newell, 1948; Bentley 1985, 1986a, b; Bent- ley and Pacey, 1989). Spawning in both male and female Arenicola marina is a direct consequence of the maturation of the gametes. The maturation of the oocytes (entry into metaphase of meiosis I), and the breakdown of the sperm morulae to free-swimming spermatozoa, results in the immediate shedding of these from the ciliated funnels of the ne- phromixia (Howie, 196 Ib, c). Oocytes mature by the ac- tion of a maturation hormone (Howie, 1963, 1966) from the prostomium, which induces germinal vesicle break- down in v;/ro(Meijerand Durchon, 1977). The dissocia- tion of the sperm morulae in males is also brought about M. G. BENTLEY ET AL by a prostomial maturation hormone (sperm maturation factor) (Howie, 1963, 1966), which is a lipid (Howie, 1961a;Bentley, 1985). Bentley (1985) began purifying the sperm matura- tion factor (SMF) using thin-layer chromatography, in- dicating that it was a relatively polar lipid. Lipids recov- ered from the TLC plates were tested for SMF activity in an in vitro assay. Biological activity was recovered from areas of the TLC plates where a number of pharmacolog- ically active, non-steroid lipids are found. Further TLC studies led Bentley (1986a) to suggest that SMF may be a metabolite of the 20-carbon polyunsaturated fatty acid — arachidonic acid. Arachidonic acid (5,8,1 1,14- eicosatetraenoic acid) is one of a number, and probably the most important, of 20-carbon polyunsaturated fatty acids that are naturally occurring precursors of a wide range of extremely biologically active compounds. These include the prostaglandins, HETE's (hydroxy-eicosatet- raenoic acids), and leukotrienes. Roles for these com- pounds have been identified in a wide range of verte- brates and invertebrates. Their roles in invertebrates have been recently reviewed (Stanley-Samuelson, 1987) and we will not discuss them further here. However, it should be noted that arachidonic acid is metabolized by starfish oocytes ( Meijer and Guerrier, 1984; Meijer ct at. , 1984; Meijer el a/., 1986), and that this results in the breakdown of the germinal vesicle prior to fertilization. In light of the information available on the chemical nature of SMF, the present investigation examines in de- tail the possible role of arachidonic acid and the related 8,11,1 4-eicosatrienoic acid in the activation of spermato- zoa in Arenicola marina Materials and Methods Gravid individuals of Arenicola marina were collected by digging in sand during low water of spring tides at St. Andrews Bay, Fife, Scotland, and Fairlie Sands, Ayr- shire, Scotland. Specimens were maintained individually in seawater at 5°C in the laboratory until use. Sperm samples for bioassay use were removed from the coe- lomic cavity as described previously (Bentley and Pacey, 1989). In vitro assays of sperm morula suspensions were performed as described by Bentley (1985). Preparation of prostomial lipid extracts Prostomial lipid extracts were prepared from mature specimens of Arenicola marina. The prostomia were re- moved using iridectomy scissors, and were homogenized using an MSE Soniprep 150 ultrasonic disintegrator at 0°C. The lipid fraction was partitioned from the sample using an equal volume of chlorofornrmethanol (2:1 v/ v). The organic layer from several extractions was re- moved, pooled, and dried over anhydrous sodium sul- phite. The samples were then concentrated by removing the solvent mixture in a rotary evaporator. The dried lipid residues were redissolved in methanol before being assayed for biological activity or used in subsequent ana- lytical procedures. Thin-layer chromatography (TLC) A sample of total lipid was applied to 20 X 20 X 0.25 cm pre-coated silica gel F254 TLC plates (Merck) using a 100 M' disposable micropipette. Prior to applying the sample, the plates were cleaned of any lipid contami- nants by running the blank plate, in the solvent system to be used, for its full length. After allowing the solvent to evaporate, the plate was activated in an oven at 1 20°C for 30 min. The solvent system used was the upper phase of: ethyl acetate:2,2,4-trimethylpentane:acetic acid:wa- ter (45:25: 10:50 v/v) (Salmon and Flower, 1982) and the plates were run in a vertical chamber until the solvent front had moved 12 cm up the plate. The solvent was then allowed to evaporate from the plate in a fume cup- board, and the spots were visualized by spraying the plate with 10% phosphomolybdic acid in ethanol. A second plate was run simultaneously with the above, but was not sprayed with phosphomolybdic acid. Areas correspond- ing to the visualized spots on the first TLC plate were scraped off the plate and the lipids eluted in methanol and tested for biological activity as described above. Argentation TLC A sample of total prostomial lipid was spotted on to two further activated TLC plates impregnated with 5% AgNO, in acetone (see Christie, 1982). The plates were developed as for the TLC described above. Free fatty acid standards were also applied to the plates. When the sol- vent front had reached the 12-cm mark, the plates were removed, the solvent evaporated, and the plates washed with distilled water to remove the AgNO,. One of the plates was sprayed with phosphomolybdic acid and the second plate was used for recovering the lipids for bio- assay. In vitro assay of 20-carbon fatty acids Free fatty acids: eicosanoic, 1 1-eicosenoic, 1 1,14-eico- sadienoic, 8,1 1,1 4-eicosatrienoic acid, 1 1,14, 17-eicosa- trienoic, 5,8,1 1,1 4-eicosatetraenoic (arachidonic), and 5,8,1 1,14, 1 7-eicosapentaenoic acids, were obtained from Sigma Chemical Co., and 1 X 10~2 M stock solu- tions prepared in HPLC grade methanol (BDH). For use in bioassay, aliquots of these stock solutions were diluted 100 fold to give a final free acid concentration of 1 X 10~4 A/ and a negligible residual solvent concentration. Double dilutions of the free acids were then used to de- FATTY ACID ACTIVATION OF SPERMATOZOA termine the biological activity of each acid in the activa- tion of spermatozoa in vitro. Effect ofcydooxygenase and lipoxygenase pathway inhibitors Stock solutions ( 1 0 mM) of the cyclooxygenase inhibi- tors aspirin, indomethacin, and tolazoline, were pre- pared in TFSW (triple filtered seawater). A 1-mM solution of butylated-hydroxytoluene, a lipoxygenase in- hibitor, was also prepared. Prostomia were then homoge- nized in solutions of each inhibitor before bioassay for SMF activity. Control experiments were carried out in which the inhibitors of cyclooxygenase or lipoxygenase were added to prostomial extract after homogenization. or TFSW prior to bioassay. This permits the distinction to be made between metabolism of fatty acid substrate by the prostomial homogenate and metabolism by the spermatozoa themselves. Incubation of biologically active fatty acid with cyclooxygenase and lipoxygenase (A) Arachidonic acid was incubated with fresh bovine lung homogenate to obtain products from the cyclooxy- genase pathway as described by Powell (1982). One gram of bovine lung tissue was homogenized on ice in 5 ml 0.05 A/ Tris-HCl buffer, pH 7.4. One ml of the homoge- nate was incubated with arachidonic acid at a final con- centration of 1 X 10~2 Mat 37°C for 5 min. The reaction was terminated by adding 5 ml ethanol, then adding 16 ml H2O, and centrifuging at 400 X g for 10 min. The supernatant was removed and assayed for biological ac- tivity. (B) One-mi aliquots each containing 1.8 mg (c. 250,000 units) of soybean lipoxygenase was incubated with arachidonic acid at a final concentration of 5 X 10 3 M at 25°C for 1 5 min. After incubation, the reaction was terminated by heating, the extract was centrifuged, and the supernatant was assayed for biological activity. Incu- bations containing denatured lipoxygenase and lacking lipoxygenase were also carried out. Incubation of prostomial extract with lipoxygenase One-mi aliquots each containing the equivalent of 0.36 prostomium were incubated with 1.8 mg (c. 250,000 units) of lipoxygenase, for 60 min at 20°C. After incubation, the reaction was stopped and the sample treated as described above. Incubations containing dena- tured lipoxygenase and lacking lipoxygenase were car- ried out in parallel. Incubations of biologically active fatty acid with BSA Prostomial homogenate and 8,1 1 , 1 4-eicosatrienoic acid were bioassayed in the presence of dissolved BSA (bovine serum albumin). BSA solutions were freshly pre- pared in TFSW to give a final concentration in the assay ofO, 100, 1000 Mg- ml"1, and 10 mg- ml ', respectively. Extraction of SMF by organic solvents and aqueous buffers Extraction of SMF from biologically inactive lipid constituents of prostomial extracts were carried out as described by Jouvenaz el al. (1970) and Van Dorp ( 197 1 ). This allows larger quantities of starting material to be purified, and permits the separation of free fatty acids from their often biologically active, but extremely labile, metabolites. This procedure involves the initial preparation of ethanolic lipid extract, washing the resi- due with ethanol:diethyl ether (1:1 v/v), followed by add- ing saline. The extract is then reduced in volume to 2.5 ml, acidified to pH 4 with citric acid, and contaminating lipids removed with petroleum ether. The remaining lip- ids are then taken up into ethyl acetate concentrated to about 5 ml total volume. Tris buffer (1.5 ml, pH 7.8) is then added to take up any prostaglandins present. All the organic and aqueous fractions obtained throughout this procedure were bioassayed for SMF activity. Extraction of SMF on ODS silica cartridges Freshly prepared prostomial homogenate and free fatty acid standards were separately applied to pre-wet Sep-Pak® Ci8 Cartridges (Waters Associates) in 10% aqueous ethanol with the pH adjusted to 4.0 using a 1- M stock solution of citric acid. The Sep-Pak® was pre- wetted using 2 ml of methanol followed by 5 ml of H2O before applying the sample or standard. Fractions were partitioned using the following solvent mixtures (Powell, 1982): aqueous ethanol (20 ml, 10%), 20 ml H2O, 10 ml petroleum ether, 10 ml petroleum etherxhloroform (65: 35 v/v), and 10 ml methyl formate. The Sep Pak® was regenerated using 10 ml of 80% aqueous ethanol. The fraction obtained using each solvent was collected and prepared for bioassay as described above. Gas-liquid chromatography of prostomial lipids Prostomial lipid extracts were prepared as described above, and methylated using a method modified from Christie (1982). The sample was dissolved in 1 ml of di- chloromethane, and refluxed for 2 h with 2% methanolic H2SO4. After cooling, 4 ml of saturated NaCl was added, and the fatty acid methyl esters extracted with 2 ml pe- troleum ether (40°-60°C). GC analysis was performed using a Hewlett Packard 5 890 A gas chromatograph fitted with a flame ionisation detector. Samples were separated on a capillary non-polar column (fused silica, 25 m X 0.25 mm i.d., 0.12 df, CP-Sil 5CB) following on-col- M. G. BENTLEY ET AL. 1.0 0 g w 49 49 ^^ SMF activity Rf value 0.6 0.4 0.2 0.0 1 : 11, 14 - eicosadienoic acid 2 8, 11, 14 - eicosatrienoic acid 3 11, 14, 17 - eicosatrienoic acid 4 8, 11, 14, 17, - eicosatetraenoic acid 5 5, 8, 11, 14. 17, - eicosapentaenoic acid 6 : Prostomial lipids Figure 1. Separation of 20-carbon fatty acids (spots I to 5 with 2. 3, 3, 4, and 5 double bonds, respectively) and prostomial lipids by ar- gentation thin-layer chromatography. The fatty acid standards with the greatest degree of saturation interact least with the silver nitrate on the TLC plate and therefore migrate furthest. The prostomial lipid extract shows a spot with an identical Rf value to the eicosatrienoic acids. This spot, when scraped off a washed plate, shows sperm maturation factor (SMF) activity in vitro umn injection. A linear thermal gradient program from 90°-300°C at 20°C-min ' was used with helium as the carrier gas (25 cm • s~ ' ). Results Analysis of prostomial lipid extract and €20 fatty acids by TLC TLC on activated silica gel plates, using the upper phase of: ethyl acetate:2,2,4-trimethylpentane:acetic acid:water (45:25: 10:50 v/v) as a solvent, allows the sepa- ration of C20 fatty acids from their metabolites. The bio- logical activity associated with prostomial lipid samples is associated with a region of the TLC plate that is identi- cal to the position where free fatty acids are found (Rf 0.78-0.82). This chromatographic separation cannot distinguish between fatty acids with varying degrees of unsaturation. Argentation TLC (using the same solvent system and TLC plates impregnated with 5% AgNO3) allows fatty acids of the same carbon number to be separated accord- ing to the number of double bonds in the molecule. Figure 1 illustrates the results of this separation. SMF ac- tivity was recovered from a region of the plate which cor- responds to the position of the C20:3 acids (8,11,1 4-eico- satrienoic acid and 1 1,14,17-eicosatrienoic acid). This technique is unable to separate these two isomers be- cause they are identical in their degree of unsaturation. Sperm activation by C20 fatty acids In vitro bioassay of eicosanoic, 1 1-eicosenoic, 11,14- eicosadienoic, 8,1 1,14-eicosatrienoic acid, 1 1,14,17-ei- cosatrienoic, 5,8,1 1,1 4-eicosatetraenoic (arachidonic), and 5,8,1 1,14, 1 7-eicosapentaenoic acids for the ability to induce sperm activation showed that both 8,11,1 4-ei- cosatrienoic acid and arachidonic acid displayed biologi- cal activity. The results are summarized in Table I. Con- centration ranges for biological activity of 8,1 1,14-eico- satrienoic acid and arachidonic acid are based on nine replicate experiments producing mean minimum con- centrations required for a response of 4.47 X 10 5 At and 2.28 X 10~4 M, respectively. These data indicate that 8,1 1,14-eicosatrienoic acid is about five times more ac- tive in this system than in arachidonic acid. Studies of cyclooxygenase and lipoxygenase pathways The preparation of prostomial extract in the presence of inhibitors of cyclooxygenase activity (aspirin, indo- methacin, tolazoline) or lipoxygenase (butylated hy- droxytoluene) did not effect the SMF activity of the ex- tract. Aspirin at concentrations of 5 mM and 10 mA/ caused sperm lysis (the reasons for this are not clear, but this effect is unlikely to be related to the cyclooxygenase inhibitory property of the aspirin). These results suggest that there is no conversion of parent fatty acid to a bio- logically active metabolite via either the cyclooxygenase or lipoxygenase pathway. However, polychaete enzymes metabolizing fatty acids may differ from vertebrate cyclooxygenases and lipoxygenases, and substances used as inhibitors may not effect enzyme activity. Quercetin, another lipoxygenase inhibitor, could not be used in this Table I Sperm activation by C20 fatly acids Threshold concentration for activation Fatty acid Activity (Mean ± S.E.; n = 9) A eicosanoic - B 1 1 -eicosenoic - C 1 1.14-eicosadienoic - D 8.1 1.14-eicosatrienoic + 4.47 ± 1.46 X 10'5 M E 11,14,17-eicosatrienoic - F 5,8,11,1 4-eicosatetraenoic + 2.28 ± 1.78 x 1(T4 M G 5,8, 11,14,1 7-eicosapentaenoic - H TFSW control — FATTY ACID ACTIVATION OF SPERMATOZOA x \Q-*M X \Q-*M Table II I'lic c/lccts i>/'cvclt>o\yKi'nu.ti' und lipoxygenase on \pcrm aclivalion hy (. '- 0 tally iiculx Threshold concentration Activity for activation a. Incubation with cyclooxygenase Arachidonic acid incubated with bovine lung cyclooxygenase Arachidonic acid + I.25X10'5M Bovine lung homogenate (cyclooxygenase) TFSW b. Incubation with lipoxygenase Arachidonic acid incubated with soybean lipoxygenase + 2.5 Arachidonic acid + 4.0 Soybean lipoxygenase TFSW study because of a non-specific effect on spermatozoa, which will be reported elsewhere. Incubation of arachidonic acid with bovine lung ho- mogenate (cyclooxygenase) or soybean lipoxygenase was carried out to examine whether there was (a) a reduction, (b) enhancement, or (c) the same level of sperm activa- tion after converting the fatty acid substrate to metabo- lites. Table II shows that both incubation with bovine lung homogenate and soybean lipoxygenase brought about a reduction in the fatty acid incubate's ability to activate spermatozoa. This suggests that the fatty acid has been largely converted to cyclooxygenase and lipoxy- genase metabolites, which cannot activate the spermato- zoa. Thin layer chromatographic analysis of the incu- bates confirm that most of the fatty acid is converted dur- ing incubation (Fig. 2). Thin layer chromatography of prostomial homogenate shows that most of the fatty acid remains unmetabolized. This indicates that the fatty acid is not normally converted to a metabolite by endogenous polychaete enzymes, which may be outcompeted for substrate during incubations with exogenous cyclooxy- genase or lipoxygenase. Incubation of prostomial homogenate with soybean li- poxygenase results in a total loss of SMF activity. This indicates the conversion of a fatty acid in the prostomial homogenate to non-active metabolites, and also suggests that it is this fatty acid component of the prostomial ho- mogenate that causes sperm activation in vitro. Incubations of 8, 11,14-eicosatrienoic acid and prostomial extract with BSA BSA (bovine serum albumin) was incubated with 8,1 1,14-eicosatrienoic acid and prostomial homogenate to investigate the possible interference of BSA on the ability of both the fatty acid and prostomial extract to activate spermatozoa of Arenicola marina. It has long been known that fatty acids interact strongly with serum albumin (Goodman, 1958). Figure 3 shows that the abil- ity of both 8,1 1,14-eicosatrienoic acid and prostomial homogenate to activate spermatozoa is markedly re- duced by the addition of BSA. This evidence lends fur- ther support to the suggestion that it is a fatty acid com- ponent of the prostomial homogenate that causes sperm activation in vitro. Further purification of SMF from prostomial homogenate Figure 4 shows the purification steps employed for the purification of SMF from crude prostomial homogenate, using the method developed by Jouvenaz et ai, (1970) and Van Dorp ( 197 1 ) for the extraction of prostaglan- dins from biological tissues. The figure also traces the bi- ological activity through the purification steps. SMF ac- tivity is finally recovered in an ethyl acetate fraction. 1.0 0.8 0.6 Rf value 0.4 0.2 0.0 1 : Arachidonic acid incubated with bovine lung 2 : Arachidonic acid 3 : Prostomial lipids Figure 2. TLC analysis of prostomial lipids. arachidonic acid, and arachidonic acid following incubation with cyclooxygenase. Arachi- donic acid can be seen at an Rf value of about 0.78. The cyclooxygenase products are clearly visible with Rf values lower than that of arachi- donic acid itself. Prostomial lipid extract shows no spots which corre- spond to cyclooxygenase products, and which may have arisen as a result of action by endogenous cyclooxygenases. M. G. BENTLEY ET AL 1.0-] o x |1 I 1 1 0 100 1000 10000 100000 BSA Concentration (ng/ml) Figure 3. Minimum concentrations of (a) 8.1 1,14-eicosatnenoic acid, and (b) prostomial homogcnatc required to bring about sperm activation in the presence of bovine serum albumin (BSA). The fatty acid concentration, or the concentration of prostomial extract required to bring about sperm activation //; wm>. increases with the concentra- tion of dissolved BSA. Data shown are the mean (±SE) minimum con- centrations required to bring about sperm activation in three replicated experiments. Prostaglandins remain in the pH 7.8 Tris buffer and would be recovered only in an ethyl acetate fraction from Tris buffer at pH 4.0. This indicates that SMF activity is not recovered with prostaglandins but is recovered in the fatty acid fraction. A parallel approach to the purification of SMF has been carried out using Sep-Pak® cartridges and a succes- sion of aqueous and organic solvents. Table III shows that SMF activity is recovered in the same fractions as the 8,1 1,14-eicosatrienoic acid standard. Gas chromaiographic analysis n/'prnstumial lipids The results of separation of methyl esters of prostomial lipid extracts are shown in Figure 5. Three peaks with retention times corresponding to methyl esters of 5,8,1 l,14-eicosatetraenoicacid{8.72 min), 8,1 1,14-eico- satrienoic acid (8.81 min), 1 1.1 4,1 7-eicosatrienoic acid (8.93 min), can be identified. This clearly indicates the presence of 8,1 1,14-eicosatrienoic acid in prostomial lipid extracts obtained from prostomia showing SMF ac- tivity in vitro. Discussion The results obtained from thin layer chromatography of prostomial total lipid extracts described above showed that SMF activity co-migrated with 20-carbon fatty acid standards. In particular, it is associated with eicosatrie- noic acids (demonstrated by argentation TLC). The bio- assay of C20 fatty acids show that only two of the fatty acids tested brought about the activation (dissociation of the morulae and the acquisition of motility) in vitro: ara- chidonic and 8,1 1,14-eicosatrienoic acids. Arachidonic acid, while capable of activating spermatozoa, does not co-migrate with prostomial SMF in the argentation TLC. Clearly, then, arachidonic acid and SMF are not the same substance. All eicosatrienoic acids co-migrate in this TLC system but only 8,1 1,14-eicosatrienoic acid causes sperm activation in vitro. While 8,1 1,14-eicosa- trienoic acid co-migrates with SMF, and has biological activity identical to SMF, this is insufficient evidence to propose that they are the same. Arachidonic acid, 8,1 1,14-eicosatrienoic acid, and ei- cosapentaenoic acid are all naturally occurring 20-car- bon fatty acids that differ in the number of double bonds, having 4, 3, and 5 double bonds, respectively. They are all precursors for a range of pharmacologically active molecules, the eicosanoids. Each of these three fatty 1.97g prostomia Homogenize in 2ml dislilled water \- • bioassay Add 10 ml ethanol Centrifuge & wash precipitate with a) 5ml ethanol b) 10ml ethanol -diethyl ether (l:lv/v) Pool supernatant bioassay Add 2. 5ml saline, reduce volume to 2.5ml Ad]ust to pH4 with citric acid extract with 7ml petroleum ether Extract aqueous phase with 3x2 volumes ethyl acetate Reduce volume of organic fraction to 5ml, Add 1.5ml Tris buffer at pH7,8 bioassay of organic fraction bioassay of aqueous fraction bioassay of organic fraction bioassay of aqueous fraction bioassay of organic fraction bioassay of aqueous fraction -ve +ve +ve -ve + ve -ve Ethyl acetate fraction stored under helium at -20 C Figure 4. Extraction procedure used for sequential purification of sperm maturation factor (SMF) using organic solvents and aqueous buffers (after Jouvenaz el ai. 1970). Following each purification step, aqueous and organic phases were dried under helium, resuspended. and tested for their ability to activate sperm //; vitro. The response is repre- sented here as +ve or -ve, where +ve indicates the presence of SMF activity evidenced by sperm morula breakdown and the presence of free-swimming spermatozoa, and -ve indicates no activation of sper- matozoa. FATTY ACID ACTIVATION OF SPERMATOZOA Table III ofprostomialSMFon ODS silica cartridges Eluent from cartridge Activity of prostomium extract Activity of 8. 1 1,14- eicosatrienoic acid 1. 20 ml 30% ethanol 2. 20mlH:Odist. 3. 10 ml petroleum etherchloroform (65:35 v/v) 4. 10 ml methyl formate 5. 10 ml 80% ethanol acids gives rise to a series of prostaglandins (PGs): arachi- donic acid, which is the best known and probably the most important, is converted to series 2 PGs; 8,1 1,14- eicosatrienoic acid is converted to series 1 PGs. Eicosa- pentaenoic acid, which is the most important C20 fatty acid in marine organisms, gives rise to series 3 PGs. The use of the principal enzymes involved in the me- tabolism of the fatty acids to their respective prostaglan- dins (cyclooxygenase) and other metabolites (lipoxygen- ase) combined with the use of selective inhibitors permits the possible pathways involved to be elucidated. Evi- dence shown above, by using bovine lung homogenate and soybean lipoxygenase, which both caused a marked reduction of SMF activity of prostomial homogenate, suggests strongly that a fatty acid present in prostomial homogenate is responsible for the SMF activity. The use of inhibitors of cyclooxygenase and lipoxygenase sug- gests that there is no conversion of fatty acid in prosto- mial homogenate to metabolite(s), which may have po- tent biological activity as previously suggested (Bentley, 1986a). Compared to other invertebrate groups, notably the insects, and the Crustacea, little is known of the chemical nature of polychaete hormones. To date, no hor- mone from polychaete tissues has been completely puri- fied or its structure elucidated. Grothe el al. (1987) identified catecholamines in the nervous system of Ophryotrocha puerilis, which may have an endocrine re- lated function. Numerous vertebrate-like peptides have been identified in the nervous system of polychaetes (Dhainaut-Courtois el al.. 1985), but functions have yet to be ascribed to these putative hormones. The possible action of a fatty acid as a hormone may seem unlikely. a- 0s- Figure 5. Gas chromatograph of fatty acid methyl esters (FAMEs) of biologically active prostomial lipids showing identified peaks corresponding to 5,8,1 1,14-acid (C20:4), 8,1 1,14-eicosatrienoic acid (C20: 3w6). and 1 1 , 14, 1 7-eicosatrienoic acid (C20:3w3) with retention times of 8.72 min, 8.8 1 min, and 8.93 min, respectively. These peaks correspond in absolute retention times, and relative distances to FAME standards of the three acids. M. G. BENTLEY ET AL. but the certain presence in the prostomium ofArenicola marina of a fatty acid that acts on distant target cells (sperm morulae in the coelomic fluid) may well be an example of such a "hormone." Its hormonal role is fur- ther supported by the cyclical nature of its appearance in prostomial extracts. Bentley (1985) showed that SMF activity of prostomial extracts is maximal around the breeding season of given populations and is non-existent during the post-spawning period. Fatty acids and prostaglandins are present in a wide range of lower animals (Srivastava and Mustafa, 1984; Stanley-Samuelson, 1987). These essential fatty acids and their metabolites also have an effect on many aspects of reproduction in marine invertebrates. The endocrine control of oocyte maturation in asteroid echinoderms is now well understood and involves the action of a peptide gonad-stimulating substance (GSS), 1 -methyl adenine, and an intracellular maturation-promoting factor (MPF) (see Giese and Kanatani, 1987 for review). One- methyl adenine acts on the oocytes to bring about matu- ration. However, Meijer ct al. ( 1986) demonstrated that arachidonic acid mimics the action of 1 -methyl adenine on starfish oocytes in vitro. It is possible that 1 -methyl adenine acts as a "second messenger" or that the arachi- donic acid mimics some hitherto unidentified fatty acid. A tri-hydroxy metabolite of arachidonic acid has been identified as the hatching factor in the barnacle Semibal- tinns (Balamis) balanoidcs (Clare ct al.. \ 982, 1985; Hol- land el al., 1985). Prostaglandins also cause spawning of the abalone, Haliolis nifi'scenx, and the mussel, Mytilus edidis ( Morse et al.. 1977). Pharmacologically active metabolites of arachidonic and related fatty acids are characteristically short-lived substances produced close to, or at, their site of action. The parent fatty acids are often metabolized by the target cells themselves. This may occur at the cell surface or intracellularly. Typically this metabolism occurs as a re- sult of the action of lipoxygenases or cyclooxygenase (PG synthetase). The precise nature of the enzymes may vary between phyla, and there is evidence that those found in some invertebrates (e.g.. Lymnaea stagnalis) may be different to those occurring in vertebrates (Clare et al.. 1986). In the starfish oocyte, arachidonic acid is con- verted to HETEs at the plasma membrane (Meijer et al., 1986). It may be that 8,1 1 , 1 4-eicosatrienoic acid is me- tabolized by the sperm morulae ofArenicola marina and this will be investigated by the use of radiolabeled precur- sors. The maturation of starfish oocytes by arachidonic acid is inhibited in a dose-dependent manner by the pres- ence of BSA, however, maturation induced by 1 -methyl adenine (the natural inducer) is not. The activation of spermatozoa of A. marina by prostomial extract or 8, 1 1 , 1 4-eicosatrienoic acid are both inhibited in a similar dose-dependent manner by BSA. This may be further ev- idence to suggest that the fatty acid from the prosto- mium, causing sperm activation in A. marina, is a pri- mary inducer rather than a "second messenger." Purification procedures, followed by structural analy- sis, must be performed to identify the chemical nature of any endocrine substance with certainty. For example, the barnacle hatching factor was identified by organic ex- traction and subsequent GC-MS analysis (Holland et al., 1985). One of the problems often encountered is obtain- ing sufficient starting material for purification. The sepa- ration procedures described in this paper show that SMF of Arenicola marina has identical chromatographic properties to 8,1 1,1 4-eicosatrienoic acid, and that 8,1 1,1 4-eicosatrienoic acid is present in the fatty acid component of prostomial extract. The use of bonded- phase C18 cartridges as a purification stage should per- mit sufficient quantities of SMF to be extracted to com- plete mass spectrometrical analysis. Acknowledgments The authors gratefully acknowledge the support of a Royal Society European Programme Fellowship to M.G.B., under the tenure of which this work was com- menced; the award of support from the Royal Society Browne Fund to S.C.; and the award of an SERC post- graduate studentship to A. A. P. The authors also thank Prof. F. D. Gunstone and Mr. K. Black (Department of Chemistry, University of St. Andrews) for their assis- tance with GC analysis. Literature Cited Ashworth, J. H. 1904. Arenicola. Mem Liverpool Mar. Biol. Comm II: 1-118. Bentley, M. G. 1985. Sperm maturation response in Arenicola ma- rina L.: an in vilro assay tor sperm maturation factor and its partial purification. Inl J Invertebr. Reprod. Dev. 8: 139-148. Bentley, M. G. 1986a. Sperm maturation in Polychaeta. Pp 2 15-220 in Advances in Invertebrate Reproduction, I ol 4. M. Porchet, J-C. Andriesand A. Dhainaut, eds. Elsevier, Amsterdam. Bentley, M. G. I986b. Infrastructure of experimentally induced sperm maturation in Arenicola manna L. P. 492 in Advances in Invertebrate Reproduction, I'ol. 4. M. Porchet, J-C. Andries, and A. Dhainaut, eds. Elsevier, Amsterdam. Bentley, M. G., and A. A. Pacey. 1989. A scanning electron micro- scopical study of sperm development and activation in Arenicola manna ( L. ) Inl J Invertebr. Reprod. Dev. 15: 2 I 1 -2 1 9. Christie, VV. \V. 1982. Lipid Analysis. Pergamon Press, Oxford. 207 PP. Clare, A. S., R. van Elk, and J. H. M. Feyen. 1986. Eicosanoids: their biosynthesis in accessory sex organs of Lymnaea stagnalis (L.). Int. J Invenchr. Reprod Dev 10: 125-131. Clare, A. S., G. Walker, D. L. Holland, and D. J. Crisp. 1982. Barnacle egg hatching: a novel role for a prostaglandin like compound. Mar. Bwl. Lett 3: 1 13-120. Clare, A. S., G. Walker, D. L. Holland, and D. J. Crisp. 1985. The hatching substance of the barnacle Balamis balanoid.es (L.). Proc R Soc Lond B224: 131-147. FATTY ACID ACTIVATION OF SPERMATOZOA Dhainaut-Courtois, N., M.-P. Duhois, G. Tramu and M. Masson. 1985. Occurrence and coexistence in Nereis iliverxicolor O. F. Miillcr (Annelida Polychaeta) of substances immunologically re- lated to vertebrate neuropcptides. Cell Tissue Res 242: 97-108. Giese, A. O, and II. kanatani. 1987. Maturation and spawning. Pp 252-329 in Reproduction in Marine Invertebrates, I HI V, General Aspects: Seeking Unity in Diversity, A. C. Giese. J. S. Pearse, and V. B. Pearse, eds. Blackwell Scientific Publications, Palo Alto, CA, and Boxwood Press, Pacific Grove, CA. Goodman, Dc\V. S. 1958. 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I WO) Development of Nerve Cells in Hydrozoan Planulae: III. Some Interstitial Cells Traverse the Ganglionic Pathway in the Endoderm VICKI J. MARTIN Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556 Abstract. Hydrozoan planulae of Pennaria tiarella possess migratory stem cells — interstitial cells — that are capable of self renewal and can differentiate into either ganglionic nerve cells or nematocytes. The commitment and differentiation of a subpopulation of larval endoder- mal interstitial cells to the neural pathway were exam- ined using light immunocytochemistry and transmission electron microscopy. Embryos of different ages, from 8 to 96 h, were tested for their ability to bind rabbit antise- rum raised to the neuropeptide FMRFamide. A subpop- ulation of interstitial cells in the anterior endoderm of the planula begins to express a FMRFamide-like antigen between 48 and 72 h postfertilization. Concurrent with this endodermal interstitial cell expression, a subset of ectodermal ganglionic cells with FMRFamide-like im- munoreactivity appears in the anterior end of the plan- ula. Ultrastructural examination of the interstitial cell population in the anterior planular endoderm, at 48 h in development, indicates that, based upon morphology, there are at least three subsets of interstitial cells in this region: undifferentiated interstitial cells, interstitial cells traversing the nematocyte differentiation pathway, and interstitial cells traversing the neural differentiation pathway. The endodermal interstitial cells entering the neural pathway form a Golgi complex, electron-dense droplets, dense cored vesicles, and microtubules. Neurite formation does not occur in the endoderm; rather, neu- rites are only found in association with ectodermal gan- glionic cells. Furthermore, planulae lack fully differenti- ated endodermal neurons. This study demonstrates that, during embryogenesis, some interstitial cells destined for neural differentiation are committed in the endoderm before their emigration to the ectoderm, begin to express Received 5 June 1984; accepted 17 November 1989. cytochemical and morphological features of neural differentiation while in the endoderm, and migrate to the ectoderm as neuroblasts. Introduction The hydrozoan planula larva is an especially good sys- tem with which to examine the commitment and differ- entiation of cells during development: the number of cell types in the larva is small; their arrangement is simple; and neither the variety nor the arrangement of larval cells are very far from those of the adult (Martin and Thomas, 1980; Martin el al., 1983; Thomas et al., 1987; Martin. 1988a, b,c). The hydrozoan planula contains a population of mi- gratory undifferentiated cells: interstitial cells. Interstitial cells are capable of self-renewal, and can differentiate into either ganglionic nerve cells or nematocytes ( Martin and Thomas, 198 la, b; Martin, 1988a). Interstitial cells arise in the endoderm. later migrate into and populate the ectoderm, and eventually differentiate into the two classes of cells (Martin and Archer, 1986). Do the inter- stitial cells ( 1 ) migrate as uncommitted cells and become committed by some sort of positional cues upon arrival in the ectoderm, or (2) are committed before they leave the endoderm, and migrate into the ectoderm to com- plete differentiation? The second alternative is correct for nematocytes, (Martin and Archer, 1986), and in this pa- per I show that it is also correct for neurons (i.e.. gangli- onic cells). This research describes a series of histological experi- ments designed to determine whether interstitial cells in a hydrozoan planula develop neuronal characteristics (ganglionic features) before arriving at their final destina- tion in the ectoderm. The numbers and locations of in- 10 NEURAL DIFFERENTIATION 11 terstitial cells and ganglionic cells in hydrozoan embryos of different ages were determined by light microscopy and transmission electron microscopy (TEM). The abil- ity of these embryos to bind a rabbit antiserum raised to the neuropeptide FMRFamide [such immunoreactivity has been demonstrated in planular sensory cells ( Martin, 1988b)] was tested to determine whether the antigen is expressed by ganglionic cells or interstitial cells differen- tiating along the ganglionic pathway. Anti-FMRFamide was used in this study because when it is applied to cni- darians, the peptides bound are likely to be related to pGlu-Gly-Arg-Phe-amide, which is present in large amounts in nervous systems of adult anthozoans and probably also in hydrozoans and scyphozoans (Graff and Grimmelikhuijzen, 1988). The planular results show that a subpopulation of interstitial cells in the anterior endoderm of 48 h planulae expresses morphological and cytochemical features of ganglionic cell differentiation. Thus, at least some interstitial cells for the neural differ- entiation pathway are committed in the endoderm and actually traverse the ganglionic pathway in the endo- derm. Materials and Methods Mature colonies of Pennaria tiarella were collected from pier pilings in Morehead City, North Carolina. Fronds from male and female colonies were placed to- gether in the dark at 6:00 pm. At 9:00 pm the bowls were returned to the light and, within an hour, early cleavage embryos were found in the bottoms of the dishes. Em- bryos were collected, placed in small finger bowls of fil- tered seawater, and reared at 23°C. Embryos of seven different ages: 8-, 10-, 16-, 24-, 48-, 72-, and 96-h, were prepared for transmission electron microscopy. Animals were fixed for 1 h in 2.5% glutaral- dehyde, pH 7.4, in 0.2 M phosphate buffer. They were subsequently postfixed for 1 h in 2% osmium tetroxide (pH 7.2, in 1.25% sodium bicarbonate), dehydrated in an ethanol series, infiltrated, and embedded in Spurr's embedding medium. Serial thick and thin sections were cut with a Porter-Blum MT-2B ultramicrotome. Thick sections were mounted on gelatin-coated slides, stained with 0.5%. toluidine blue in 1% sodium borate, and ex- amined with a Zeiss research microscope. Thin sections were placed on 1 50-mesh copper grids and stained with 3.5% uranyl acetate in ethanol followed by lead hydrox- ide. Grids were examined and photographed with a Hi- tachi H-600 transmission electron microscope. Wholemounts and paraffin sections of the selected em- bryonic stages were tested for their ability to bind a rabbit antiserum raised to FMRFamide (Immuno Nuclear Corporation). Twenty-four-hour planulae, treated for 2 h with 0.2% colchicine in seawater and subsequently al- lowed to recover for 24 h, were also exposed to the FMRFamide antiserum. Such colchicine treatment eliminates the entire interstitial cell system i.e.. intersti- tial cells, nematoblasts, nematocytes, and ganglionic cells (Martin and Thomas, 1981b). To visualize the binding of FMRFamide antiserum on wholemounts, the procedure presented by Martin ( 1988b) was followed with some modifications. Animals were fixed for 1 h in 10% formalin in seawater and subse- quently washed 3 times, for 15mineach, in 10mA/ phos- phate-buffered saline (PBS, pH 7.2). Incubation with the FMRFamide antiserum was for 1-4 h, with the primary antibody diluted 1:200 with 10 mM PBS, pH 7.2, con- taining 0.1% sodium azide, 0.3% Triton X-100, and 2% fetal calf serum. Incubations were carried out with plan- ulae in lid-covered 96 well tissue culture plates placed on a rotating shaker platform set at 60 rpm. At the end of the first incubation period, the primary antibody was re- moved, and animals were washed 3 times, for 15 min each, in 10 mM PBS, pH 7.2. Incubation with the second antibody was for 1 h in fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobin (Boehringer Mannheim). The FITC-tagged antibody was diluted 1:120 in 10 mM PBS, pH 7.2, containing 0.1% sodium azide, 0.3% Triton X-100, and 10% fetal calf se- rum. The second incubations were also done in 96 well plates rotated at 60 rpm. After the second incubation, animals were washed for three 15-minute changes in 10 mM PBS, pH 7.2. Wholemounts were examined for fluorescently labeled cells with a Zeiss microscope equipped with epi fluorescence. To visualize binding of FMRFamide antiserum to par- affin sections of embryos, samples fixed in formalin were dehydrated through an alcohol series, infiltrated and em- bedded in paraffin, and serially sectioned at 8 nm. Nine sections were mounted in the center of a glass slide, three rows one above the other, and each row containing three sections. Slides were rehydrated to distilled water, and the sections were surrounded by an outer ring of vacuum grease. Grease application was done in a moist chamber to prevent the sections from drying. FMRFamide antise- rum was placed in the grease-created wells, thus immers- ing the sections. The slides were placed in a covered moist chamber and rotated at 20-40 rpm for 1-4 h. PBS rinses and incubation in the second antibody were car- ried out in the moist chamber. After incubation, the grease was removed from the slides; the sections were covered with mineral oil and examined for fluorescently labeled cells. Binding specificity of the FMRFamide antiserum was determined by preincubating a 1 :200 dilution of the anti- serum with 10 Mg/ml synthetic FMRFamide (Peninsula Lab) for 24 h at 4°C before using it to stain the embryos. 12 V. J. MARTIN Results Mature planula (72-96 h postfertilization) The mature planula consists of an ectoderm, an acellu- lar mesoglea, and an endoderm (Fig. 1). The ectoderm contains epithelial cells (epitheliomuscular, glandular, and sensory), interstitial cells, and their derivatives (nematoblasts, nematocytes. and ganglionic cells), whereas, the endoderm has gastrodermal epithelial cells, interstitial cells, and nematoblasts. Interstitial cells, nem- atoblasts, nematocytes, and ganglionic cells are easily identified in planular tissue at the light microscopic level (Figs. 1-3). Interstitial cells — small round cells measur- ing 7.5 jiin in diameter — contain a centrally located nu- cleus with one to several nucleoli. They possess few cy- toplasmic organelles and are scattered among the epithe- lial cells in both the ectoderm and the endoderm along the entire anterior-posterior axis of the planula (Fig. 2). Nematoblasts (developing nematocytes) range from 10 to 12.5 /urn in diameter and have distinctive dark- or light-staining capsules (Figs. 1, 2). Each capsule houses a nematocyst thread that may possess barbs and spines. Nematoblasts are located in both the ectoderm and the endoderm and are mostly confined to the anterior and middle two-thirds of the planular axis. Mature nemato- cytes are found only at the ectodermal surfaces of planu- lae and exhibit the same distribution pattern as that of the nematoblasts. Ganglionic cells are 5 //m in diameter, exhibit a spindle shape, and are positioned all along the planular anterior-posterior axis at the base of the ecto- derm just above the mesoglea (Fig. 3). The ganglionic perikaryon, its long axis oriented parallel to the meso- glea, contains a Golgi complex, microtubules, mitochon- dria, electron-dense droplets, and dense cored vesicles. Neurites project from either side of the cell bodies and form an extensive ectodermal neural plexus above the mesoglea (Figs. 3, 4). Such neurites are filled with micro- tubules, mitochondria, electron-dense droplets, and dense cored vesicles ( Fig. 4 ). Electron-dense droplets and dense cored vesicles are found exclusively in differenti- ating and full-differentiated nerves. The endoderm lacks ganglionic cells and a neural plexus. Interstitial cells and nematoblasts are migratory, whereas, nematocytes and ganglionic cells are not (Mar- tin and Archer, 1986). These migratory cells move as sin- gle cells, and migration has been observed from the endo- derm to the ectoderm and, once in the ectoderm, up and down the planular axis. FMRFamide-like immunoreactivity is detected in a subpopulation of ganglionic cells in the planular ecto- derm at 72 h postfertilization, just before metamorphosis (Fig. 5). Such immunopositive nerve cells are located at the base of the ectoderm above the mesoglea and are con- fined to the anterior head and anterior sides of the plan- Figure 1 . Longitudinal section of a 72 h planula showing ectoderm ( EC), mesoglea ( M ) and endoderm ( E ). Endodermal nematoblasts (sin- gle arrow) and interstitial cells (double arrows) and an ectodermal gan- glionic cell (triple arrows) are visible. X250. Figure 2. Endodermal interstitial cells (arrows) in a 72 h planula. Each cell contains a large nucleus with a prominent nucleolus and few other cytoplasmic organelles. x620. Figure 3. Ectodermal ganglionic cell (arrow) in a 72 h planula. The cell body is oriented parallel to the mesoglea and neurites (double ar- rows) extend from each side of the perikaryon. x620. NEURAL DIFFERENTIATION 13 Figure 4. Ectodermal neurites of ganglionic cells. These neurites form a plexus just above the mesoglea and are rich in microtubules, mitochondria, electron-dense droplets (single arrows) and dense cored vesi- cles (double arrows), xl 9.000. ula. Cell bodies of the immunopositive ganglionic cells are stained, whereas their neurites (processes) are not. Nematoblasts and nematocytes do not produce the Figure 5. Wholemount of a 72 h planula. A subpopulation of gan- glionic cells (arrow) in the ectoderm of the planula exhibits FMRF- amide-like immunoreactivity. Such ectodermal ganglionic cells are confined to the anterior head and anterior sides of the planula. X200. FMRFamide-like peptide, as indicated by their lack of staining. Furthermore, the majority of planular intersti- tial cells do not stain with the antibody. There is, how- ever, a small subset of interstitial cells in the anterior en- doderm of 72-h planulae that does express a FMRFami- de-like peptide. Such positive cells first produce the neuropeptide at 48 h in development and are described below (see Forty-eight hour planula). Gastrulating embryo (8-10 h postfertilization) Embryos gastrulate between 8-10 h postfertilization resulting in the formation of an immature. 10-h planula. This young planula consists of an ectoderm, an acellular mesoglea, and an endoderm (Figs. 6, 7). The ectoderm contains epithelial cells (dark-staining epitheliomuscular cells and light-staining glandular cells) and is devoid of interstitial cells, nematoblasts, nematocytes, and gangli- onic cells. The endoderm consists of an outer epithelial layer of gastrodermal cells surrounding a central core of tightly packed interstitial cells (Figs. 6, 7). This core of interstitial cells extends the entire length of the planular 14 V. J. MARTIN Figure 6. Cross section of a 10-h planula. The embryo consists ot'an ectoderm (EC), an acellular meso- glea (M), and an endoderm (E). The endoderm is composed of an outer columnar epithelial layer (G) surrounding a central core of lightly staining interstitial cells (arrows). The ectoderm contains epithelio- muscular cells (dark-staining cells) and glandular cells (light-staining cells), but is devoid of interstitial cells, nematoblasts, nematocytes, and ganglionic cells. X320. Figure 7. Endodermal region of a 10-h planula. Clusters of interstitial cells (arrows) occupy the central endoderm. EC, ectoderm; G, epithelial layer of endoderm: M, mesoglea. • 320. anterior-posterior axis. These lightly staining, oval- shaped interstitial cells possess a large, centrally located nucleus with one to several nucleoli. Dark-staining gran- ules occupy the cytoplasm of these young interstitial cells, however, such granules disappear as the cells ma- ture. Interstitial cells of late planulae possess few granules (see Fig. 2). Interstitial cells traverse the nematocyte differentia- tion pathway in the endoderm (see Figs. 1, 2, 8). Such cells are distinguished by the appearance of either a dark- or light-staining nematocyst capsule. The capsule en- larges to an extent that it displaces the nucleus to one side of the cell. A few endodermal nematoblasts, confined to the anterior and middle two-thirds of the endoderm. have been observed in the 10-h planula. Interstitial cells traversing the neural differentiation pathway have not been seen in the immature planula. Interstitial cells and nematoblasts emigrate as single cells from the endoderm to the ectoderm. Interstitial cells migrate out from all locations along the planular endo- dermal axis, whereas outward nematoblast migration is confined to the anterior and middle endodermal regions. Interstitial cells and nematoblasts first appear in the planular ectoderm at 14 h postfertilization (Martin and Archer, 1986). Their ectodermal distribution corre- sponds to their above-mentioned migration patterns. Immature planulae (10 h) do not express a FMRF- amide-like antigen as indicated by their lack of staining. Yininxphimtla (24 h postfertilization) By 24 h, the planular ectoderm contains epithelial cells (epitheliomuscular, glandular, and sensory), interstitial cells, nematoblasts, a few nematocytes, and ganglionic cells; the endoderm has gastrodermal epithelial cells, in- terstitial cells, and nematoblasts. Both interstitial cells NEURAL DIFFERENTIATION 15 Figure 8. Longitudinal section of a 24-h planula. Differentiating nematohlasts (single arrows) are visible in both the endoderm (E) and the ectoderm (EC). A young ganglionic cell (double arrows) is seen at the base of the ectoderm above the mesoglea (M). Its neurites are not yet fully formed. Triple arrows, endodermal interstitial cells. X250. Figure 9. Ectodermal ganglionic cell (arrow) in a 24 h planula. Note its spindle shape and extending neurites. X620. and ganglionic cells occupy the entire anterior-posterior axis of the planula, whereas, nematoblasts and nemato- cytes are confined to the anterior and middle regions of the animal (Figs. 8, 9). In the ectoderm, ganglionic cells and nematoblasts are positioned in close proximity to the mesoglea, and interstitial cells are located slightly above these cells (i.e., toward the outer ectodermal sur- face). In the endoderm, interstitial cells and nemato- blasts may be found in the central core or out closer to the mesoglea. As planulae mature (24-96 h) the numbers of ectodermal and endodermal interstitial cells, ectoder- mal and endodermal nematoblasts, ectodermal nemato- cytes, and ectodermal ganglionic cells increase. At 24 h, the nervous system begins to form (Martin, 1988a, b). This neural system is entirely ectodermal and consists of ganglionic cells (interstitial cell derivatives) and sensory cells (epithelial derivatives). Ganglionic cells form a neural plexus composed of cell bodies and their neurites; this plexus extends the entire length of the plan- ula and is located just above the mesoglea (Fig. 9). These ganglionic cells have originated from interstitial cells that have migrated from the endoderm to the base of the ecto- derm. Once in this ectodermal position, they elaborated morphological features characteristic of ganglionic cell differentiation. Interstitial cells traversing the ganglionic pathway in the endoderm have not been observed at this stage. Sensory cells first arise in the anterior end of the planula (later in development they appear all along the length of the planula) and extend from the free surface of the planula to the ganglionic plexus where they insert neurites into the plexus (Martin, 1988b). Twenty-four hour ganglionic cells do not produce a FMRFamide-like peptide, however, the sensory cells do (Martin, 1988b). The FMRFamide-like peptide is first observed in the apices of sensory cells and only later in their mid to basal regions. FMRFamide-like positive sen- sory cells are observed throughout the remaining larval period (Figs. 10, 11). Interstitial cells and nematoblasts lack immunostaining at this stage, as does the entire en- doderm. Forty-eight hour planula The distribution of interstitial cells and their progeny in the 48-h planula is similar to that observed in the 24- h planula. By 48 h, the numbers of these cells have dra- matically increased in both germ layers. At 48 h, a subpopulation of interstitial cells in the ante- rior endoderm begins to express a FMRFamide-like anti- gen(Figs. 10, 1 1). These positive-staining interstitial cells are found exclusively in the anterior-most endodermal region of the planula and are present in the central endo- dermal core and in the outer endodermal periphery (Figs. 10, 11). Depending upon the plane of section, the FMRFamide-positive interstitial cells exhibit either a mesenchymal shape or an oval morphology. Interstitial cells located in the mid to posterior endodermal regions do not express the FMRFamide-like peptide, as indi- cated by an absence of staining (Fig. 12). Just after the endodermal appearance of these FMRFamide-like posi- tive interstitial cells, a few immunopositive ganglionic cells are detected in the ectoderm above the mesoglea confined to the anterior head and anterior sides of the planula. Their cell bodies are stained whereas their neu- rites are not. This is a full day after ganglionic cells first appear in the planular ectoderm. Nematoblasts and nematocytes do not stain for the FMRFamide-like pep- tide at this stage. Between 48 and 72 h, the number of immunopositive endodermal interstitial cells and immunopositive ecto- dermal ganglionic cells increase. Their distribution is limited to the anterior end of the planula. 16 V. J. MARTIN Figure 10. Longitudinal paraffin section of a 48-h planula. A suhpopulation of interstitial cells (arrows) in the anterior endoderm of the planula begins to express a FMRFamide-like antigen at this stage in devel- opment. A. anterior; E. endoderm; EC, ectoderm; M, mesoglea; P, posterior. • 250. Figure II. Longitudinal paraffin section of a 48-h planula. This section is taken from a deeper region of the same planula shown in Figure 10. A subset of interstitial cells in the anterior endoderm expressing a FMRFamide-like antigen is visible, as are FMRFamide-positive sensory cells (double arrows). A. anterior; E, endoderm; EC. ectoderm; M. mesoglea; P, posterior. >250. NEURAL DIFFERENTIATION 17 Ultrastructural examination of interstitial cells in the anterior endoderm of 48-h planulae indicates that, based upon morphology, at least three subsets of interstitial cells are found in this region: undifferentiated interstitial cells, interstitial cells traversing the nematocyte differen- tiation pathway (nematoblasts), and interstitial cells traversing the ganglionic differentiation pathway (neuro- blasts) (Figs. 14-19). All three subpopulations can be found in the central endodermal core and at the periph- ery of the endoderm. These three subsets are also founc1 in older planulae (72 h) in the same endodermal posi tion. Undifferentiated interstitial cells are characterized by a centrally located nucleus with a nucleolus, and a cytoplasm containing free ribosomes, a few mitochon- dria, and a few segments of rough endoplasmic reticulum (Fig. 14). These interstitial cells, as of yet, show no spe- cific organelles indicative of a particular differentiation pathway. Interstitial cells committed to the nematocyte differentiation pathway have a cytoplasm rich in rough endoplasmic reticulum and form a distinctive nemato- cyst capsule (Fig. 15). This capsule is in close proximity to the nucleus and often displaces it to one side of the cell. Interstitial cells undergoing neural differentiation (ganglionic cell pathway) form a Golgi complex, elec- tron-dense droplets, dense cored vesicles, and microtu- bules (Figs. 16-19). These electron-dense droplets and dense cored vesicles occupy the cell bodies of the devel- oping endodermal ganglionic cells and are morphologi- cally identical to the droplets and vesicles found in the ectodermal ganglionic cell bodies and neurites (see Figs. 4, 16, 17, 18, and 19). These developing endodermal neuro- blasts do not form neurites in the endoderm, as neurites have only been observed in the ectoderm of the planula. Colchicine-treated embryos Embryos treated with colchicine and subsequently al- lowed to recover for one to two days lack all interstitial cells and their differentiated progeny (ganglionic cells, nemato- blasts, neuroblasts, and nematocytes) (Martin and Thomas, 1 98 1 a). When such epithelial planulae are exposed to FMR- Famide antiserum, they show no immunostaining (Fig. 13). There are no immunopositive interstitial cells, immunoposi- tive neuroblasts, or immunopositive ganglionic cells. Discussion Research presented here, as well as past work (Martin and Archer, 1986), indicates that at least some larval in- terstitial cells are committed within the endoderm to the differentiation of either nerve cells or nematocytes. These restricted cells enter a differentiation pathway in the endoderm, and most probably migrate as nemato- blasts or neuroblasts to a position in the ectoderm where differentiation is completed. This process probably ac- counts for the spatial distribution of the interstitial cell system in the larval ectoderm. With regard to ganglionic cell formation, this study demonstrates a subpopulation of anterior endodermal interstitial cells that shows early signs of neural cyto- chemical differentiation by expressing a FMRFamide- like antigen. Concurrent with the appearance of these immunopositive interstitial cells, TEM indicates that a subset of interstitial cells in the same anterior endoder- mal region develops morphological features indicative of neural differentiation: formation of a Golgi complex, electron-dense droplets, dense cored vesicles, and micro- tubules. This subset of interstitial cells probably includes the FMRFamide-positive interstitial cells. Furthermore, a subset of FMRFamide-positive gangli- onic cells appears in the anterior ectoderm between 48- 72 h. Because this occurs just after the endodermal ap- pearance of the immunopositive interstitial cells, and be- cause both populations are confined to the same anterior head region, the immunopositive interstitial cells have probably migrated to the base of the ectoderm where they differentiated into ganglionic cells. Alternatively, the interstitial cells traversing the neural pathway in the endoderm might never migrate to the ectoderm but sim- ply remain and complete their differentiation, or die, in the endoderm. The alternative is unlikely because the planular endoderm lacks fully differentiated ganglionic cells [i.e.. they do not form neurites in the endoderm; neurite formation constitutes the last step in ganglionic cell differentiation (Martin, 1988a)], and TEM studies reveal no signs of degenerating cells in the endoderm at any stage of planular development. The movements of interstitial cells, nematoblasts, and neuroblasts in planulae appear to be coordinated, as evi- denced by their final placement within the ectoderm. In- terstitial cells, which divide and possibly remain as stem cells, migrate out from all regions of the endoderm and distribute themselves along the whole planular axis in the ectoderm. Developing nematoblasts emigrate from the endoderm in a specific region of the planula (anterior to mid endoderm) and concentrate in an ectodermal area extending from the anterior end of the planula to the Figure 12. Paraffin section of the posterior (P) region of a 48 h planula. The posterior endoderm (E) is devoid of FMRFamide-positive interstitial cells. X250. Figure 13. Paraffin section of a mature "recovered" colchicine-treated planula. Such epithelial planu- lae lack FMRFamide-like activity as indicated by the absence of staining. X250. 18 V J. MARTIN WP'i •-» NEURAL DIFFERENTIATION 19 Figure 18. Golgi region of a "neural" endodermal interstitial cell in a 48 h planula. Several mitochon- dria and microtubules are seen in close proximity to the Golgi. x37,400. Figure 19. Electron-dense droplets (arrows) in the Golgi region of a developing endodermal ganglionic cell. Such droplets are characteristic of neural differentiation. X20.400. mid-region of the planula. Interstitial cells destined to form ganglionic cells migrate out from all regions of the central endoderm and are evenly distributed along the planular anterior-posterior axis in the ectoderm. Since the interstitial cells and their progeny exhibit a rather precise positioning within the ectoderm, some mecha- nism of directed migration may be operating in the plan- ula. The FMRFamide findings support the notion of di- rected migration. FMRFamide-positive endodermal in- terstitial cells and FMRFamide-positive ectodermal Figure 14. Endodermal interstitial cell in the anterior region of a 48-h planula. This undifferentiated cell contains a centrally located nucleus (N), a few segments of rough endoplasmic reticulum (arrow), a few mitochondria, and numerous free ribosomes. Although not visible in this plane of section, the intersti- tial cell also contains a prominent nucleolus. This interstitial cell has migrated from its site of origin in the central endoderm to the outer endoderm (E) and is in close proximity to the mesoglea (M). x 14.400. Figure 15. Developing nematoblast in theanteriorendodermofa48-h planula. Interstitial cells travers- ing the nematocyte differentiation pathway are characterized by the appearance of large amounts of rough endoplasmic reticulum (arrow) and by the formation of a nematocyst capsule (C). Such cells eventually emigrate to the ectoderm. N, nucleus. X 10,800. Figure 16. Interstitial cell traversing the neural differentiation pathway in the anterior endoderm of a 48-h planula. Such interstitial cells committed to the ganglionic pathway form small electron-dense drop- lets (arrows) and dense cored vesicles (see Fig. 17), develop a Golgi (see Fig. 18), and accumulate microtu- bules in their cytoplasm (see Fig. 18). These cells do not develop neurites in the endoderm. N, nucleus. X9.600. Figure 17. Developing ganglionic cell in the endoderm of a 48-h planula. The cytoplasm of the differ- entiating interstitial cell becomes filled with electron-dense droplets (single arrow) and dense cored vesicles (double arrows). Similar droplets and vesicles are abundant in the cell bodies and the neurites of ectodermal ganglionic cells (see Fig. 4). N. nucleus. X2 1,600. 20 V. J. MARTIN ganglionic cells are confined to the same anterior head region of the late planula. As stated previously these posi- tive interstitial cells probably emigrated to the ectoderm and formed the positive ganglionic cells. The fact that the FMRFamide-positi ve ganglionic cells are confined to a specific region in the ectoderm and not distributed at random suggests directed migration. Acknowledgments This research was supported by National Science Foundation Grants DCB-8702212, Career Advance- ment Award DCB-871 1245, and DCB-8942149. Literature Cited Graff, D., and C. J. P. Grimmelikhuij/en. 1988. Isolation of 76** 23 ± 4 33 ± 4 42 ± 4 22 ± 2 47 ± 12* 53 ± 10* 1 Number of cases/total number within group. : Mean number ot days ± 1 S.D. *P<0.01;**P<0.001. ated. Total irradiation was either 2 kRad (whole-body and limb) or 2.2 kRad (whole body only). Irradiation was provided using a Picker-Gemini 320 kV industrial x-ray unit equipped with an aluminum filter. Output intensi- ties of 1 25 or 1 60 kV were employed for 17.5 min to yield 2 and 2.2 kRad irradiation, respectively. Animals were positioned 1 5 cm from the source and radiation was ad- ministered dorsally. Shielding was provided by a 6 mm thickness of lead plate. Skin grafts Subsequently, one group of nontreated newts and two groups of newts receiving whole-body irradiation re- ceived skin allografts. These animals were used to assess effects of irradiation in cellular immunity. Reciprocal al- lografts consisted of small pieces of skin, approximately 2 mnr, implanted into wound sites created by removal of skin used as grafts for other animals. Thus each newt served as both a donor and a recipient. In addition, a small group of control and irradiated animals received autografts. Amputations The remaining control and irradiated animals were used to evaluate effects on regeneration. All regeneration groups were subjected to unilateral forelimb amputation through the distal stylopodium. In all instances, any por- tions of humerus extending beyond the wound surface were carefully trimmed. Results and Discussion Effects on regeneration Regeneration occurred among all control animals and progressed to the early digital stage in approximately 42 days (Table I). Whole-body irradiation suppressed regen- eration of shielded limbs in 3 of 10 newts and signifi- cantly (P < 0.01 ) slowed the rate of regeneration among REGENERATION AND GRAFT REJECTION 23 Table II Effect of irradiation (ingraft Treatment Retained skin graft' Interrupted circulation2 Loss of pigment Rejection Autografts: None 3/3 >76 — — 2kRad 3/3 >76 — — Allografts: None 3/14 12± 1 18±8 27+7 2 kRad 1/12 18±6* 24 ±6 33 ± 10 >2 kRad 0/10 19 ± 7* 30 ±6* 36 + 6* 1 Number of cases/total number within group. : Mean number of days ± I S.D. *F<0.05. the remainder (Table I). Thus, there was an adverse effect on forelimb regeneration of whole-body irradiation. In contrast to the results of the shielded-limb group, irradiation of limbs caused total suppression of regenera- tion in all cases. This is in complete accord with observa- tions from other laboratories (reviewed in Wallace, 1981). Moreover, irradiation of the limbs led to severe and persistent inflammation and ultimately to resorp- tion of the limbs in 85% of the cases. These events ap- peared strikingly reminiscent of those described by Schotte and Butler (1941) in larval Ambystoma follow- ing limb denervation and by Butler (1933) in larval Am- bystoma following irradiation. These investigators as- cribed the resorption phenomenon to a failure of de- differentiation to stop and the progressive stages of regeneration to commence. They inferred that apparent resorption of the stump occurred because of an inability to retain an appendage of dedifferentiated tissues. How- ever, the aggravated initial inflammation followed by loss of pigmentation, and subsequent resorption of soft tissues observed in this study resembled more the rejec- tion of a foreign graft (Cohen, 1966). The extent to which this similarity can be pursued and its implications are the objects of additional studies. Effects on allograft rejection Skin autografts were tolerated by those few control and irradiated animals used for that purpose (Table II). In addition, rejection of skin allografts occurred in 1 1 of 14 controls and 21 of 22 irradiated animals (Table II). However, the rate of rejection appeared to be sensitive to irradiation. In particular, irradiation above 2 kRad sig- nificantly delayed (P < 0.05), but did not suppress, allo- graft rejection. These results suggest that irradiation affected the immunological status of the newts; however, at the dosages used, this effect did not cripple the newts' immune system. Demonstration of delays in the rate of regeneration of shielded limbs of otherwise whole-body irradiated ani- mals suggest that one or more factors outside of the am- putation site had been adversely affected by irradiation. To this observation, the resorption of amputated irradi- ated limbs presents an intriguing counterpoint. Further- more, the persistence of inflammation and subsequent erosion (resorption) of the stump seems reminiscent of graft rejection. Therefore, it is tempting to suggest that the factors extrinsic to the limb that were affected by irra- diation are associated with the immune system. In fact, the occurrence of both of these manifestations is consis- tent with interactions between the immune system and the amputated limb. Speculations Similarities between these data and previous observa- tions of limb resorption following irradiation (Butler, 1 933) or limb denervation (Schotte and Butler, 1 94 1 ) in larval Ambystoma prompt the speculation: Following amputation, dedifferentiation of local tissues occurs. If dedifferentiated cells are stabilized and activated (e.g., by neurotrophic factors), they modulate immunological ex- pression favoring blastema formation and possibly con- tributing to promoting blastemal growth. However, if stabilization and activation does not occur, presumptive blastemal cells are eliminated by activated immunologi- cal defenses (Prehn, 1970; Coleman el al. 1989). More- over, the absence of a pool of accumulating blastema cells (e.g.. following irradiation) might lead to limb re- sorption in a futile attempt to establish such a pool. Conclusions The design of this investigation does not enable the means through which x-irradiation induced the particu- lar effects observed to be known with certainty; conse- quently, alternative interpretations of our observations might be equally tenable. Nevertheless, the results of this investigation demonstrate that regeneration of shielded forelimbs by newts otherwise receiving whole-body irra- diation occurs in animals that are still immunocompe- tent, at least in terms of allograft rejection. In addition, these data suggest that x-irradiation can affect the expres- sion of epimorphic regeneration through central, as well as local, effects. Furthermore, this impairment of regen- eration appears to occur in parallel to retardation of the rate of allograft rejection. Consequently, a relationship between immunological expression and epimorphic re- generation is suggested. Moreover, these results remove the potentially devastating challenge to this hypothesis presented by earlier investigations of limb regeneration in which x-irradiation was used. 24 R. E. SICARD AND M. F. LOMBARD Acknowledgments The technical assistance of Kara Anderson, Katherine Costello, and Laurie Peluso is gratefully acknowledged. Literature Cited Brunst, V. V., and E. A. Cheremetieva. 1936. Sur la perte locale du pouvoir regenerateur chez le triton et 1'axolotl causee par 1'rradia- tion avec les rayons x. Arch. Zool. Expil. Gen. 78: 57-67. Butler, E. G. 1933. The effects of x-radiation on the regeneration of the forelimb of Amhlysloma larvae. ./ Exp /no/. 65: 27 1-316. Butler, E. G. 1935. Studies on limb regeneration in x-rayed Amhly- stoina\arvae.Amil. Rec 62: 295-307. Butler, E. G., and J. P. O'Brien. 1942. Effects oflocalized x-radiation on regeneration of the urodele limb. Anal Rec 84: 407-4 1 3. Cohen, N. 1966. Tissue transplantation immunity in the adult newt, Diemtctvliis viruleseen.s II. The rejection phase: first- and second- set allograft rejections and lack of sexual dimorphism. J Exp. /.mil 163: 173-190. Coleman, R., M. Lombard, H. Sicard, and N. Rencricca. 1989. Fundamental Imiiiitnulnuy. Unit 8: Cancer and Transplantation. Wm. C. Brown Publishers. Dubuque. IA. Pp. 427-504. Prehn, R. T. 1970. Immunosurveillance, regeneration, and oncogen- esis. Progr. Exp. Tumor Res 14: 1-24. Prehn, R. T. 1972. The immune reaction as a stimulator of tumor growth. Science 176: 1 70- 171. Prehn, R. T., and M. Lappe. 1971. An immunostimulation theory of tumor development. Transplant. Rev 7: 26-54. Schotte, O. E., and E. G. Butler. 1941. Morphological effects of de- nervation and amputation of limbs in urodele larvae. J. Exp. Zool. 87: 279-322. Schotte, O. E., and R. E. Sicard. 1982. Cyclophosphamide-induced leukopenia and suppression of limb regeneration in the adult newt, Notophlhalniits viruiescens. J. Exp. Zool. 222: 199-202. Sicard, R. E. 1981. The effects of putative immunological manipula- tions upon the rate oflimb regeneration in adult newts, Notophthal- niii.s viriilexcens. IRCSMed. Sci.i. 9: 692-693. Sicard, R. E., and W. T. Laffond. 1983. Putative immunological in- fluence upon amphibian forelimb regeneration. I. Effects of several immunoactive agents on regeneration rate and gross morphology. Exp. Cell Bin/ 51: 337-344. Sicard, R. E., and M. F. Lombard. 1989. Epimorphic regeneration and the immune system. Pp. 107-1 19 in Reccnl Trends in Regener- ation Research. V. Kiortsis. S. Koussoulakos, and H. Wallace, eds. Plenum Publ. Corp.. New York. Wallace, II. 1981. Vertebrate Limb Regeneration Wiley and Sons, Chichester. Reference: Biol. Hull. 178: 25-32. (February, 1 WO) Correlation of Abnormal Radular Secretion with Tissue Degrowth During Stress Periods in Helisoma trivolvis (Pulmonata, Basommatophora) DAVID A. SMITH1 AND W. D. RUSSELL-HUNTER23 1 \Vabash College, Department of Biology, Crawfordsville, Indiana 47933: 2 Syracuse University, Department of Biology, Syracuse, New York 13244-1270: and* Marine Biological Laboratory, Woods Hole, Massachusetts 02543 Abstract. Laboratory experiments on starvation stress in Helisoma trivolvis elucidate a relationship between modifications of radular secretion and tissue degrowth resulting from stress. Tissue losses in starved adults ranged from 4.5% at 40 days to 27.7% at 160 days, with negligible mortality (<2%). Modifications in radular se- cretion that paralleled tissue loss involved not only ab- normal secretion of individual teeth and of tooth rows, but especially an increased "packing" of radular rows per unit ribbon length. Radular length remained constant during experimental trials, however the mean number of tooth rows increased by almost 47% after 120 days of food deprivation. Radular patterns reflecting degrowth observed in these experiments were paralleled in radulae taken from overwintered animals sampled from natural populations. Rates of radular turnover averaged between 2.3% new growth per day (43 days to turnover) and 4.0% new growth per day (25 days to turnover). Radular sam- ples could provide for post hoc detection of recent peri- ods of tissue degrowth in snails, just as evidence of longer periods of tissue degrowth can be detected in the shells of long-lived bivalves. Introduction Natural populations of aquatic molluscs can experi- ence tissue loss during winter. This phenomenon in- volves complex shifts in metabolism and has been called "degrowth" (Russell-Hunter, 1985). Previous reports have demonstrated that physiological stress, both short- term and of longer duration, can temporarily affect radu- lar secretion (Isarankura and Runham, 1968; Kerth, Received 6 July 1 989; accepted 14 November 1989. 1971; Fujioka, 1985; Smith, 1987). The laboratory ex- periments reported here were designed to clarify the rela- tionship between abnormal radular secretion and con- current tissue degrowth resulting from starvation stress. Applied to field populations, these observations could provide an independent (short-term) method for detect- ing periods of starvation that had occurred shortly before sampling. This would complement the long-term detec- tion of stress-induced degrowth based on "oversized" shellsfRussell-Hunterrffl/., 1984), or on modified catab- olism (Russell-Hunter el al. 1983). The Ramshorn snail of eastern North America, Helisoma trivolvis (Say, 1817), was particularly suitable for these experiments be- cause it performs well in laboratory culture, and because recent studies have documented not only the biometry and mechanics of its radula (Smith, 1987, 1988, 1989), but also its actuarial bioenergetics, including its capacity for degrowth (Russell-Hunter and Eversole, 1976; Rus- sell-Hunter et al, 1983, 1984). Detailed analysis of radula-tooth biometry in Heli- soma has shown significant levels of interpopulation variation in this species (Smith, 1987, 1989). As with similar studies on Lymnaeid pulmonates by Berrie (1959) and Hunter (1975), in Helisoma there are no ob- servable ecophenotypic effects on tooth shape. Despite this constancy (within individuals, and within populations) more general aspects of radular secretion, including the number and density of tooth rows, can be modified by envi- ronmental stress. Short exposures to near-freezing tempera- tures will produce a zone of modified tooth rows on the rad- ula ribbon (Isarankura and Runham, 1968; Kerth, 1971; Fujioka, 1985; Smith, 1987), and longer exposures to the stress of starvation will produce "bunching" or "packing" of radular rows, as described below. 25 26 D. A. SMITH AND W. D. RUSSELL-HUNTER Work on the bioenergetics of tissue degrowth in Heli- sorna is also cognate to these experiments. Held in the laboratory in a metabolic framework simulating that of natural overwintering, a representative cohort of Hcli- soma showed a 50% loss of tissue biomass (involving per- haps 20% loss of protein) with only 10%> mortality over 132 days (Russell-Hunter and Eversole, 1976). Meta- bolic shifts during the degrowth process were studied by Russell-Hunter el al. ( 1983) using nearly concurrent as- sessments of oxygen consumption and of nitrogenous excretion. There was a clearly controlled differential ca- tabolism of protein resources during degrowth. One of the first quantitative reports of direct field evidence for tissue degrowth during winter is for natural populations ofHelisoma trivolvis and ofLymnaea palustris in central New York state (Russell-Hunter ct al.. 1 984). The existence of all these recent reports not only made stocks of Hclisoma appropriate for these experiments, but also made it likely that a history of recent degrowth could be detected by detailed examination of the radula. In field studies this might come to parallel evidence (Clark, 1976; Mallet et al.. 1987; Peterson el al.. 1985)of longer periods of degrowth and regrowth which can be detected in the shells of long-lived bivalves. Materials and Methods Hclisoma trivolvis is one of the more common gastro- pod molluscs of central New York state. This euryoecic, pulmonate snail is found primarily in eutrophic environ- ments including lakes, ponds, streams, farm ponds, and drainage ditches. Mature adults used for laboratory anal- ysis of tissue degrowth were taken from a small pond in Ithaca, New York (76°22.96'W, 42°25.78'N). To study the effects of overwintering on radula secretion, as well as the patterns of radular regrowth during early spring, snails were sampled in April and May, 1985, from four additional field sites (Eaton Reservoir, 75°42.27'W, 42°51.10TSf; Meadowbrook Pond, 76°07.08'W, 43°01.59'N; Otter Pond, 76°32.83'W, 43°09.52'N; and Silver Lake, Remsen, 75°08.19'W. 43°20.97'N). Methods used to quantify tissue degrowth were modi- fied from those of Russell-Hunter and Eversole (1976), and protocols for radula preparation are detailed by Smith (1987). To initiate an investigation of tissue de- growth, more than 400 adult snails were collected at the Ithaca field site in October 1985. This sample reflects the natural variation in a single generation of Helisoma as it moves into winter conditions. All shells were measured with dial calipers (±0. 1 mm) for maximum shell diame- ter (MD). On the basis of MD, individuals were then di- vided into three size classes: <14.0, 14.1-16.9, and >17.0 mm. Two individuals from each class were then cultured together in translucent plastic beverage cups (n = 6 per container) in approximately 400 ml of filtered pond water. At this time, groups, each containing six snails, were randomly designated (using Japanese icosa- hedral dice) as "fed" or "starved" experimentals or as baseline controls. Fed animals were provided fresh let- tuce for the duration of the experiment; starved animals were starved for 1 20 days and then fed lettuce for the last 40 days of the trial. The experiment was run in a B.O.D. chamber at 8°C. Cold fluorescent lights illuminated the cultures on a 14L/IOD cycle. Cups were cleaned, and provided with fresh, filtered, water each week. Samples were taken at 0, 40, 80, 120, and 160 days both for analysis of tissue degrowth and for radular prep- arations. Of 264 animals used in this study, 72 were des- ignated controls and 192 were experimentals. Of these 192, 144 were used for tissue analysis and 48 were used for estimating radular degrowth. At 40-day intervals, samples of 18 snails from each treatment were assessed for shell and tissue dry weight. Individuals were oven dried at 65°C, treated with an excess of 8.5%. HNO, ( 1 2% v/v nitric acid), washed, and then redried, giving two dry weights, whole snail and tissue, and, by subtraction, a value for dissolved calcium carbonate. Tissue degrowth was then calculated as the difference between actual tis- sue dry weight (TDW) and that predicted from initial tis- sue-to-shell regressions established at the start of the trial (TDWp). To determine the effects of food deprivation on radu- lar secretion, 6 specimens from each of the above treat- ments were sampled every 40 days. Individuals were sac- rificed in boiling water and removed from their shells. The buccal mass of each individual was removed, soft- ened in saturated KOH for 2-5 seconds, and transferred to distilled water. Radulae were then removed with fine forceps, placed onto clean glass slides, arranged, and cov- ered with coverglasses. Preparations were then held in distilled water for 24 hours, dehydrated in 70%> EtOH and air dried. New coverglasses and mounting fluid were then applied. Abnormal radular secretion was quantified by first dividing each radula into three equal sectors on the basis of overall length. The total number of tooth rows per sector was then counted. To study the natural patterns of return to normal rad- ular secretion following overwinter stress, adult Hcli- soma were collected in early spring at four field sites. Sampling continued until evidence of abnormal secre- tion (row-packing) was no longer present. Radular growth rates were calculated as length of new growth as a fraction of ribbon length. Methods used to study radular turnover in the labora- tory follow Isarankura and Runham (1968). Pond water was cooled to approximately PC. Snails were placed in this bath for 24 hours. Individuals were then returned to room temperature (18°C) and were provided fresh let- tuce. Individuals were sacrificed daily until regions of radular malformation were absent. RADULAR CORRELATES OF DEGROWTH 27 Table I Tissue tifgrowlli in Helisoma trivolvis A. 40d 80d I20d I60d Fed -1.9 ±1.63 -6.5 ±1.41 -8.7 ±1.50 -11.3+1.91 Unfed -2.2+1.54 -8.6+1.62 -13.1 + 1.69 -14.6+1.80 B. 40 d 80 d 12()d 160 d Fed* -3.5 + 2.58 -1I.O±2.46 -15.7 + 2.29 -19.0 ±3.05 Unfed** -4.5 ±2.57 -15.6 + 3.44 -23.8 ± 2.05 -27.7 + 2.83 * ANOVA F,.68 = 7.438, P < 0.00 1 ** ANOVA F3.6g = 1 3.878, P< 0.001 Data were subject to arcsin-square root transformation before analysis. A. Change in tissue dry weight in milligrams (as TDW-TDWp, n = 18) over 160 days. B. Change in tissue dry weight as a percentage of predicted tissue dry weight [((TDW-TDWp)/TDWp)- 100, n = 18], over 1 60 days. Results At the start of the laboratory trial 72 control individu- als had been sacrificed. For these, analysis showed that tissue dry weight (mg) related to shell dry weight (mg) as TDW = 0.254- SOW + 1.335 (r = 0.956, n = 72, P < 0.001). With each set of known values of SOW, this relationship was then used as a predictor of TDW. The deviation of predicted (TDWp) from expected TDW for each individual was used as an indicator of tissue growth or of tissue degrowth. These values for each of four sam- pling periods are set out in Table I. Two-hundred and sixty-four individuals began the trial; four died (<2%) and were replaced with parallel experimental animals. Tissue degrowth clearly occurred by 80 days in both sets of experimental animals, and this had nearly doubled by 160 days (Table I). Degrowth over 160 days of food de- privation ranged from 4.5% tissue loss at 40 days to 27.7% tissue loss at 1 60 days. These values correspond to 2.2 mg below predicted tissue dry weight and 14.6 mg below TDWp, respectively. Degrowth in animals belong- ing to the fed treatment ranged from 3.5% at 40 days to 19.0% at 160 days. Levels of tissue loss in fed and unfed treatments did not differ (P > 0.05) at 40 and at 80 days. At 120 and at 160 days, however, treatments did show significantly different mean levels of degrowth (ti:o days = 2.414, n = 36, P < 0.05; tlw) days = 2.098, n = 36, Tissue degrowth in the experimental snails was paral- leled by abnormal radular secretion (Fig. 1 ). This was manifest not only in malformations of individual radular rows (including smaller lateral, marginal, and rachidian teeth; irregular lateral, marginal, and rachidian teeth; and missing marginal teeth), but also, and most consis- tently, by an increased number of radular rows (packing) per unit ribbon length. Radular length remained con- stant during experimental trials, however the mean number of tooth rows increased by almost 47% after 1 20 days of food deprivation (Table Ha). Observations showed this increase was associated with the generative (posterior) end of the radular ribbon. Although secretory activity of the odontoblasts continued during the trial, secretion by the membranoblasts (which produces lengthening of the radula ribbon) proceeded at a much reduced relative rate (Fig. 2). [The precise mechanism of post-secretory radular transport remains uncertain. The topic has been reviewed by Runham (1963), Mischor and Miirkel (1984), and Mackenstedt and Markel ( 1987).] This differential activity of odontoblasts and of membranoblasts resulted in an increased density of tooth rows at the posterior end of the radula ribbon (Fig. 2). During the last 40 days of the trial (refeeding), radular transport was restored and the proper pattern of radular se- cretion was again established (Table lib). Once a normal pat- tern of secretion was established, the region between the tightly compressed rows (generated during stress) and the normally deposited rows (generated during refeeding) pro- vided a marker which could be used to quantify radular turnover rates either in experimental or in natural popula- tions. Patterns of abnormal radular secretion observed in the laboratory were paralleled in radulae taken from animals sampled from five field sites (Fig. 3). Weekly sampling in early spring allowed a unique opportunity to estimate radular growth and turnover rates under natural condi- tions. Values among five sites ranged between 3-4% new growth per day. This figure corresponds to approxi- mately 5-7 rows per day, and to 225-315 teeth per day. Radulae from Meadowbrook Pond showed the slowest turnover (2.3% growth/day, 43 days to turnover) while radulae from Ithaca turned over most rapidly (4.0%/day, 25 days to turnover). Radulae from the other three sites turned over in approximately 30 days (Eaton, 2.9%/day, 34 days turnover; Otter, 3.6%/day, 28 days turnover, Remsen, 3.5%/day, 29 days turnover). These rate data agree with laboratory trials, which showed ribbon turn- over in 30 days at room temperature. Field rates also agree with those determined by Isarankura and Runham (1968) who reported average radular production of ap- proximately 3.2 rows per day (minimum 0.5, maximum 7.5, average minimum 2.8, average maximum 4.2 rows/ day) for a variety of molluscs including Helix and Lym- naea. At one field site (Ithaca) the progress of return to normal radular secretion correlated with changes in tis- sue regrowth (as tissue/shell) (r = 0.937, n = 5, P < 0.05) indicating that radular growth is associated with in- creases in early spring tissue biomass. Discussion There is no doubt that, for Helisoma trivolvis at least, our experimental conditions closely match those of over- 28 D. A. SMITH AND W. D. RUSSELL-HUNTER Figure I. Untouched photographs to show radular malformations in Ilcli^onui A. Normal radula. B. Radula alter 7? days of food deprivation. In both A and B. the most recently secreted radular rows are at the top of each photograph, and the rows in use are near the bottom. C. Enlarged view of rachidian and lateral-tooth region from B. Note lateral tooth malformations and row packing. D. Enlarged view of margi- nal-tooth region from B. Note that marginal teeth are absent from the region of degrowlh. wintering in natural populations. Degrowth in three field populations measured by Russell-Hunter el al. (1984) showed average losses in tissue biomass of 24.7%, 28.3%, and 41.3%. The maximum loss of 27.7%. over 160 days in these experiments is appropriate. The results of the present investigation confirm that the physiological stress of starvation in Hclisnma results not only in tissue degrowlh but also in concurrent changes in radular secretion. The significance of this con- currence is twofold, involving first, possible insight into the fundamental control mechanisms of stress response in molluscs, and second, the possibility (for applied stud- ies) of post hoc detection, in natural populations, of ear- lier periods of starvation or similar stress. Before review- ing these two aspects, however, it is necessary to set out certain strengths and weaknesses in laboratory starvation experiments. In general, quantitative studies of any kind of stress on Table II Modification <>l nuluhir •iccrctmn in Helisoma tnvolvis A. Od 40 d 80 d 120d 16()d Length (mm)* 2.7 ± 0.04 2.9 + 0.16 2.7 ±0.17 2.7 ± 0.19 2 4± 0.29 Total rows** 138 + 1.9 169 ±9.4 192 ±9.8 202 ± 13.7 194 ±27.1 Rows/mm*** 52 ± 0.9 59 + 1.3 70 +2.0 77 + 4.1 82 ± 9.7 * ANOVA F4.20 = 0.707. P > 0.5. "ANOVA F4.;o = 3.072. P< 0.05. ***ANOVA F4 :il = 6 724./><0.01. B. Od 40 d 80 d 120d 160d Anterior 34.6 + 0.40 30.6 + 0.51 28.8 + 0.92 26.0 ± 1.48 31 4+ 3.23 Middle 32.2 ±0.37 29.2 + 0.97 27.6 ± 0.40 24.4 ± 1.17 32 8+ 4.89 Posterior 33.2 + 0.49 40.6 + 0.87 43.4+ 1.03 49.2 + 2.25 36 ,2± 2.82 Row number 138 ±1.9 169 ±9.4 192 ±9.8 202 + 13.7 194 ±27.1 A. Basic statistics for abnormal radular secretion in unfed snails over 160 days (n = 5). B. Sector analysis as percent per sector based on total number of rows (average total on last line). RADULAR CORRELATES OF DEGROWTH 29 100 90 35 - 25 80 70 60 50 - 15 o - 5 40 80 DAYS 120 160 Figure 2. Patterns of radular modification and tissue degrowth in laboratory stocks of Helisoma trimlnx. Main plot (A) shows correla- tion of tissue degrowth and abnormal radular secretion. Insert plot (B) shows pattern of radular packing (see text for further explanation). Ver- tical bars are 95% confidence limits ol each mean. animals in laboratory culture must not involve high rates of mortality. Our survivorship rate (>98%) in the experi- mental groups is clearly satisfactory. Evidence of de- growth in snails and in other shelled molluscs is based on the permanence of the calcareous shell as a record of previous tissue biomass. In a review of molluscan de- growth studies, Russell-Hunter (1985) emphasizes an important caveat, that ratio measurements of tissue-shell relationships and, hence, predicted values of tissue bio- mass should be obtained only from those species that de- monstrably show no shell resorption. Helisoma trivolvis has been well studied in this respect, and its shell does not change in mass or in composition (Russell-Hunter andEversole, 1976; Russell-Hunter et al., 1983, 1984). A more immediate difficulty is that, with experimental groups set up as in the present series, it is empirically impossible to provide polar trophic conditions. Under our experimental conditions, "fed" snails are not sati- ated, while "unfed" snails are not totally starved (micro- organisms are present in 7-day-old water and on shells). Our controls represent unstressed snails, the fed snails represent some nutritional stress, and the unfeds greater stress. In similar experiments, which assessed the control of differential catabolism (by measuring oxygen con- sumption and nitrogenous excretion) during degrowth (Russell-Hunter^ al., 1983), highly stressed snails estab- lished an effective regime of metabolic compensation (by reducing the proportion of protein catabolism) more rapidly than less stressed snails. Unlike shell mass, tissue biomass is not a static value (see Russell-Hunter and Buckley. 1983, for discussion of this in the actuarial bio- energetics of molluscan productivity). While any indi- vidual organism remains alive, its tissue biomass contin- ues to be in turnover. Thus, growth represents a positive- value (and degrowth a negative value) for a combined net rate that involves both inputs and outputs as rate functions (Russell-Hunter and Buckley, 1983; Russell- Hunter et al., 1983). Tissue degrowth in our experiments was paralleled by abnormal patterns of radular secretion. This was mani- fest in several ways. Smaller lateral, marginal, and rachi- dian teeth; irregular (malformed) lateral, marginal, and rachidian teeth; and missing marginals were readily ap- parent (Fig. Ic). Most consistently, tissue degrowth was correlated with an increase in the number of radular rows per unit ribbon length. At 40 days it was apparent that either ( 1 ) production of subradular membrane by the membranoblasts and transport by the inferior epithe- lium had slowed, or (2) the production of radular teeth by the odontoblasts had hastened. Regardless of the rela- tive contributions of these alternative processes, the re- sult is the same. An obvious zone (Figs. 1, 4) of denser row-packing has been created. These observations, after confirmation from field analysis, suggest that activity of membranoblast and odontoblast cell lines is differen- tially impaired during periods of food deprivation. The nature of the control mechanism regulating these cells is still uncertain so it is not possible to determine how food deprivation influences the results documented here. However, membranoblast activity is reduced to a greater extent than that of the odontoblasts during periods of sustained stress, and this differential secretory response produces the characteristically packed rows. The fact that there are no observable ecophenotypic effects on tooth shape (thought to be under rigid genetic control in 1.0 o o; o O 0.8 0.6 0.4 0.2 4.15 4.25 5.05 DATE 5.15 Figure 3. Return to normal radular secretion in spring in five natu- ral populations of Helisoma, demonstrating recovery from radular row-packing overwinter (and from presumptive overwinter tissue de- growth). Vertical bars are 95% confidence limits of each mean. 30 D. A. SMITH AND W. D. RUSSELL-HUNTER Eaton B 1 mm C. Remsen 1 mm Figure 4. Llnlouched photographs showing patterns of return to normal radular secretion in two natu- ral stocks of lltiiMiiiui irivolvis. Note that the most recently secreted radular rows are at the top of each photograph. Radulae from Eaton Reservoir (top, A-D. left to right) represent 29%, 40%, 61%, and 80% regrowth (as fraction new rows of total rows). Radulae from Silver Lake. Remsen (bottom. E-H. left to right) represent 17%, 36%', 53% and 76%' regrowth. Samples from both sites were taken at weekly intervals beginning 4. 22. 85. Actual sizes are: Eaton (top, left to right), 3.1. 3.7, 3.2, and 3.8 mm, Remsen (bottom, left to right). 3.7, 4.3. 3.8. 3.4 mm. each stock or population; Smith, 1987, 1989) empha- sizes the unique significance of row-packing as a predict- able response to environmental stress. In one respect, that of the time sequence of return to normal tissue growth after stress, the radular record of row-packing can be more useful than any assessments based on shell-tissue ratios. In field studies of tissue de- growth (Russell-Hunter el a/., 1984), one pond stock of Hclisoma recovered from 41.3% average degrowth to only 32.8% over three months in spring. In another stock RADULAR CORRELATES OH DEGROWTH 31 (from a highly eutrophic lake), overwinter degrowth was eliminated (47.1% net growth) in two spring months. The data on radular recovery (corresponding to re- growth) from the five sites sampled during this study not only show that the process was complete within 25-43 days, but also indicated its temporal sequence in stages. As noted above, there are two significant aspects to the concurrence of abnormal radular secretion and of tissue degrowth as consequences of starvation stress. The first concerns a matter of fundamental biology in attempting to deduce the control mechanisms involved and, ulti- mately, the sequence of causality. All patterns of re- sponse to environmental stress have evolved to increase the fitness of individuals, and all basically require (i) re- ceptors monitoring changes in the rate of abiotic and physiological parameters, (ii) some system capable of in- tegrating such inputs, and (iii) effector tissues that carry out the response. In the case of the response to starvation in gastropods, we have quantified for (iii) several kinds of effects, we can deduce something of (ii), but we are almost completely ignorant of (i) in specific terms. At the very least we know that the simultaneous effects include both abnormal radular secretion and general tissue de- growth. The former involves both absolute and relative reductions of the secretory activity of membranoblasts. The latter involves not only highly reduced levels of gen- eral catabolic activity but also a metabolic shift towards relatively higher turnover of nonprotein carbon. As has been noted (Russell-Hunter, 1985), such controlled differential catabolism can be considered an appropriate parsimony in the net flow through of amino acids (rather than as the defense of a static protein biomass). There are obvious elements of adaptive conservation in the fact that odontoblast activity is less reduced than membranoblast activity, and in the preservation (rela- tively) of structural proteins in the tissues. Both differen- tial processes are adaptive in their potential to accelerate return to normal secretion and tissue regrowth when the period of stress has ended. Parenthetically, it should be noted that this capacity for controlled tissue degrowth [increasing individual survivorship under certain envi- ronmental conditions by a decrease in individual energy content (Russell-Hunter, 1985; and references therein)] compels reconsideration of certain fitness predictions from simple models of age structure and energy parti- tioning between growth and reproduction (see for exam- ple, Williams, 1966; Tinkle and Hadley, 1975; Browne and Russell-Hunter, 1978). It seems likely that the ganglia of the snail's central nervous system are involved in integration after the on- set of starvation stress. It is unlikely that the integrating system is linked neurally to the rate-controlling cells for membranoblast secretion and those of differential pro- tein catabolism, and barely possible that a specialized en- docrine tissue if involved. It can be postulated that the most likely link is through neurosecretory cells. Other systems of integrated control in molluscs involve neu- rosecretion. For example, sex change in Crcpidida (Rus- sell-Hunter et ai, 197 1 ), and cyclic reproductive behav- ior in high littoral snails (Price, 1979) involve neurose- cretion. Despite the degree of integration of the response to overwinter starvation, it may not be appropriate to term this a diapause, since it is less obligate and more plastic in these snails than in those nematodes and insect larvae from similar habitats for which an innate and es- sential seasonal diapause has been described. However, there is integration of responses (probably involving neu- rosecretion), and there can be no question either of ab- normal radular secretion (row-packing) causing tissue degrowth or even of tissue degrowth causing row-pack- ing directly. Although the common cause of both sets of responses appears to be the stress of starvation, these statements belong within David Hume's ( 1 748) regular- ity theory of causation, which remains appropriate for the logical description of such biological sequences, de- spite being currently unfashionable among many profes- sional philosophers. Conclusions from these experimental data have a sec- ond significance to applied biology: the possibility of a retrospective detection, in the field, of earlier periods of stress affecting natural populations. Just as the trunks of long-lived forest trees can record in their rings the histori- cal sequence of drought years and of minor forest fires, so the shells of long-lived bivalve molluscs (Clark, 1976; Mallet et ai, 1987; Peterson et al., 1985) can record, in their growth rings, a history of severe winters. Radular records of degrowth periods as zones of modified tooth- row secretion may provide a history of more recent envi- ronmental stress. This may be of applied value in some gastropod stocks by using comparative spring samples of radulae from known populations to assess relative levels of overwinter starvation, and thence to predict produc- tivity for the rest of the year. In addition, similar radular records could be useful in assessing the metabolic stress of a transient period of pollution (such as an oil spill) on populations of freshwater or marine littoral gastropods, even if no records had been obtained before the popula- tions were stressed. Acknowledgments Work was supported by grants from the Senate Re- search Committee of Syracuse University (D.A.S. and W.D.R-H.) and the Theodore Roosevelt Memorial Fund (D.A.S.). Preparation of this manuscript was supported by the Treves and Carscallen Funds of Wabash College. This is contribution #102 of the Upstate Freshwater In- stitute. 32 D. A. SMITH AND W. D. RUSSELL-HUNTER Literature Cited Bcrrie, A. D. 1959. Variation in the radula of the freshwater snail Lymnaea pcrcgra (Muller) from northwestern Europe. Ark Zoo/. 12: 391-404. Browne, R. A., and VV. D. Russell-Hunter. 1978. Reproductive effort in molluscs. Oecologiu (Berlin) 37: 23-27. Clark, G. R., II. 1976. Shell growth in the marine environment: ap- proaches to the problem of marginal calcification. An: '/.mil 16: 617-626. Kujioka, Y. 1985. Seasonal aberrant radular formation in Thai* bronni (Dunker) and T clavigera (Kiister) (Gastropoda: Murici- dae). J. Exp. Mar. Biol. Ecol. 90: 43-54. Hume, D. 1748. An Inquiry Concerning Human Understanding. (Original title: Philosophical Essays Concerning Human Under- standing.). London, [republished in 1888, Oxford University Press (Clarendon). London and New York]. Hunter, R. D. 1975. Variation in populations of Lyinnaea palnslris in upstate New Y'ork. Am. Midi. Nat 94: 401-420. Isarankura, K., and N. VV. Runham. 1968. Studies on the replacement of the gastropod radula. Afulucologiti 7: 7 1 -9 1 . Kerth, K. 1971. Radula-ersatz und zahnchenmuster der weinberg- schnecke im winterhalbjahr. ~/.ool. .Ih Anal. Bd. 88: 47-62. Mackenstedt, II., and K. Market. 1987. Experimental and compara- tive morphology of radula renewal in pulmonates (Mollusca, Gas- tropoda), /.oomorphology 107: 209-239. Mallet, A. L., C. E. A. Carver, S. S. Coffen, and K. R. Freeman. 1987. Winter growth of the blue mussel Mytilus edulis L.: impor- tance of stock and site. ./ /i'.v/) \ltir liiol Ecol 108: 217-228. Mischor, B., and A. Market. 1984. Histology and regeneration of the radula ofPomacca hridgesi (Gastropoda. Prosobranchia). /.oomor- phology 104: 42-66. Peterson, C. H., P. B. Duncan, II. C. Sumnierson, and B. F. Beal. 1985. Annual band deposition within shells of the hard clam, Mercenaria mercenaria: consistency across habitat near Cape Lookout. North Carolina. Fishery Hull. N'.O.A.A. (U.S.) 83: 257- 260. Price, C. H. 1979. Physical factors and neurosecretion in the control of reproduction in Melampus (Mollusca: Pulmonata). ./. Exp. Zoo/. 207: 269-282. Runham, N. W. 1963. A study of the replacement mechanism of the pulmonate radula. Q J. Microsc. Sei. 104: 27 1 -277. Russell-Hunter, VV. D. 1985. Physiological, ecological and evolution- ary aspects of molluscan tissue degrowth. Am. Malac Bull V "M3- 221. Russell-Hunter, VV. D., and D. E. Buckley. 1983. Actuarial bioener- getics of nonmarine molluscan productivity. Pp. 464-503 in The Mollusca, Vol. 6, K. M. Wilbur, ed. Academic Press, Orlando, New Y'ork. and London. Russell-Hunter, VV . D., and A. G. Eversole. 1976. Evidence for tissue degrowth in starved freshwater pulmonate snails (Helisoma tn- volvis) from tissue, carbon and nitrogen analysis. Camp. Biocheni. Phyxiol. 54A: 447-453. Russell-Hunter, VV. D., M. L. Apley, and J. L. Banner III. 1971. Preliminary studies on brain implants and sex change in Crepidulafornicata(L.). liiol. Hull 141:400. Russell-Hunter, VV. D., D. VV. Aldridge, J. S. lashiro, and B. S. Payne. 1983. Oxygen uptake and nitrogenous excretion rates during overwinter degrowth conditions in the pulmonate snail, Helisoma trn-olvis. Comp. Biochcm. Physio/. 74A: 49 1-497. Russell-Hunter, W. D., R. A. Browne, and D. VV. Aldridge. 1984. Overwinter tissue degrowth in natural populations of fresh- water pul monate snails (Hell 'soma involvis and L ymnaca palustris). Ecology 65: 223-229. Smith, D. A. 1987. Functional adaptation and intrinsic biometry in the radula of Helisoma involvis Ph.D. Dissertation, Syracuse Uni- versity, Syracuse, New Y'ork (Entire dissertation available from Dis- sertation Ahstracis 49: 26B, Order #88-05 1 83: or protocols can be supplied by D. A. S.). Smith, D. A. 1988. Radular kinetics during grazing in Helisoma tri- vo/vw (Gastropoda: Pulmonata)./ K.\p. Biol. 136:89-102. Smith, D. A. 1989. Radula-tooth biometry in Helisoma trivohis (Gastropoda. Pulmonata): interpopulation variation and the ques- tion of adaptive significance. Can. J. Zoo/. 67: 1960-1965. Tinkle, D. VV., and N. F. Hadley. 1975. Lizard reproductive effort: calorific estimates and comments on its evolution. Ecology 56: 427- 434. Williams, G. C. 1966. Natural selection, the costs of reproduction, and a refinement of Lack's principle. Am. Nat. 100: 687-692. Reference: Biol. Bull 178: 33-45. (February. 1990) A Decapod Hemocyte Classification Scheme Integrating Morphology, Cytochemistry, and Function JO ELLEN HOSE, GARY G. MARTIN, AND ALISON SUE GERARD Department of Biology. Occidental College, Los Angeles. California 90041 Abstract. We have examined the hemocytes of three decapod crustaceans (Homarus americanus, Panulirns interruptus, and Loxorhynchus grandis) and propose a classification of these cells based on morphology, cyto- chemistry, and studies of cell functions. In all species, hyaline cells and granulocytes were identified. Although we have retained the widely used names for these cells, we show that traditional morphological features alone do not accurately differentiate between these categories. Historically, the term hyaline cell refers to hemocytes that contain no or only a few cytoplasmic granules, whereas granulocytes contain abundant granules. How- ever, the size and number of granules in hyaline cells vary greatly between species and therefore are not useful criteria for identifying these cells. Since morphological identification alone is inadequate and misleading, espe- cially with regard to hyaline cells, a combination of mor- phological, cytochemical and functional methods is nec- essary to identify decapod hemocytes. Features of hya- line cells include: a higher nucleocytoplasmic ratio than that of granulocytes, the presence of abundant small (~50 nm), round, electron-dense deposits in the cyto- plasm, and their accumulation of trypan blue dye prior to cytolysis. Granulocytes do not take up trypan blue or lyse during a 5-min incubation, and they contain pro- phenoloxidase and hydrolases. Hyaline cells are involved in the initiation of hemolymph coagulation whereas granulocytes are involved in defense against foreign ma- terial by phagocytosis and encapsulation. We propose that these criteria be applied to other crustacean species and expect that they will facilitate our understanding of the physiological roles of their hemocytes. Introduction In crustaceans, circulating hemocytes are thought to be involved in hardening of the exoskeleton, prevention Received 3 April 1 989; accepted 30 November 1989. of blood loss and the confinement of invasive organisms by clot formation, recognition of non-self, phagocytosis, and encapsulation (Bauchau, 1981; Ratnerand Vinson, 1983). Although recent research has expanded the vari- ous physiological roles played by crustacean hemocytes, extention of this information from one species to an- other is difficult because of the lack of a unified classifi- cation scheme for the hemocytes of all Crustacea. Prior hemocyte classification systems rely on tinctorial proper- ties of the cells, which are often subtle or subjective, and seldom apply to other species ( Martin and Graves, 1985). Using the penaeid shrimp Sicyonia ingentis as a proto- type for decapod crustaceans, a hemocyte classification system was developed, which relates cellular morphology at the light and electron microscope levels, cytochemis- try, and three essential functions: clotting, phagocytosis, and encapsulation (Martin et a!., 1987; Hose etal., 1987; Omori et al., 1989; Hose and Martin, 1989). The choice of this species proved serendipitous because the three types of hemocytes are morphologically distinct and clot- ting occurs by explosive cytolysis (Tail's type C coagula- tion; Tait, 1911), making identification of the clotting cell type relatively easy. At the electron microscope level, the cells that initiate clotting are readily identified by sev- eral features typical of hyaline cells (small size, a high nucleocytoplasmic ratio, and scarcity of cytoplasmic granules) and by the presence of numerous, small (~50 nm diameter), electron-dense deposits in the cytoplasm. In addition, the hyaline cells selectively stain with Sudan black B. as does coagulogen extracted from cell-free hemolymph of Paniilirus interruptus and Astacus lepto- clactyhts (Durliat, 1985). During lysis of these cells, the deposits appear to extend through breaks in the plasma membrane and hydrate to produce the clot (Omori et al.. 1989). The granulocytes are larger cells with a lower nucleocytoplasmic ratio and contain numerous small (0.4 ^m diam.) or large (0.8 ^m diam.) granules. Granu- locytes (small and large granule hemocytes) show no 33 34 J. E. HOSE ET AL morphological changes during coagulation and are capa- ble of phagocytosis of bacteria and encapsulation of fun- gal hyphae. Phagocytosis is accomplished primarily by small granule hemocytes (Hose and Martin, 1989); they contain many vesicles and occasional granules that stain for acid hydrolases (acid phosphatase, /3-glucuronidase, and nonspecific esterase) (Hose et al., 1987). Encapsula- tion is initiated by large granule hemocytes and, to a lesser extent, by small granule hemocytes (Hose and Martin, 1989). Prophenoloxidase (PPO), an enzyme in- volved with melanization of encapsulated material and possibly the recognition of non-self items (Soderhall. 1982), is most abundant in large granule hemocytes and to a lesser degree in some small granule cells. In contrast, hyaline cells, which do not phagocytize bacteria or assist in capsule formation, do not contain PPO and only rarely acid phosphatase (Hose el al.. 1987). Thus the di- vision of shrimp hemocytes into two functional groups, hyaline (or clotting) cells and granulocytes, is supported by morphology, cytochemistry, and function. This paper extends the results of the shrimp studies to other decapods and attempts to develop a unified hemo- cyte classification system for the diverse assemblage of crustaceans. This diversity is exemplified by the exis- tence of multiple coagulation mechanisms. In contrast to clotting via explosive cytolysis as in the shrimp (type C according to Tait, 1911), other decapods exhibit type- A coagulation which is distinguished by the formation of a dense hemocyte network which seals oft' the injury and plasma coagulation is not apparent or type-B coagula- tion in which hemocyte aggregation is followed by plasma coagulation (Tait, 191 1). In the present study, we examined the hemocytes in one species with type-A co- agulation (a crab, Loxorhynchus grandis), one species with type-B coagulation (the Maine lobster, Homarm anicriainus), and one species with type-C coagulation (the spiny lobster, Pamdiriis interniptus). Light and elec- tron microscopic features of hemocytes from these three decapods are compared to those identified in the shrimp and correlated with a suite of cytochemical characteris- tics (Sudan black B, acid phosphatase, and PPO) and a group of essential physiological functions (clotting, phagocytosis, and encapsulation). The methods pre- sented here should facilitate study of decapod hemocytes by providing a framework for practical hemocyte classi- fication. Materials and Methods Animals Spiny lobsters (P. interruptus] and sheep crabs (L. grandis) were collected in less than 1 0 m of water at King Harbor Marina, Los Angeles, California. Maine lobsters (H. americtiHiis) were purchased commercially. Crusta- ceans were maintained in flow-through aquaria at 18°C and only intermolt animals were studied. Microscopic examination of hemocytes Freshly fixed hemocytes were examined by light mi- croscopy (LM) (brightfield and phase contrast optics) to determine cell size, cell shape, granule size, and differen- tial hemocyte counts. An aliquot of hemolymph (usually 0.2 cc) was withdrawn from the ventral sinus or heart into a 1 cc syringe containing 0.4 cc of fixative (2.5% glu- taraldehyde in 0. 1 A/sodium cacodylatepH 7. 8 contain- ing 12% glucose). Excess fixative was added to a second 0.2 cc hemo- lymph aliquot, and the cells were processed for examina- tion by electron microscopy. The cells were fixed for 2 h at room temperature and pelleted (10,000 X g for 1.5 min). Following a 10-min wash in 0.1 M sodium caco- dylate (pH 7.8) containing 24% sucrose, the cells were post-fixed in 1% OsO4 in 0.1 M sodium cacodylate for 1 h at room temperature. Each sample was stained en bloc for 1 h with 3% uranyl acetate in 0. 1 M sodium acetate, dehydrated in a graded series of ethanol, and infiltrated and embedded in Spurr's (1969) low viscosity plastic. Thin sections (90 nm) were cut on a Porter Blum MT2B ultramicrotome, stained with lead citrate and exam- ined in a Hitachi HU 1 1 A transmission electron micro- scope (TEM). Nucleocytoplasmic ratios were determined by divid- ing the area of the nucleus by the area of the cell. For hyaline and small granule hemocytes, both areas were clearly identified in light micrographs of immediately fixed cells and measured using a digitizing tablet and Sig- ma-Scan® computer software (Jandel Scientific). Be- cause the nucleus is difficult to visualize in phase contrast images of large granule hemocytes, measurements were made from thick plastic sections. To ensure that cells were sectioned through their greatest axis, only large granule hemocytes showing typical length and width measurements were used. There was no difference in size measurements of fixed cells examined in wet mounts by phase optics and cells embedded in plastic and sectioned. Identification of cell-type initiating coagulation Two previously used approaches helped to identify the type of hemocyte initiating coagulation of the hemo- lymph: ( 1 ) visual examination of hemocyte types accu- mulating trypan blue, an event we have previously shown to be a direct precursor to cytolysis and ensuing clot formation and (2) ultrastructural examination of he- mocytes fixed at stages during clot formation (Omori et al.. 1989). For the first technique, 0.1 cc of freshly drawn hemolymph was gently mixed on a glass slide with 0. 1 cc of a 1.2% solution of trypan blue in seawater. Within 1- 2 min, certain hemocytes accumulate the blue color in DECAPOD HEMOCYTE CLASSIFICATION 35 both the cytoplasm and nucleus. By 5 min these cells lyse and the cytoplasm is lost, but the blue staining nuclei remain. Individual cells may be identified and observed as they accumulate the dye and lyse. After 5 min, the number of blue stained nuclei and the cells remaining intact and colorless were counted. Six hundred cells were evaluated for each species. The second method provided ultrastructural informa- tion on the type of hemocyte that initiates coagulation as well as changes in these cells during cytolysis. Aliquots of hemolymph (0. 1 cc) and seawater (0. 1 ml) were mixed for times ranging from 1 5 s to 5 min and then fixed by the addition of an excess amount of gluaraldehyde fixative and prepared for TEM examination as described above. Phagocytosis of bacteria by hemocytes In vitro phagocytosis experiments were performed as described by Hose and Martin (1989). A glass micro- scope coverslip was placed into each of two sterile plastic Petri dishes and each covered with 20 ml of shrimp cul- ture medium (SCM, Brody and Chang, in press). Ap- proximately 0.3 cc of hemolymph was added over each coverslip, and hemocytes were allowed to settle and at- tach to the coverslips for 15 min. Approximately 100,000 cells of a Gram-negative marine bacterium (Cy- top/iaga sp.; Occidental College Isolate 1 ) were added to one of the dishes. Cultures were incubated at 12°C for 3 h. Coverslips were fixed in methanol for 5 min and stained with May Grunwald-Giemsa. Differential counts of approximately 200 hemocytes were performed and the numbers of phagocytic cells (hemocytes containing at least 1 bacterium within a vacuole) were recorded. Dead hemocytes were differentiated from viable cells by the presence of nuclear degeneration (karyolysis, pycnosis). Fungal encapsulation The method of Hose and Martin (1989) was used to determine the types of hemocyte that attached to fungal hyphae and initiated capsule formation. Approximately 1 ml of hemolymph was added to 1 2 ml SCM in a 1 5 ml plastic centrifuge tube. Small cubes (0.5 mm3) of Sabou- raud-dextrose agar containing primarily hyphae of Fu- sarium solani (University of Arizona strain 1623C) were added to the tube; the culture was incubated at 1 2°C. Af- ter 1. 2, and 5 min, a cube was removed and washed gently in SCM to remove nonadherent hemocytes. The cell types attached to the fungus were identified using phase contrast microscopy (total of 200 cells for each species). Hemocyte cytochemistry Hemolymph (0.2 cc) was withdrawn into 0.2 cc of 12.5% unbuffered citrate anticoagulant (which prevents lysis of hyaline cells), spread on three glass microscope slides, and allowed to air dry. Constituents of hemocyte granules and cytoplasm were visualized using methods given in Hose et al. (1987). Smears were prepared from six individuals of each species. Where possible, 200 cells per slide were evaluated using brightfield microscopy ( 1000X); each hemocyte was categorized and individual cellular reactions were recorded. Lipids and lipoproteins were demonstrated using a commercial Sudan Black B kit (Sigma Chemical Co. Kit #380). Glutaraldehyde-fixed hemocytes were processed according to provided directions except that the nuclear counterstain was not used. Cytoplasmic staining was differentiated from staining of granule or plasma mem- branes and was termed a positive reaction (Hose et al., 1987). Occasionally entire granules were stained by Su- dan Black B; these are noted in the results. Prophenoloxidase (PPO) activity was evaluated in smears fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 h at 4°C. The smears were rinsed three times in phosphate buffer ( 1 5 min each ), then incu- bated in 0.1% L-DOPA in phosphate buffer for 16 h at room temperature. Black staining of the granules was in- terpreted as a positive reaction (Hose el al., 1987). Acid phosphatase in glutaraldehyde-fixed hemocytes was visualized using a commercial research kit (Sigma Chemical Co. Kit #386). Naphthoi AS-B1 phosphate was used as the substrate, yielding a red-violet reaction prod- uct (naphthol AS-Bl-fast garnet GBC complex). Al- though the location and abundance of acid phosphatase was species-specific, the rating system previously used for the shrimp (Hose el al., 1 987) was acceptable for use with the lobsters. In the shrimp we recognized the following categories: rare (0 to 3 positive foci per cell), few (4 to 10 foci per cell), intermediate ( 1 1 to 30 foci per cell), and many (>30 foci per cell). Because L. grandis contained more acid phosphatase foci than the shrimp, the rating system was slightly modified for evaluation of crab he- mocytes. A distribution of the number of positive foci per cell was constructed and the limit for the "rare" cate- gory placed between the groups containing rare and few foci. Thus while the rare category consisted of less than four foci for shrimp and the lobsters, it was enlarged to include less than six foci for the crab. A rare response was interpreted as negative. Remaining limits were identical for all four species. Two hundred cells were evaluated for each test; each hemocyte was identified and individual cellular reactions were recorded. Results Description of hemocyte types Using morphological criteria previously developed for the shrimp (Martin et al., 1987), three basic cell types were observed in each of the decapods studied; one type 36 Comparative hemocytc morphology J. E. HOSE ET AL Table I Large granule cells Small granule cells Hyaline cells Homarus americanus Cell size (length x width) Granule diameter Number ot granules Nucleocytoplasmic ratio Panulirus interruptus Cell size (length • width) Granule diameter Number ot granules Nucleocytoplasmic ratio Loxorhynchus grandis Cell size (length • width) Granule diameter Number of granules Nucleocytoplasmic ratio 23.4±2.2 x 12. 3± 1.3(11) 1.3 ±0.1 (30) 72. 7 ±4.6 (30) 20.3 ± 1.8(10) 22.6 ± l.3x 14.1 ±0.5(12) 1 .4 ±0.1 (30) 34.0 + 2.4(30) I8.3± 1.3(8) 20.4 ±0.5 < 13. 7 ±0.6 (1 9) 1.4 + 0.1 (30) 28.0 ±4. 1 (30) 17.5+ 1.5(1 1) 20.9 ± 0.6 x 13.9 ±0.3 (18) 0.9 + 0.1 (30) 27. 3 ±5.6 (30) 27.5 ±2.4(10) 1 9. 7 ±0.9 x 10.0 ±0.3 (18) 0.8 ±0.1 (33) 10.8 ±4.3 (30) 24.6 ±2.1 (16) I 7.9 ±0.5 X 10.6 ±0.4 (15) 1.0 + 0.1 (30) 8.0 ± 1.6(30) 23.5 ±1.0(11) 14.1 + 0.8X 11.2±0.6(16) 0.9 ±0.1 (30) 13.9 + 3.0(30) 40.0 ±2.5 (13) 14.2 + 0.5 X 10.6 ±0.2 (18) 1.2 ±0.1 (30) 5.6 + 0.8(30) 40.2 ± 1.5(16) 14.5 ± 1.0 x 9.1 ±0.3(12) 1.0 + 0.1 (30) 38.3 ±5.6 (30) 36.0+ 1.5(16) Measurements are mean ± standard error (number ot measurements). Cell sizes are presented in length • width. Cell and granule sizes are in /jm. Number ot'granules is the number per sectioned cell. Nucleocytoplasmic ratios are percentages. T-tests showed significantly smaller hyaline hemocyte size for all three species compared to either small or large granule hemocytes (P < 0.05). Nucleocytoplasmic ratios for hyaline hemocytes of all three species were significantly larger than those of small or large granule hemocytes (P < 0.05). of hyaline cell and two subgroups of granulocytes (small and large granule hemocytes) (Table I). Hyaline Cells (Figs. 1A-6A). Hyaline cells were the most morphologically diverse type of hemocyte. When examined by phase contrast microscopy, they were gen- erally ovoid in shape, smaller than granulocytes and with a higher nucleocytoplasmic ratio (Table I), and either contained few large granules (P. interruptus) or numer- ous smaller granules (//. americanus and L. grandis). Most hyaline cells of //. americanus measured 21X11 Atm although smaller hemocytes (from 12 ^m in the larg- est dimension) were occasionally observed. The smaller cells may represent immature hyaline cells because they were continuous in size with the hyaline cell and proba- bly correspond to the prohyalocyte category recognized by Cornick and Stewart (1978). Hyaline hemocytes of H. americanus had numerous (14/section) small, ovoid granules, 0.9 jum long, the contents of which appeared homogeneous and electron dense at the EM level. The cytoplasm contained Golgi bodies, abundant rough en- doplasmic reticulum (RER), a circumferential band of microtubules, a few vesicles, mitochondria, and small (~50 nm diam.), round, electron-dense deposits (Fig. 7). Hyaline cells of the crab (L. grandis) resembled those of the Homarus, except they contained more granules (40/ section) that, at the EM level, were ovoid, homogeneous, and electron dense. Hyaline hemocytes of the spiny lob- ster (P. interruptus) were distinctive in that only a few (6/section) large (1.2 ^m diam.) granules were present. Ultrastructurally the granules had a punctate pattern. Otherwise, features of the cytoplasm were similar to those described for the other species. Granulocytes. Granulocytes could be easily differen- tiated into two groups using phase contrast microscopy: small (Figs. 1B-6B) and large (Figs. 1C-6C) granule he- mocytes. Small granule hemocytes contained few to many, round, dark, small (usually <1.0^m diam.) gran- ules and a relatively small, centrally located nucleus, whereas the cytoplasm of large granule hemocytes was packed with larger (1.3-2.0 nm diameter), refractile granules that obscured the eccentrically placed nucleus (Table I). However, it was sometimes difficult using TEM to distinguish between small granule hemocytes containing numerous granules and large granule hemo- cytes because the sectioned granules appeared similar in size. These cells may be part of a single line of maturation in which the number and size ofgranules in small gran- ule hemocytes increase until the cell is recognized as a large granule hemocyte. To distinguish between small and large granule hemocytes, we relied on ( 1 ) the loca- tion of the nucleus (centrally or eccentrically placed) and (2) the presence of only large granules (> 1.2 ^m diam.) in large granule hemocytes while small granule hemocytes may contain both large and small granules. Both types of granules were often surrounded by a clear (artifactual?) space (see Figs. 6B and C), and in P. interruptus, granules in the large granule hemocytes often did not section cleanly but appeared fractured (see Fig. 4C), unlike those present in small granule cells. The cytoplasm of granulo- cytes, both small and large granule hemocytes, contained DECAPOD HEMOCYTE CLASSIFICATION 37 Figures 1-3. Light micrographs of hemocytes from Panulirux interruptus (Fig. 1), Htmiarus ameri- canus (Fig. 2), and Loxorhynchus grandis (Fig, 3) showing hyaline cells (column A), small granule (column B), and large granule (column C) types. Note the small size of the hyaline cells compared to the granulo- cytes. The large granule cells are highly refractile and it is difficult to observe the nucleus. All figures at 2600X; scale bar = Golgi bodies, RER, vesicles, mitochondria, ribosomes, and microfilaments scattered between the granules (see Fig. 7). Microtubules, typically in a band adjacent to the plasma membrane, were commonly seen. Hemocyte differential counts Differential counts were performed using phase con- trast LM and TEM (Table II). Although individual paired counts are similar, we consider the TEM counts more accurate for comparison because of the inherent greater resolution. P. interruptus had the highest percent- age of hyaline cells at 56%, whereas L. grandis and //. americanus were considerably lower at 2 1 % and 27%, re- spectively. Large granule granulocytes constituted be- tween 10% and 13% of the total with small granulocytes comprising about 65%. in H. americanus and L. grandis and 31%. in P. interruptus. Clotting Patterns of coagulation. The species studied represent the three coagulation patterns described by Tail (1911). In L. grandis (Tail category A), the bulk of the clot con- sisted of long cellular aggregations linked by strands of clot material. The clot produced by H. americanus (Tail category B) contained isolated islands of coagulated hemolymph with intervening areas of packed hemo- cytes, whereas in P. interruptus (Tail category C), cell ag- Figures 4-6. Transmission electron micrographs of hemocytes from Panulints inierrupiu* (Fig. 4). Homarus americanus (Fig. 5), and Loxorhynchusgrandis(F\g. 6) showing hyaline (column A), small gran- ule (column B) and large granule (column C) types. Granules are present in hyaline cells although not abundant. Small granule hemocytes are characterized by small granules in a relatively large amount of cytoplasm and in large granule hemocyles the granules till much ol the cytoplasm. All figures at 5500X; scale bar = 5 ^m. 38 Figure 7. Transmission electron micrograph of a hyaline hemocyte from Homarus americanux show- ing cytoplasmic deposits (arrows), RER (R), a granule (G). vesicles (V), nucleus (N). and edge of Golgi body (GB). 70,000x; scale bar = 0.5 ^m. Figure 8. Light micrograph of a large granule hemocyte from Pamdinis inlemiptux 2 min after mixing equal volumes of hemolymph and seawater containing trypan blue. Note the cell is intact and has spread on the glass substrate. Nucleus (N); filopodia(F). 2000X; scale bar = 10 ^m. Figure 9. Light micrograph of two hyaline cells from P. inlcrrupius from the same preparation as cells in Figure 8. These cells have not attached to the substrate and they have lysed, leaving a light (blue-stained) nucleus (N), blebs (B), and thin rim of residual cytoplasm (arrows). 2000 X; scale bar = 10 ^m. Figure 10. Light micrograph of hemocytes from P. inlvrruplus during early clot formation (2 min after mixing with seawater). Note the clusters of hemocytes between the large circular areas of coagulated hemolymph (C). Large granule hemocytes (L); small granule hemocytes (S); and hyaline cell (H). 700X; scale bar = 25 ^m. Figure 11. Light micrograph of hemocytes from Loxorhynchus ,i,'*w/ ; scale bar = 2.5 fim. DECAPOD HEMOCYTE CLASSIFICATION Table III 41 Comparative liemocyte lunctiun.t Large granule cells (% positive) Small granule cells (% positive) Hyaline cells (% positive) Homarus americanus Clotting: % accumulate trypan blue Phagocytosis: % phagocytic %dead(+ bacteria) %dead(- bactena) Encapsulation: % of adherent cells Painilirus interrupt us Clotting: % accumulate trypan blue Phagocytosis: % phagocytic % dead (+ bacteria) %dead(- bacteria) Encapsulation: % of adherent cells Loxorhynchus grandis Clotting: % accumulate trypan blue Phagocytosis: % phagocytic %dead(+ bacteria) %dead(- bacteria) Encapsulation: % of adherent cells 0.0 39.3 9.3 0.0 63.3 32.0 0.0 0.0 68.4 0.3 70.0 4.8 16.7 67.2 1.2 93.1 11.5 0.0 21.2 83.1 16.2 0.0 25.3 0.7 95.9 6.1 1.7 28.7 100.0 0.0 83.0 29.7 0.0 100.0 0.0 31.6 1.6 6.3 100.0 0.0 95.0 24.3 4.1 Mean percentage of each category which accumulates trypan blue, is phagocytic, or initiates encapsulation by adhering to fungal hyphae. In the clotting experiments. >100 hemocytes in each category were evaluated from each of 5 animals. In the phagocytosis experiments, >100 hemocytes in each category were evaluated from a single animal. Percentages of dead hemocytes were compared in the presence ( + ) and absence ( - ) of bacteria. In the encapsulation experiments, > 100 hemocytes in each category were evaluated from each of 5 animals. became concentrated around the nuclear envelope. As the plasma membrane over the blebs ruptured, cyto- plasm containing the deposits and disrupted organelles was released. Surrounding the degenerating hyaline cell, long strands were formed in the hemolymph, which ap- parently hydrated into typical clot material. Concur- rently, granules developed a scalloped margin, their con- tents became grainy, and adjacent granules sometimes fused. Granules released their constituents either by exo- cytosis or lysis into the cytoplasm. As hyaline cells of P. interniptus lysed, spheres of coag- ulated hemolymph developed around each cell (Fig. 10). The spheres expanded and fused with adjacent spheres to produce a continuous hemolymph clot with clusters of granulocytes scattered between roughly spherical ar- eas of coagulated hemolymph. Hemolymph clots of Ho- marus contained fewer areas of coagulated hemolymph and larger intervening granulocyte clusters. In contrast, the clotted hemolymph of L. grandis was composed of masses of aggregated granulocytes (Fig. 1 1 ) often adher- ing to long strands of clot material. The granulocytes in all three species did not lyse during the 1-h time period examined in this study, although they extended filo- podia. Large and small granule hemocytes rarely dis- played exocytosis of granules. Phagocytosis of bacteria Phagocytosis of the Gram-negative bacterium Cyio- phaga sp. was performed by most small granule hemo- cytes, some large granule hemocytes, and none of the hy- aline hemocytes (Table III). The percentage of phago- cytic small granule hemocytes ranged from 83% to 96%, whereas only 30% to 67% of the large granule cells were phagocytic. In contrast to incubation in seawater, where cytolysis of hyaline cells was observed, most hyaline cells and granulocytes remained viable when incubated in SCM (shrimp culture medium) for the 2-h duration of the phagocytosis experiments. However, enhanced au- tolysis was observed when hyaline cells and granulocytes were cultured in the presence of bacteria (Table III). Hya- line cells that did not lyse during the experiments did not 42 J. E. HOSE ET AL. Table IV Comparative hemocyte cytochemistry Large granule cells Small granule cells Hyaline cells Hninuni.'iiimi'riainii.'i Acid phosphatase 48.3 ± 8.5 23.6 ±8.0 0.5 ± 0.5 (25.0-80.0) (6.7-59.5) (0.0-2.9) Prophenoloxidase 86.6 ±5. 2 10.8 ± 1.3 0.0 ± 0.0 (71.4-100.0) (5.0-14.3) (0.0-0.0) Sudan Black B 0.0 ± 0.0 0.4 ± 0.4 99.4 ±0.6 (0.0-0.0) (0.0-2.5) (96.1-100.0) Piiiiulirit.t inti'mi/Hits Acid phosphatase 72.1 ±6.4 69.4 ± 7.2 4.9 ± 1.7 (44.4-90.0) (4 1.0-9 1.5l (0.0-1 1.1) Prophenoloxidase 96.8 ± 3.4 35.2 ± 4.9 0.2 ±0.2 (91.7-100.0) (20.7-55.7) (0.0-1.0) Sudan Black B 0.0 ±0.0 (i.o ±0.0 99.6 + 0.2 (0.0) (0.0) (98.8-100.0) Loxorhynchus grandi \ Acid phosphatase 88. 7 ±5. 8 76.6 ±7. 7 11.4 ±2. 8 (61.5-100.0) (51.9-96.5) (1.9-22.6) Prophenoloxidase 100.0 ±0.0 53.4 ± 4.2 1.0± 1.0 (100.0) (41 8-66.7) (0.0-5.9) Sudan Black B 0.0 ± 0.0 0.4 ±0.2 100.0 J no (0.0) (0.0-1.3) (100.0) Percentage of positive hemocytes ± standard error. Minimum and maximum values are in parenthesis. Twenty large granule. 50 small granule, and 50 hyaline hemocytes were examined from each of 6 ani- mals. attach to the glass and spread as did the granulocytes, but often adhered to the granulocytes and remained ovoid. Total phagocytosis rates (denned as the number of phagocytic cells divided by the total number of surviving hemocytes) were 79% and 88%, respectively, for //. americanus and L. grand is (the two species with no sur- vival of hyaline hemocytes) and 54% for P. internipius which had higher hyaline cell survival. Encapsulation of fungal hyphac LM observations of initial hemocyte contact with fun- gal hyphae showed that approximately two-thirds of ad- herent cells were large granule hemocytes, between 20% and 30% were small granule hemocytes and only small percentages were hyaline hemocytes (Table III). For all three species, percentages of adherent large granule cells were enriched 7 to 15 times over those found in hemo- lymph. Hemocyte cytochemistry Hemocyte smears were stained to identify sites of lipid (with Sudan black B), or acid phosphatase, or PPO activ- ity (Table IV). Sudan black B, which stains lipids and lipoproteins, produced diffuse cytoplasmic staining in all hyaline cells of each species. However, the staining inten- sity was less than that previously reported for deposit- containing hyaline cells of the penaeid shrimp in which the entire cytoplasm is darkly stained in a clumpy pat- tern (Hose el al. 1987). In the present species, the light grey, homogeneous staining of the cytoplasm was diffi- cult to detect without prior experience with the stain. The most distinctive feature in Sudan black-stained cells was that the nucleus in hyaline cells was obsurred by the stain, similar to that observed in the shrimp (Hose el al., 1987). Granules in the hyaline cells of all three species did not accumulate the stain, although membranes around the larger granules in some hyaline hemocytes of //. americanus displayed intense staining. The cyto- plasm of the granulocytes remained unstained by Sudan black B and the nucleus was always visible. Except for intense staining of granule membranes in large granule hemocytes of//, americanus, staining reactions of granu- locytes were identical to those reported for shrimp hemo- cytes. Reaction sites demonstrating acid phosphatase were rare in hyaline cells and more abundant in granulocytes (Table IV). For each species, ranges of the percentages of positive cells did not overlap between the two categories. Most small granule hemocytes had few to an intermedi- ate number of foci. As observed in the shrimp (Hose el al., 1987), not all large granule hemocytes contained re- action sites, but positive cells had numerous foci. In L. grandis, acid phosphatase was primarily located in the granules, although some vesicles and tubules (most likely RER) stained positive as well. In the lobsters, only a few granules contained acid phosphatase and most of the re- action sites were located in vesicles and tubules of RER. PPO activity was restricted to granulocytes (only one out of approximately 300 hyaline hemocytes of the crab and spiny lobster appeared positive). From 11% (//. americanus) to 53% (L. grandis) of small granule hemo- cytes had positively stained granules, whereas most (>87%) large granule hemocytes contained numerous dark-staining granules. The cytoplasm of large granule cells also contained PPO while staining in small granule hemocytes was confined to the granules. Discussion Our results suggest that hemocytes of decapod crusta- ceans are composed of two major groups, hyaline cells and granulocytes, which have distinct functional and cy- tochemical differences. Most investigators have histori- cally recognized these two categories and separated them using morphological criteria (see Martin and Graves, 1985, for review). However, our work demonstrates that the morphological features traditionally used to identify these categories do not reliably correlate with cellular DECAPOD HEMOCYTE CLASSIFICATION 43 functions. Although granulocytes of the three species studied in this paper, the penaeid shrimp (Sicyonia in- gentis) used to develop the system, and several other spe- cies described in the literature (Bauchau, 1981) are morphologically almost indistinguishable, hyaline he- mocytes constitute a heterogeneous group. Our observa- tions may explain much of the confusion in the literature regarding hemocyte morphology and function. Such dis- crepancies have prevented information obtained on a particular species to be readily interpreted with regard to other decapods. In some cases, functional studies have not identified cell types involved in hemolymph coagula- tion and phagocytosis. For example, both Schapiro cl al. (1977) and Goldenberg et al. (1986) presented quantita- tive data on phagocytosis of bacteria by H. americaniis hemocytes, but neither group could identify the phago- cytic hemocytes. In other cases, morphological identifi- cation did not correspond to functional roles. For in- stance, Soderhall cl al. (1986) refer to the hyaline cell as the main phagocytic hemocyte in the crab Carcinus maemis whereas in the crayfish Pacifastacus leniiisculus phagocytosis is performed by both hyaline cells and semigranular cells. Such lack of consistency in ascribing similar functions to apparently similar hemocyte types stems from difficulties of using a classification system based on traditional morphological interpretations of hy- aline and granular hemocytes (i.e., granule number and size). Our data show that, for the four species investigated thus far, function is correlated with other morphological features such as cell size, nucleocytoplasmic ratio, and the presence of cytoplasmic deposits. Historically, a second area of confusion is the identity of the type of hemocyte that initiates coagulation. Re- sponsible cells have been described as either "explosive corpuscles" and "hyaline cells" (Wood and Visentin, 1967;Woodrta/., 1971; Ravindranath, 1980) or granu- locytes (Toney, 1958; Hearing and Vernick, 1967; Mengeot et al., 1977; Madaras et al., 1981). Our studies provide an answer for the apparent confusion regarding the identity of clotting hemocytes. Hyaline cells lyse and initiate coagulation in all species; however in different species, these cells exhibit variations in the abundance and size of granules. For example, the granules of Loxo- rhynchus grandis are so abundant that the hyaline cells are easily confused with granulocytes while in Panulirus interruptiis, the granules in large granule and hyaline he- mocytes are approximately the same size. Hyaline hemo- cytes do have in common numerous, 50-nm diameter cytoplasmic deposits. These deposits can be detected us- ing TEM, a technique rarely included in previous classi- fication schemes, and by their propensity to stain with Sudan Black B. We avoided the use of the term "hyaline cell" in our previous publications (Martin and Graves, 1985; Martin et al., 1987; Hose et al.. 1987; Hose and Martin, 1989; Omori et al., 1989) in an attempt to avoid bias in devel- oping a classification scheme and instead referred to de- posit cells (with and without granules), small granule and large granule hemocytes. We now consider that shrimp deposit cells are equivalent to hyaline cells. Therefore, in an attempt to simplify the classification of crustacean hemocytes, we suggest the following categories of hemo- cytes: hyaline, small and large granule hemocytes. It is very important to recognize that morphology alone is in- sufficient for assigning any cell to one of these categories; instead the following criteria for hemocyte identification are suggested. Hyaline cells have a nucleocytoplasmic ratios of >0.35 and lyse during clot formation. Because lysis is rapid, identification of these cells, especially in species with rel- atively low numbers of hyaline cells, is facilitated by mix- ing a trypan blue-seawater solution with hemolymph. Hyaline cells turn blue prior to lysis, thereby allowing morphological identification of the cell and observation of changes in cell morphology during coagulation. At the TEM level, these cells contain tiny cytoplasmic deposits that appear to be involved with the clotting process be- cause they are only present in the hyaline cells and their release from the lysing cell precedes hemolymph coagu- lation. In addition, hyaline cells in the species we have studied (this paper and Hose et al., 1987), selectively stain with Sudan Black B. Although this is a general stain for lipid. it has also been shown to stain coagulogen iso- lated electrophoretically (Durliat, 1985). Coagulogen may be contained within hyaline hemocytes or perhaps produced but not stored in high levels by these hemo- cytes. In crustacean hyaline cells, the cytoplasmic depos- its are sudanophilic, with the most intense staining ob- served from the clustered deposits present in shrimp (Hose et al.. 1987). Although the test is useful, interspe- cific variations in the intensity of Sudan Black B staining are subtle and require careful interpretation. The less subjective criterion for a positive reaction is the obscu- rance of the nucleus by the stain. Our results suggest that coagulation in decapods in- volves a common mechanism; the release of cytoplasmic material through breaks in the plasma membrane, possi- bly including the granules. The identity of the materials released is not clear. It has been suggested that ( 1 ) coagu- logen, the clotting protein, is found in the plasma and activated by chemicals released from hemocytes and (2) coagulogen and its activators are released from cells (see Omori et al., 1989). Ghidalia el al. (1981) reviewed this topic and demonstrated the presence of coagulogen in the plasma of decapods representing Tail's (1911) three patterns of coagulation. Although the presence of coagu- logen in plasma could result from lysis of hyaline hemo- cytes during cell separation, these investigators used an anticoagulant (1:9, hemolymph: 10% sodium citrate, v:v) which we have shown to be effective in preventing hya- 44 J. E. HOSE ET AL. line cell lysis. They conclude that differences between the three coagulation patterns are probably due to the man- ner in which the clot-initiating materials are released. From the present study we show that decapods placed in Tail's category C (characterized by rapid gelation of the plasma) have twice the percentage of hyaline cells as in species where hemocyte aggregation occurs followed by slight gelation of the plasma (Tail's category A). What remains unclear is Ihe localizalion of Ihe clolling prolein coagulogen (in cells, plasma, or both) and an idenlifica- lion of Ihe material released from Ihe hyaline cells lhal iniliales coagulalion. The most abundanl cyloplasmic malerial released during coagulalion is Ihe eleclron- dense deposils. These deposils were idenlified in Ihe hya- line cells of all decapods we examined using TEM and appear similar lo published micrographs of coagulocyles in some insecls (Ralcliffe and Rowley, 1979). Clearly a specific labelling lechnique for Ihese deposils and coagu- logen is needed, as in Bohn ct al.'s ( 198 1 ) immunocylo- chemical study of insecl coagulogen. Granulocyles, Ihe second major category of decapod hemocyles, have a nucleocyloplasmic ralio of <0.35 and Ihey do nol accumulate Irypan blue or lyse rapidly in cullure. They are identified by the presence of numerous cytoplasmic granules, positive staining reactions for acid phosphatase and PPO, and in vitro phagocytosis of bacle- ria and allachmenl to fungal hyphae. The two subdivi- sions of granulocyles may be dislinguished by ( 1 ) cenlral location of nuclei in small granule hemocyles and eccen- Iric location of nuclei in large granule hemocytes, (2) Ihe presence of only large granules in large granule hemo- cytes whereas in small granule hemocyles there is a mix- lure of granules wilh varying sizes, and (3) Ihe refraclile nalure of granules only in large granule hemocyles when examined by phase conlrasl microscopy. The functional roles of granulocyles correlale well wilh observed cylochemical fealures. Granulocyles are Ihe primary defensive cells of Ihe hemolymph and Ihe Iwo sublypes perform overlapping funclions. Small granule hemocyles are Ihe main cells involved in phago- cylosis and conlain many lysosomes. while large granule cells, which mosl frequently iniliale encapsulation of fungi, show more intense staining for PPO (Hose and Martin, 1989). The funclional and cytochemical crileria for recogniz- ing two categories of hemocytes (hyaline cells and granu- locyles) are further supported by observalions of hemo- cyte maturation within Ihe hemalopoielic lissue of the shrimp ( Martin etui., 1987). In this species, we observed mitosis only in agranular hyaline cells and small granule hemocyles. Clusters of hyaline cells and granulocyles were segregaled within Ihe hemalopoielic lissue (Martin el ul., 1987). We propose that the Iwo hemocyle catego- ries represent two cell lines. Cell size is significantly smaller in hyaline cells and is disconlinuous belween hy- aline cells and small granule hemocyles. The nucleocy- loplasmic ratios of hyaline cells of shrimp and the three species considered here are significanlly higher lhan Ihose of granulocyles. The ralios of Iwo granulocyle cate- gories overlap and decrease in large granule hemocyles coincidenl wilh increases in granule number and size (Table I). Granulocyles Ihus appear as a conlinuum of differenlialion from the less mature small granule hemo- cyles lo the large granule hemocyles. To summarize, a combinalion of morphological, cylo- chemical and funclional melhods musl be used lo iden- lify decapod hemocyles, because Iradilional morpholog- ical fealures are inadequate and misleading, especially with regard to hyaline cells. Further sludies by invesliga- lors ulilizing other decapods are necessary to lest the use- fulness of Ihis classificalion scheme and lo offer improve- menls by developing more specific crileria. Acknowledgments We want to thank Heidi Parker and Laura Targgart for collecling and mainlaining Ihe cruslaceans; Sidne Omori, Calhy Corazine, Celesle Chong, and Erin Camp- bell for lechnical support; and Dr. Don Lightner and Le- ona Mohney for supplying the cullures ofFusariwn so- luni. The projecl was supported by NSF granl DCB- 85021 50 loGM and JEH. Literature Cited Bauchau, A. G. 1981. Crustaceans. Pp 386-420 in Imrrtchruic Blood C'r//v. Vol. 2. Academic Press. New York. Bohn, H., B. Barwig, and B. Bohn. 1981. Immunochemical analysis of hemolymph clotting in the insect LeuL'opliaca medarae (Rlatta- ria). J Cmnp. /Virv/o/ I43B: 169-184. Brody, M., and K. Chang. (In press). Ecdysteroid effects on primary cell cultures. //;/ ./. Invcnchr, Rcpro Dev Cornick, J. \\ ., and J. E. Stewart. 1978. Lobster (Hoinunts nincih canux) hemocytes: classification, differential counts and associated agglutmin activity. J. Invcnchr. 1'alhol. 31: 194-203. Durliat, M. 1985. Clotting processes in Crustacea Decapoda. Biol. KIT 60:473-498. Ghidalia, \V., R. Vendrely, C. Montmory, V. Coirault, and M. O. Brou- ard. 1981. Coagulation in decapod Crustacea. / Comp I'/iyxiol. 142:473-478. Goldenberg, P. Z., A. H. Greenberg, and J. M. Gerrard. 1986. Activation of lobster hemocyles: cytoarchitcctural aspects. J. Invcr- ichr I'athol 47: 143-154. Hearing, V. J., and S. H. Vernick. 1967. Fine structure of the blood cells of the lobster. Homarus americanus. Cites. Sci. 8: 170-186. Hose, J. E., G. G. Martin, V. A. Nguyen, J. Lucus, and I . Rosenstein. 1987. Cytochemical features of shrimp hemocytes. Biol Bull. 173: 178-187. Hose, J. E., and G. G. Martin. 1989. Defense functions of granulo- cytes in the ridgeback prawn Sicyoniu ingenue Burkenroad 1938. J. Invcnchr Pallwl. 53: 335-346. Madaras, F., M. Y. Chew, and J. D. Parkin. 1981. Purification and characterization of the sand crab (Ovalipes bipustulalus) coagulo- gen (nbnnogen). Thromb. Haemosl. 45: 77-81. Martin, G. G., and B. L. Graves. 1985. Fine structure and classifica- tion of shrimp hemocytes. J. Moiphol 185: 339-348. DECAPOD HEMOCYTE CLASSIFICATION 45 Martin, G.G..J. E. Hose, and J.J. Kim. 1987. Structure of hemato- poietic nodules in the ridgeback prawn Sicynnia mgentis: light and electron microscopic observations. ./ Morphol 192: 193-204. Mengeot, J. C., A. G. Bauchau, M. B. DeBrouwer, and E. Passelecq- Gerin. 1977. Isolement des granules des hemocytes de Homarus vulgaris. Examens electrophoretiques du contenu proteique des granules. Coinp. Kiochem Phy\iol 58(A): 343-403. Omori, S. A., G. G. Martin, and .J. E. Hose. 1989. Morphology of hemocyte lysis and clotting in the ridgehack prawn, Sicyonia m- genlis. Cell Tissue Res. 255: 1 17-123. Ratcliffe, N. A., and A. F. Rowley. 1979. Role of hemocytes in de- fense against biological agents. Pp 332-414 in Insect Hemocytes: Development, Form, Functions and Techniques. A. P. Gupta, ed. Cambridge University Press. Cambridge. Ratner, S., and S. B. Vinson. 1983. Phagocytosis and encapsulation: cellular immune responses in Arthropoda. Am. Zoo/. 23: 185-194. Ratindranath. M. 11. 1980. Haemocytes in haemolymph coagulation of arthropods. Biol. Rev 55: 139-170. Shapiro, H. C., J. F. Steenbergen, and /. A. Fitzgerald. 1977. Hemocytes and phagocytosis in the amencan lobster. Homarus amcncanm. Pp 126-134 in Comparative I'alhology, Vol. 3. Ple- num Press, New York. Soderhall, K. 1982. Prophenoloxidase activating system and melani- zation — a recognition mechanism of arthropods? A review. Dev. Co/up Immunol 6: 60 1 -6 1 I . Soderhall, K., V. J. Smith, and M. W. Johansson. 1986. Exocytosis and uptake of bacteria by isolated haemocyte populations of tun crustaceans evidence for cellular co-operation in the defense reac- tions of arthropods. Cell Tissue Rex. 245: 43-49. Spurrs, A. 1969. A low viscosity epoxy embedding medium for elec- tron microscopy. J. Ultraxtnicl. Res. 26:31-43. Tail, J. 1911. Types of crustacean blood coagulation. J. Mar Biol. Assoc. U. A'. 9: 191-198. Toney, M. E. 1958. Morphology of the blood cells of some Crustacea. Growth 22: 35-50. Wood, P. J., and L. P. Visentin. 1967. Histological and histochemical observations of the hemolymph cells in the crayfish, Orconectes vir- ilis J Morphol. 123:559-568. Wood, P.J., J. Podlewski, and T. E. Shenk. 1971. Cytochemical ob- servations of hemolymph cells during coagulation in the crayfish. Ommeaes virilis. J Morphol 134: 479-488. Reference: Bio/ Bull 178: 46-54. (February, 1990) Respiratory Responses of the Blue Crab Callinectes sapidus to Long-Term Hypoxia PETER L. DEFUR1*, CHARLOTTE P. MANGUM2, AND JOHN E. REESE2 ^ Department of Biology, George Mason University, Fairfax, Virginia 22030 and2 Department of Biologv. College of William and Mary, Williamsburg, I'irginia 23185 Abstract. Blue crabs (Callinectes sapidus) were held in hypoxic (50-55 mm Hg) water for 7-25 days. Post- branchial blood PO2 fell by about 80% within 24 h and then remained unchanged. Postbranchial blood total CO: increased within 24 h and remained elevated for the duration of the experiment. There was no change in post- branchial blood pH, osmolality, or Cl. Lactate, urate, and Ca0 all raise the O: affinity of blue crab hemocya- nin; by 25 days, blood lactate and urate had risen slightly, but Ca+: had increased dramatically. Hemocyanin con- centration had also increased by 25 days. At both 7 and 25 days there was an intrinsic increase in hemocyanin-O2 affinity and a change in subunit composition. The highly adaptive homotropic change is believed to be due to an attendant shift in the proportions of two of the three vari- able monomeric hemocyanin subunits. Thus, both het- erotropic and homotropic adaptations enhance blood oxygenation at the gill during long-term hypoxia. Introduction The respiratory response to long term hypoxia, de- fined here as exposure for three or more days, has been examined in six species of aquatic crustaceans: three crayfish (McMahon el a/., 1974; Dejours and Armand, 1980; Wilkes and McMahon, 1982a, b), a lobster (Mc- Mahon et a/., 1978), a crab (Burnett and Johansen, 1981), and a prawn (Hagerman and Uglow, 1985). In all cases, the initial response was hyperventilation, which resulted in a respiratory alkalosis. Subsequently, how- ever, the response in different species became diverse. Received 1 November 1988; accepted 30 November 1989. * Present address: Environmental Defense Fund. 1 108 E. Main St.. Richmond. VA 232 19. Blood pH either returned in full (Wilkes and McMahon, 1982a) or in large part (Butler et ai, 1978; McMahon et ai, 1978) to the normoxic level, or remained decidedly alkalotic for as long as 3-8 days (Dejours and Armand, 1980; Burnett and Johansen, 1981). Crustacean hemocyanins (Hcs) typically have very large normal Bohr shifts; the quantity A log Pso/ApH is commonly near -1 (Mangum, 1980). Thus, the alkalo- sis, which had also been observed during acute hypoxia (Truchot, 1975; Burnett, 1979), would have the impor- tant respiratory consequence of raising blood O: affinity. The increases were observed, but were attributed by pre- vious workers to the rise in blood pH. We now know that at least three other allosteric effectors, viz.. L-lactate (Truchot, 1980; Booth et ai. 1982), Ca+: (Mangum, 1985) and urate (Morris et ai. 1985; Lallier et ai. 1987), also may increase HcO2 affinity during acute hypoxia. The levels of these effectors in the blood during pro- longed exposure, however, are not known. Intrinsic changes in O2 affinity of He in response to prolonged changes in environmental factors have re- cently been observed in both crayfish (Rutledge, 1981) and crabs (Mauro and Mangum, 1982; Mason et ai, 1983; Mangum and Rainer, 1988). In the blue crab, Cal- linectes sapidus Rathbun, salinity-induced changes are accompanied by shifts in the concentrations of two of the 5-6 subunits of the He polymers (Mason et ai. 1983). The changes in one of the two subunits fully explains the attendant shift in O: affinity (Mangum and Rainer, 1988). Although an intrinsic molecular change would not be expected to occur during acute hypoxia, it might occur during long-term hypoxia. In the shrimp Crangon cran- gon. He levels increase sharply during prolonged hypoxia 46 HYPOXIA IN BLUE CRABS 47 (Hagerman, 1 986); a similar increase in the blue crab ap- pears to hasten intrinsic molecular adaptation to a salin- ity change (Mason el a/., 1983). Therefore, we have ex- amined the possibility that a change in net synthesis or degradation during hypoxia produces additional or re- placement molecules that differ from those in normoxic animals. The blue crab inhabits many bodies of water that are not invariably normoxic (Carpenter and Cargo, 1957; May, 1973; Carlo, 1979; Harpers al., 1981; Turner and Allen, 1982). Lethal levels (PO2 < about 50 mmHg) of- ten kill animals that cannot escape from pots (Carpenter and Cargo. 1957). Free-ranging animals may even emerge into air (Loesch, 1960; Officer el al., 1984), de- spite limited tolerance of it. In the Chesapeake Bay sys- tem, sublethal O: levels, still well below normoxia, are so widespread that crabs must encounter them for long periods. Water PO2 in the range 50-100 mmHg is char- acteristic of the Chesapeake Bay for several months dur- ing the spring and fall (Officer el al., 1984; Seliger el al, 1985). In the summer, cyclical destratification in the channels produces sublethal hypoxia throughout the wa- ter column for several weeks at a time (Webb and D'Elia, 1980). Processes ranging from tidal flushing of the marshes, to seiching of the water in the channels, pro- duce sublethal hypoxia in extensive areas of shallow wa- ter as well (Carpenter and Cargo, 1957; Axelrad el al., 1976; Kemp and Boynton, 1980; Taft el al, 1980; Ma- lone el al, 1986; MacKiernan, 1987). We have determined the response of blood respiratory and osmotic variables of the blue crab Callinectes sapi- dus Rathbun to sublethal hypoxia. The treatments in- clude acute exposure, more prolonged exposures similar to those employed in previous studies, and still more pro- longed exposures designed to elicit intrinsic changes in the He molecule. We have measured all of the known physiological effectors of HcO2 binding, both hetero- and homotropic. Materials and Methods Animals Large (ca. 1 20-220 gm wet wt.), male, intermolt crabs were obtained from commercial watermen or collected by the first author near the Rhode River or the mouth of the Patuxent River in Maryland. They were returned to Fairfax. Virginia, and maintained in open containers (100-200 1) of natural, aerated water (500-530 mOsM, 2 1 -23°C) for 4-7 days prior to the experimental hypoxia. Water osmolality was monitored frequently and distilled water added as needed; water pH was also monitored and kept above 7.9 by the addition of NaHCO,. Crabs were fed thawed smelt twice a week throughout the control and experimental periods, but not within 24 h of sam- pling. Design The experimental protocol consisted of taking blood from the same crabs before, during, and following expo- sure to hypoxia. Insofar as possible, the design of paired observations on the same individuals was maintained, and the data were analyzed accordingly. The significance of changes in blood pH. PO:, total CO2, lactate, Cl, and osmolality was tested according to Student's /-test for un- grouped (paired) data as the mean of the differences of each value from the control for the same individual, the null hypothesis being that there was none. Because the same individuals were sampled repetitively, the blood samples were necessarily small (0.5 ml), thus insufficient volumes of many samples remained for the other mea- surements. The paired observations design could not be maintained for the analysis of Ca+2, urate, and He con- centrations; these values were analyzed by Student's t- test for grouped data (two samples). For measurements such as O2 binding, which require a total of more than 0.5 ml of material, samples were pooled; the results were analyzed by regression. In these measurements, pooled samples for —1 and 7-day exposure were made up of blood from the same individuals, whereas that for 25-day exposure was composed of blood from different animals. Hypoxia Nitrogen gas was bubbled into the water to reduce the PO2 from 140-155 to approximately 55 mm Hg in 3-4 h, and then the bubbling was stopped. Thereafter a slow, steady air flow was maintained, and N2 was bubbled into the water only as needed to offset the air. N2 flow was regulated by a metering system that bal- anced N2 against rising O2. The system consisted of an O2 electrode and meter (Instrumentation Laboratories Models 1703B and 113, respectively), the output of which provided the signal for a logic circuit that con- trolled an electric gas valve. The circuit was set to evalu- ate the output of the meter every 5 min and to open the valve if the PO2 had increased above the set point of 50 mmHg. Thus, once the initial PO2 of 50 mmHg was reached, further changes were confined to the ranges 50- 55 mmHg and occurred slowly. The continuous airflow stirred the water and ensured that O2 uptake by the crabs did not reduce water PO2 below 50 mmHg. Blood sampling Postbranchial hemolymph samples for the determina- tion of in vivo respiratory variables were withdrawn through holes in the carapace dorsolateral to the heart. 48 P. L. DEFUR ET AL. The holes had been drilled four or more days prior to the control period, and covered with latex rubber affixed with cyanoacrylate cement. On occasion, prebranchial hemolymph was also withdrawn from the base of one of the legs for the measurement of Cl, osmolality, and lactate. Blood samples were withdrawn into iced syringes and immediately placed on ice to slow clotting. After deter- mination of blood gas and acid-base variables, these sam- ples were frozen for the remaining analyses. The samples for HcO: binding were kept cool and, with the exception noted below, unfrozen; O: binding measurements on blood from normoxic and hypoxic animals were made within a few days. In vivo variables Hemolymph pH was measured with a thermostatted glass capillary electrode (Radiometer G299A) and meter ( Radiometer PHM 84). PO2 was measured with a polaro- graphic electrode (Radiometer E5046) and acid-base an- alyzer (PHM 72). Total CO: was determined in 50 n\ samples with a Corning Model 965 CO2 analyzer. Lac- tate was measured enzymatically (Sigma Procedure No. 826), with the modifications for He-containing blood de- veloped by Graham el al. ( 1983). Osmolality was deter- mined with a vapor pressure osmometer ( Wescor Model 5100C). Ca1" activity was determined with a Radiometer elec- trode and PHM 84 meter, following 1:99 dilution with 0.05 Tris Maleate buffer, pH 7.6 (Mangum and Lyk- keboe. 1979). Chloride was measured by electrometric titration (Corning Model 920). We determined urate as the quinoneimine produced by digestion with uricase (Sigma Procedure No. 685), af- ter first verifying that 100% of the urate added to test samples of blood could be recovered. Because He ab- sorbs at 685 nm, the absorbance was measured in repli- cate, once with and once without the analytical reagents, and the interference of He was subtracted. HcO: binding and He concentration Hemolymph was declotted with a tissue grinder, cen- trifuged, and then dialyzed at 4°C for 24-28 h against a saline made up according to Mason el al. (1983). HcO: binding was determined by the cell respiration method, in which the deviation from a constant rate of O? deple- tion is used to estimate fractional oxygenation at the measured PO: (Mangum and Lykkeboe. 1979). Before determining He concentration in the blood, we eliminated the effect of light scattering, dissociating the native polymers to monomeric subunits by dilution ( 1: 39) with Tris HC1 containing 50 mM EDTA. Absor- bance of He was measured at 338 nA/with a Milton Roy Spectronic 501 spectrophotometer; the concentration was calculated using the extinction coefficient for por- tunid He reported by Nickerson and Van Holde ( 197 1 ). Electrophoresis Alkaline dissociation electrophoresis (Hames and Rickwood, 1981) of He monomers on polyacrylamide gel slabs was performed as described by Mangum and Rainer (1988). In the present case, aliquots of the three pools of blood used to compare O2 binding in normoxic and hypoxic animals were run on the same gels, which were scanned with a Gelman Instruments Model 3372 integrating densitometer (modified for transparent me- dia). Changes were estimated by comparing peaks of the variable subunits with that of an invariant subunit. Results The experiment was performed three times. The first hypoxic exposure period was 7 days, and samples were taken at - 1 (control), 1, 4, and 7 days. The second and third exposure periods were 25 and 23 days, and samples were taken at -1, 7, 9, or 16, and 23 or 25 days. In the first experiment, the crabs were also sampled one day af- ter the return to normoxic water. Behavior and mortality When ambient PO2 fell to 50 mm Hg, most of the crabs became active and moved slowly around the aquarium, as reported by Lowery and Tate ( 1 986); some crabs tried to climb out of the water. Elevated activity ceased within a few hours, and the animals became qui- escent for the duration of the hypoxic exposure. They frequently buried in the sand lining the bottom of the aquarium. Mortality was low. There was none during the first ex- periment and only 20% during the longer exposures. In our experience, this level would be low under normoxic conditions. Hemolymph variables Many of the data for normoxic animals are unexcep- tional (Table I, day - 1 ), but pH and PO: are high relative to those in the literature for this species (e.g., Weiland and Mangum, 1975; Mangum el al.. 1985). Our value for blood urate in normoxic animals is also considerably lower than that reported by Morris el al. (1986) for the crayfish Austropotamobius pallipes (0.35 mM}. but it is similar to the figure (0.08 mM) found in the portunid crab Carcinus niaenas (Lallier et al.. 1987). The low urate levels in the portunid bloods may explain the ab- sence (Mangum, 1983) or small size (Truchot, 1975) of HYPOXIA IN BLUE CRABS 49 Table 1 Respiratory variables* in the heinolvmpli of blue erabs exposed lo moderate hypoxic? for 7-25 days3 (day - 1 = control) No. animals PaO, (mm Hg) pHa CaCO2 (mM) Lactate(mM) Ca+2 Urate (mM) (mM) [He] (g/ 100 ml) Duration4 (days) 7 23-25 7 23-25 7 23-25 7 23-25 7 23-25 23-25 23-25 23-25 Day -1 8 9-11 9x 70 7.81 7.71 2.3 2.3 0.01 0.94 6.73 0.05 3.11 (control) ±3 ±8 ±0.03 ±0.03 ±0.2 ±0.4 ±0.01 ±0.09 ± 0.75 ± 0.02 ±0.41 1 8 18 7.79 5.4 0.08 ±4 ±0.02 ±0.3 ±0.05 4 8 15 7.80 5.1 0.08 ±4 ±0.04 ±0.3 ±0.06 7 8 4-8 13 22 7.83 4.6 3.6 0.09 4.73 0.03 1.26 + 2 ±2 ± 0.03 ±0.4 ±0.2 ± 0.05 ± 0.75 + 0.00 ±0.11 Recovery 8 87 7.77 3.3 0.00 ± 10 ±0.02 ±0.2 ±0.00 9 6 19 7.81 2.6 0.80 ±3 ±0.06 ±0.2 ±0.09 16 4 21 ±3 23-25 5-11 21 7.76 3.2 1.79 10.1 0.14 4.40 ±4 ±0.04 ±0.3 ±0.50 ± 0.5 ± 0.02 ±0.19 1 Top no. = mean, bottom no. = S.E. : -50-55 mm Hg, 21-2.VC, 500-530 mOsM. 3 Symbols: PaO2 = postbranchial blood PO2, pH = postbranchial blood pH. CaCO2 = postbranchial blood total CO:. 4 Two columns under the first five headings represent different exposure periods, as indicated. changes in HcO: affinity following dialysis of normoxic serum against a physiological saline. Within 24 h of the onset of the 7-day hypoxic exposure in the first experiment, postbranchial blood PO; (PaO:) fell by 80% and total CO, (CaCO2) more than doubled while pH remained unchanged (Table I). The subse- quent changes in these three variables are not significant (P > .05). The apparent increase in lactate is not signifi- cant (P > .05) if the difference at each sampling period is tested against zero. If the particular sampling period is disregarded and the maximum increase for each individ- ual is tested against the null hypothesis, however, then the mean increase (0.18 ± .06 mM) is significantly greater than zero (P < .01 ). More important, this change is very small, indicating a highly aerobic condition. Within 24 h of return to normoxic water, control levels of postbranchial PO2 were restored, although total CO2 remained slightly elevated (P < .02). In the second and third experiments, the same and longer periods of exposure (23-25 days) to hypoxia re- sulted in similar patterns of PO2, pH, and total CO: (Ta- ble I). Once again blood lactate increased slightly, al- though in this case significantly (P = .05), regardless of how the data are grouped. At 25 but not 7 days, blood Ca+2 rose by a large amount (P = 0.025), blood urate rose significantly (P < .001 ), and He concentration increased by almost half (P < . 05). In none of the three experiments, did blood Cl or os- molality change, nor were there any coherent trends in these variables. The mean values (±S.E., n = 61) for all periods are 355 (±4) mA/Cl and 756 (±5) mOsm. HcO} binding and subunit composition At the end of the 7-day period, a change in HcO2 affinity had clearly occurred (Fig. 1, upper panel). The 95% confidence intervals around the regression lines de- scribing the data for -1 and 7 days in Figure 1 do not overlap at any point (Table II). The slopes of regression lines (-0.97 ± 0.21 95% C.I. for normoxic and -1.1 1 ± 0.1 1 for 7 days, hypoxic animals), and thus the Bohr shifts, do not differ significantly. The relationship between cooperativity (n) and pH of the decapod Hcs is usually quite complex, often reaching a maximum in the middle of the physiological pH range and showing lower values at the extremes. No very sensi- 50 P. L. DEFUR ET AL 60 I 40 20 (torr) 7.0 7.2 7.4 7.6 7.8 8.0 8.2 PH "so o o 7.0 7.2 7.4 7.6 7.8 8.0 8.2 Figure 1. Oxygen binding by stripped He of normoxic (O), 7 days hypoxic (•). and 25 day hypoxic (A) blue crabs. 25°C, 0.05 M Tns maleate buffered saline containing 494 mM NaCl, 1 6 mM KC1. 23 mA/ CaCI:. 23 mA/ MgCl, 25 mA/(Na):SO4 and 2 mM NaHCO,. Upper panel shows oxygen affinity (PsuK and lower panel shows the coopera- tivityat PCK = Pw (n,,,). live procedure for data analysis is available. In the lower panel of Figure 1, cooperativity seems to decrease at 7 days, but the mean values are not significantly different (P = .10). A Mann-Whitney U test also did not distin- guish a significant change. There was a clear change in the subunit composition of the Hcs (Fig. 2). Specifically, subunits 3, 5, and 6 de- creased in concentration relative to subunit 4, which has remained invariant in samples taken, by now, from more than 500 individuals (Mangum, unpubl. obs.; Rainer, 1988). After 25 days of hypoxia, HcO: affinity increased fur- ther (Fig. 1). The 95% confidence interval around a re- gression line describing O: affinity does not overlap those for control or 7-day hypoxic animals in any part of the pH range (Table II). The slope of the regression line de- scribing the 25 day data (-1.00 ±0.17 95% C.I.) does not differ from the other two. In this case, the mean value for cooperativity (1.95 ± 0.06 S.E.) of the He from 25 day hypoxic animals (Fig. 1) differs significantly (P= .02) from that for control and 7 day hypoxic animals (2.41 ±0.14). All three variable subunits decreased further in con- centration relative to subunit 4 (Fig. 2). Indeed the pres- ence of subunit 3, which is sometimes completely absent (Mason et ai. 1983), is dubious. By the end of 25 days of hypoxia, the concentration of subunit 6 had dropped from the highest in the control period to rank fourth; no. 5 had dropped from second to third; and no. 3 had dropped from clearly present to undetectable, or nearly so. The two weak bands appearing between peaks 1 and 2 of the He from hypoxic crabs (Fig. 2B, C) are usually not present: they are not copper containing and have no influence on oxygen binding (Mangum and Rainer, 1988). Although the effects of elevated Ca+2 (and L-lactate) on C. sapidus He are well known (e.g.. Booth et a/., 1982; Mangum, 1983; Mason el al., 1983; Johnson el ul., 1 984), those of urate are not. Therefore we used the small amount of (frozen) blood remaining after the measure- ment of extrinsic co-factors to examine urate sensitivity. Figure 3 shows that small quantities of urate clearly raise O: affinity of the He of animals exposed to hypoxia for 25 days, with its altered subunit composition. The positions (but not slopes) of regression lines describing the data for 0. 0.55. and 2.35 mM urate all differ at P = 0.05. Al- though the data suggest little further difference beyond 1.17 mA/. the small number of observations permitted by the volume of material available mandates some cau- tion on this point. We emphasize that, unlike the measurements in Fig- ure 1 , those in Figure 3 were made on Hcs that had been frozen for several months. As mentioned earlier (Man- gum, 1983), freezing does not usually influence Pso (see also Morris, 1988), at least if the He retains its native Table II Ninety-five percent confidence intervals around semilogarithmic (log Y) regression lines fit in P50 data in Figure I PH Control (r = 0.946) 7-day hypoxia (r2 = 0.994) 25-day hypoxia (r: = 0.965) 7.0 57.2-60.5 49.7-53.5 31.0-33.7 7.2 36.6-38.6 30.5-32.7 19.6-21.2 7.4 23.4-24.6 18.7-20.0 12.4-13.4 7.6 15.0-15.7 11.6-12.2 7.86-8.46 7.8 9.54-10.1 7.01-7.48 4.96-5.35 8.0 6.09-6.44 4.29-4.59 3.13-3.39 8.2 3.88-4.12 2.62-2.81 1.97-2.15 HYPOXIA IN BLUE CRABS 51 Figure 2. Densitometer scan of slab gels, showing native suhunits (numbered peaks) of blue crab He separated by charge (subunit 1 is at the anodal end). The He applied to the gels was from the same samples from which data were collected for Figure 1. A. Normoxic. B. 7-day hypoxic. C. 25-day hypoxic. Subunit 3 in C is dubious. optical properties. The control values in the two figures are essentially identical (95% confidence intervals around regression lines broadly overlap). Because freez- ing frequently influences cooperativity, however (Man- gum, 1983; S. Morris, pers. comm.), we did not analyze the cooperativity of the thawed samples. Morris el al. (1986) found no effect of urate on cooperativity. Discussion Blood pH.PO:. and CO: In view of the unanimity of previous reports of blood alkalosis accompanying hypoxia of virtually any dura- tion in crustaceans, we were surprised to find none in the present experiments. Hyperventilation and alkalosis are not always precisely correlated; in the crayfish O. rusti- cus, ventilation returns to control levels while blood pH is still elevated (Wilkes and McMahon, 1982a). But all reports agree that blood pH rises at some point. In fact, in severely hypoxic C. maenas, Lallier et al. (1987) re- ported a pH increase of more than 0.3 units accompany- ing an increase in lactate of 25 mA/, despite no change in base. In other investigations of C. sapidus, we have either found (Pease et al., 1986), or not found (Mangum and Weiland, 1975, and unpubl. obs.), a hypoxic alkalo- sis. The response in this species is apparently highly la- bile, for reasons that are presently unclear. The increases in lactate observed here seem too small to offset a respira- tory alkalosis brought about by vigorous hyperventila- tion (Pease and deFur, 1987). Further increases in pH and PO2 might have been precluded because ventilation was already high. Elevated ventilation during the control period could have arisen from sensory stimulation (Mc- Donald^/ al., 1977) and been unrelated to ambient PO2. Extrinsic modulation of HcO: affinity In many crustaceans L-lactate is a physiologically im- portant modulator of HcO; affinity, both during exercise and hypoxia (Booth et al., 1982; Graham et al.. 1983) and very severe environmental hypoxia (Lowery and Tate, 1986; Lallier et al.. 1987). The small increases ob- served here would raise HcO2 affinity at physiological pH by less than 1 mmHg. The increase in urate would raise HcCK affinity by a similarly small amount. In contrast, Ca+2 may be an important effector after 23-25 (but not 7) days, by which time the increase in Ca+: would raise HcO: affinity by more than 5 mmHg. 40 20 Pso (torr) 7.1 7.3 7.5 7.7 7.9 8.1 8.3 PH Figure 3. Effect of urate on O2 affinity of stripped He from hypoxic animals. Conditions as in Figure I . (O) no urale: (•)0.57 mA/;(A) 1.15 mM: and (D) 2.35 mA/. 52 P. L. DE FUR ET AL. These conclusions, inferred from the data in Figure 3 and those of Mason et at. (1983) and Johnson el at. (1984). assume that the two organic effectors act com- pletely independently and cumulatively, which is not en- tirely true (S. Morris, pers. comm.). The interaction of Ca+2 with the organic effectors is included in the present data for HcO; affinity because the actions of urate and lactate were determined in the presence of Ca+:. The in- teraction of urate and lactate would further diminish their effects, albeit by a small amount. Intrinsic adaptation ofHcO? affinity Estuarine (and also normoxic) blue crabs transferred to high salinity in the laboratory show a rapid decrease in He concentrations. Concomitantly, HcCK affinity in- creases and the levels of subunits 3 and 5 decrease, closely resembling the He in animals freshly caught at a seaside location. Subunit 6 remains unchanged. In sea- side (and normoxic) animals transferred to low salinity, the He concentration rapidly increases while the HcO: affinity decreases (Mason et ul., 1983). The intrinsic change opposes the effects of salinity-induced changes in extrinsic co-factors. In initial samples freshly taken at the estuarine and seaside localities, the vast majority of animals exhibited the molecular phenotype associated with the comparable acclimation salinity in the laboratory (C. P. Mangum andG. Godette, unpubl. obs.; Rainer et a/.. 1985). How- ever, subunit 6 was variable in both samples, implicating another environmental or physiological effector unre- lated to salinity per se. Moreover, when the field study was enlarged, no clinal variation of the three subunits was obvious along a salinity gradient between the two localities (Rainer, 1988); these findings also suggest a confounding variable. Under the same ionic conditions, the O: affinities of blue crab Hcs composed of different combinations of the variable chains indicate that the levels of subunits 3 and 6 both influence oxygen binding; the effects of variation in subunit 5 are not entirely clear (Mangum and Rainer, 1988). Changes in subunit 3 (alone) can fully explain the difference between seaside and estuarine animals. How- ever, changes in subunit 6 (alone), smaller than those ob- served here, significantly alter HcO: affinity by almost 20% at physiological pH. Although there is no difference between an He with low levels of only subunit 3, and one with low levels of both 3 and 5. an He with low levels of 5 alone has not been examined. The present results suggest that blue crab He is intrin- sically adaptable to prolonged hypoxia as well as to salin- ity. The adaptation may be expedited by an increase in He concentration, which is clear at 25 days, and it is ac- companied by changes in the same three subunits al- ready known to be variable. A decrease in concentration of subunits 3 and 6 during hypoxia has the same effect as that of decreasing either alone or in combination; i.e., increasing O2 affinity (Mangum and Rainer, 1988). The present findings suggest that, while subunits 3 and 5 respond to a change in both salinity and oxygen, subunit 6 responds only to oxygen. The changes in subunits 3 and 5 as a result of hypoxia were much smaller than the salinity-induced changes, but the changes in P5I) were about the same in the two groups, at physiological pH. The smaller changes in the amounts of subunits 3 and 5 in hypoxia may be due to lower levels at the onset of hypoxia (for comparison see fig. 1 in Mangum and Rainer, 1988). The oxygen-induced change in subunit 6, however, was much larger than observed by Mangum and Rainer (1988). A greater change in subunit 6 may offset a smaller change in subunit 3, and the intrinsic ad- aptation of HcO: affinity to hypoxia may involve the change in subunit composition. Moreover, we suggest that the variation of subunit 6 in nature is related to hyp- oxia, which does not vary along a salinity gradient in a simple fashion. Finally, the increase observed here in He concentra- tion occurs widely in hypoxic crustaceans. In different species its magnitude may be much greater, it may occur in a far shorter period, and it may occur at a much lower temperature (Hagerman and Oksama, 1985; Hagerman and Uglow. 1985; Hagerman, 1986). It will be interesting to learn whether intrinsic molecular adaptability is sim- ilarly widespread. Acknowledgments Supported by NSF Grant DCB 84-14856 (Regulatory Biology) to CPM. This work is a result of research spon- sored in part by NOA A Office of Sea Grant, U. S. Depart- ment of Commerce, under Grant No. NA85AA-D- SGO 1 6 to the Virginia Graduate Marine Science Consor- tium and Virginia Sea Grant College Program. The U. S. Government is authorized to produce and distrib- ute reprints for governmental purposes notwithstanding any copyright notation that may appear hereon. Literature Cited Axelrad. D. M., K. A. Moore, and M. E. Bender. 1976. Nitrogen, phosphorous and carbon flux in Chesapeake Bay marshes. Bull 79, Water Resources Research Center, Virginia Polytechnic Institute, Blacksburg. VA. 182 pp. Booth, C. K., B. R. McMahon, and A. VV. Finder. 1982. Oxygen up- take and the potentiating effects of increased hemolymph lactate on oxygen transport during exercise in the blue crab Callinectes sapi- dus. J Comp Physinl 148: 111-121. Burnett, L. E. 1979. 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(February. 1440) The Horseshoe Crab Tachypleus tridentatus has Two Kinds of Hemocytes: Granulocytes and Plasmatocytes PER PLOUG JAKOBSEN AND PETER SUHR-JESSEN Department ofAnatomv& Cytology, University of Odense, Campusvej 55, 5230 OdenseM, Denmark Abstract. For the first time, the fine structure of the he- mocytes from the horseshoe crab Tachypleus tridentatus is investigated by transmission electron microscopy and light microscopy serial sectioning. Two morphologically distinct, ellipsoidal, and mononucleate hemocytes — granulocytes (amebocytes) and plasmatocytes — are re- vealed. Granulocytes constitute about 97% of the hemo- cytes. They have a marginal band of microtubules, a het- erochromatic nucleus, distended but poorly developed RER, few free ribosomes, few mitochondria, and many large secretory granules. The majority of these granules have a uniform content and are mature. Structured gran- ules located in the proximity of Golgi complexes may be immature transitional stages leading to the mature uni- form granules. Upon stimulation with endotoxin from gram negative bacteria, the mature granules become transitory structured before exocytosis. In contrast, the immature granules are not exocytosed. Plasmatocytes constitute about 3% of the hemocytes. They differ from granulocytes by having an euchromatic nucleus, a well- developed RER of flattened or tubular cisternae, many free ribosomes, many mitochondria, but only few, if any, large secretory granules. Apparently, plasmatocytes are not affected by endotoxin. The relationship and possible functions of granulocytes and plasmatocytes are dis- cussed and compared with those of the horseshoe crab, Limulus polyphemus. Introduction Horseshoe crabs are "living fossils." which have un- dergone little morphological evolution during the last 360 million years; they can be traced back more than 500 million years (Sekiguchi and Sugita, 1980; Shishikura el Received 14 June 1 984; accepted 28 November 1984. al., 1982;Mikkelsen, 1988). If this stability is reflected in their physiology, studies of their immune defense system may shed light on when and how the different parts of it evolved in horseshoe crabs and possibly also in higher and more recent phyla. Inoculation of gram negative bacteria or their endo- toxins into the hemolymph of horseshoe crabs cause fatal intravascular coagulation (Bang, 1956). This involves exocytosis of the large secretory granules from the he- mocytes. These granules contain coagulogen and all other proteins necessary for the coagulation (Levin and Bang, 1964; Ornberg and Reese, 1981; Iwanaga el al., 1986; Suhr-Jessen et al., 1989). Hemocyte (amebocyte) lysates can be made from all four extant species of horse- shoe crabs, and are now extensively used to detect min- ute quantities of endotoxin (Shishikura el al., 1 983; Wat- son et al., 1987). The horseshoe crab best characterized is Limulus poly- phemus. Until recently, only one hemocyte, the granulo- cyte, had been identified in this species (Dumont et al., 1966; Levin and Bang, 1 968; Copeland and Levin, 1985; Tablin and Levin. 1988). However, a second hemocyte, the plasmatocyte, has been identified independently by light microscopical observations of live cells, by light mi- croscopical serial sectioning of fixed cells, and by trans- mission electron microscopy alone and combined with immuno-gold labeling (Suhr-Jessen eta/., 1989). Inaddi- tion, cyanocytes and cyanoblasts have been reported to be present in the sinusoids around the compound eyes (Fahrenbach, 1970). Early light microscopical studies suggested that Tachypleus tridentatus had two kinds of granulocytes (Shishikura et al.. 1977; Shishikura and Sekiguchi, 1979). The aim of the present study is to characterize the fine structure of T. tridentatus hemocytes — the cellu- lar part of the immune defense system — in the pres- 55 56 P P. JAKOBSEN AND P. SUHR-JESSEN ' RER v X • ^ , M~ \ :.;' jJHtj 3p> 9 PER RER 5 jjm •- • *• "^g PM 3 250 nm • 50 nm Figure 1. I'm/iv/'/i'ii^ initcntaius granulocyte with its heterochromatic nucleus (N), and many large secretory granules (GR). Mitochondria (M). Rough endoplasmic reticulum (RER). Marginal band (arrow- heads). T.\Cin'l'U-:i'S IRIDI.M III .S III MOO IIS 57 500 nm • ' 500 nm Figure 5. The distended rough endoplasmic reticulum (RER) from a Tacliyi>lcn.\ iridematus granulo- cyte. Golgi complex (G). Figure 6. The flattened or tubular RER from a T. Iridentatux plasmatocyte. Many free ribosomes are present (arrowheads). Golgi complex (G); nucleus (N). Figure 1. T. Iridcnlalus granulocyte with a structured (immature) large granule (IG) located in close proximity to a Golgi complex (G). Centrioles (C): mitochondria (M); uniform (mature) granule (MG); nucleus (N). ence and absence of endotoxin. We show that the gen- eral circulation of T. tridenlatm contains plasmato- cytes and a single class of granulocytes. Furthermore, a temporal relationship is described for the formation to final secretion of the large secretory granules in the granulocytes. Figure 2. T. tridenlalm plasmatocyte with its euchromatic nucleus (N), well-developed RER. and many mitochondria (M). Marginal band (arrowheads). Figures 3, 4. Longitudinal and transverse sections of marginal bands of microtubules (arrowheads) in T. indcnlalus hemocytes. Plasma membrane (PM). 58 P. P. JAKOBSEN AND P. SUHR-JESSEN 12a Figures 8-10. Differently structured immature large granules from Tachyplen\ tridcnlatus granulo- cytes. Insert: close-up of the about 17-nm tubular structures in transverse and longitudinal section (bar equals 100 nm). Apparently, a coated pit (CP) and a coated vesicle (CV) are present. Golgi complexes (G). TACHYI'LKUS TRIDENTATUS HEMOCYTES 59 Materials and Methods Six adult T. tridentatus females (males were not avail- able) (prosomal width: 30-33 cm) were collected in the Tonkin Gulf, China, and kept in seawater (3.0% NaCl) at 15°C at The Danish Aquaculture Institute, H0rsholm, for up to nine months. Throughout this period, hemo- lymph samplings from all animals gave similar results. Hemolymph was drawn by cardiac puncture at the etha- nol-cleaned prosoma-opistosoma junction. Access to the heart was made by a 19-gauge needle alone or combined with a 5-ml syringe containing fixative or, as part of a total bleed of the animal, through a large cut by a sterile (LPS-free) scalpel. The three methods gave similar re- sults. Hemolymph was floating directly into 5% glutaral- dehyde in 0.1 M sodium cacodylate buffer, pH 7.4, to give a final glutaraldehyde percentage of no less than 4. Samples were also incubated for 5 to 300 s with 10 4 10"" g E. coli endotoxin (Sigma no. L 3755)/ml hemo- lymph prior to fixation. The fixed samples were pro- cessed as described (Willumsen el ai, 1987). Transmis- sion electron microscopy sections (about 50 nm) were mounted on pioloform F-50 coated Cu- or Ni-grids, con- trasted with lead citrate, examined in a Jeol JEM-100CX electron microscope at 80 kV, and photographed using Agfa-Gevaert 23D56 film. Light microscopy serial sec- tions (about 1 .0 /urn) were stained with toluidin blue, ex- amined in a Zeiss microscope (numerical aparture: 1 .30) at 400X using immersion oil, and photographed using Kodak panatomic X film. To eliminate inaccuracies due to minor differences in the thickness of the sections, a plasmatocyte was always compared with a nearby granu- locyte starting and ending at almost the same section numbers. In the two cells, the number of cuts through mitochondria rather than the actual number of mito- chondria was determined. Assuming that the mitochon- dria are randomly oriented and approximately of the same size in the two cells, any consistent deviation from 1 in the PL/GR ratio reflects differences in numbers of mitochondria. Results General morphology of the hemocytes T. tridentatm hemocytes are spheroid, and about 1 5- 20 jum at their longest axis (Figs. 1,2, 15). A marginal band of microtubules run parallel to the longitudinal axis of the cells at least one microtubule diameter beneath the plasma membrane (Figs. 1-4). The almost parallel arrangement of the microtubules, combined with the electron-dense material seen between them, suggest that they are connected (Figs. 3, 4). Each hemocyte has a sin- gle, non-lobated nucleus containing one or a few nucleoli. The cells also contain rough endoplasmic re- ticulum (RER), free ribosomes, mitochondria with la- mellar cristae, and Golgi complexes with 3-6 layers of cisternae — the cis-ones being more distended than the trans-ones (Figs. 1-2, 5-7). The paired centrioles form an obtuse angle to each other (Fig. 7). No sign of mitosis was seen in any of the examined hemocytes. Digestive vacuoles and apparently coated pits and coated vesicles are also present (Figs. 9, 14). No cytoplasmic crystals were observed. Granulocytes About 97% of the hemocytes are granulocytes. They have a heterochromatic nucleus, a poorly developed but distended RER, few free ribosomes, and few mitochon- dria (Figs. 1, 5). However, their most prominent feature is the many large secretory granules with diameters around 1-2 ^m (see below). Large secretory granules The majority of the large secretory granules in granu- locytes have a uniform content (Fig. 1). However, one class of granules, with structures ranging from amor- phous to highly organized tubules with diameters around 1 7 nm, are seen in close proximity to Golgi complexes (Figs. 7-10). When hemocytes are stimulated with endo- toxin a second class of structured granules containing tu- bules with diameters around 10 nm become transitorily present (Fig. 1 1). A reverse relationship seems to exist between the numbers of structured granules of the sec- ond class and the uniform granules. After this, exocytosis occurs (Fig. 1 2). In contrast, structured granules of the first class are usually not exocytosed following stimula- tion with endotoxin (Figs. 13, 14). Following exocytosis, the granulocytes gain numerous pseudopodia, and the organelles collect in the center of the cell surrounded by microtubules (Figs. 13, 14). Plasmatocytes Plasmatocytes constitute about 3% of the hemocytes. This conclusion is reached by examining duplicate sam- Kigure 1 1 . Granulocyte from T. tridentatus incubated with 10 4 g endotoxin per ml hemolymph for 30 s. A stimulated large secretory granule (SG) is in close connection with the plasma membrane (PM). Its tubular structures (arrows) have a diameter around 10 nm, while 17-nm tubular structures (arrowheads) are present in the immature granule (IG) located in close proximity to a Golgi complex (G). Figure 12. Successive stages in exocytosis of the large secretory granules from T tridcnlatus granulo- cytes. Bar length: 500 nm. 60 P. P. JAKOBSEN AND P. SUHR-JESSEN - - ': «* *v*c %%*&& •' Figure 13. Granulocyte from Tachypleus tridentatus incubated with 10 4 g endotoxin per ml hemo- lymph for 300 s. The large secretory granules are exocytosed (arrow), except the immature ones (1G). and pseudopodia (P) are projected. Nucleus (N). pies from each of six animals. From all samples, at least 10 sections, each containing more than 100 he- mocytes, were examined by light microscopy; at least 10 sections were examined by transmission electron microscopy. The plasmatocyte has an euchromatic nucleus, a well-developed system of flattened or tubu- lar cisternae of RER, and many free ribosomes (Figs. 2, 6). Mitochondria are approximately three times as frequent as in granulocytes (Table I). Plasmatocytes contain few, if any, large secretory granules. These observations are confirmed by LM serial sections of 12 different plasmatocytes (Fig. 15): two plasmato- cytes contained zero, five contained one, three con- tained two, and two contained three large granules. Plasmatocytes are not affected by endotoxin stimula- tion. lACini'LEVS TRIOENTATL'S HEMOCYTES 61 Figure 14. Granulocyte from Tachyplcus tridentalus incubated with 10 * g endotoxin per ml hemo- lymph for 300 s. After exocytosis, the remaining organelles collect in the middle of the cell surrounded by microtubules (arrowheads) as observed also in Limn/us polyphemus (Tablin and Levin. 1988). Digestive vacuoles (DV). Immature granule (IG); nucleus (N). Discussion Two major groups ofhemocytes We reveal one granular and one almost agranular type of hemocyte in the general circulation of T. tridentatus (Figs. 1, 2, 15). In agreement with the terminology from other arthropods, including other chelicerates. these he- mocytes are named granulocytes and plasmatocytes, re- spectively (Gupta, 1979; Sherman, 1981; Gupta, 1985; Suhr-Jessen et al., 1989). Their main differences are summarized in Table II. The plasmatocyte has not pre- viously been observed in T. tridentatus, but it makes up about 3% of the hemocytes in all samples from the six animals studied. The plasmatocyte is not a cyanoblast or a cyanocyte (Fahrenbach, 1970), because plasmatocytes have the same size, are present in the general circulation of all ani- mals studied at all times, and do not contain cytoplasmic crystals. The plasmatocyte is not a granulocyte that exocy- tosed during sampling, because the two cells differ in amounts of hetrochromatin, RER, free ribosomes, and mitochondria (Table II). In other systems, such dra- matic changes usually takes hours. Furthermore, the plasmatocyte has the smooth ellipsoidal shape with a marginal band characteristic of the unstimulated gran- ulocyte in contrast to the pseudopodial form following exocytosis (Figs. 1, 13; Dumont etai, 1966; Armstrong, 1980; Armstrong and Rickles, 1982; Armstrong, 1985; Tablin and Levin, 1988). However, it cannot be ex- cluded that plasmatocytes are granulocytes, which have undergone spontaneous exocytosis so early prior to hemolymph sampling that the marginal band of micro- tubules have reformed. Because the production of gran- 62 P. P. JAKOBSEN AND P. SUHR-JESSEN . . N Figure 15. Serial sections of a plasmatocyte from Tachyplciis trulcntaliix. The nucleus ( N) is uniformly euchromatic: a single large granule (GR) and many mitochondria (M) are present. The neighboring granu- locytes contain many large secretory granules, but few mitochondria. ulocytes is not continuous (Cohen, 1 985), this latter in- terpretation implies either: ( 1 ) that approximately 3% of the hemocytes in all T. tridcntatus examined are con- stantly recovering from spontaneous exocytosis, and that the transition from plasmatocyte to granulocyte is so fast that intermediate stages are at least one order of magnitude less frequent than plasmatocytes; or (2) that approximately 3% of the hemocytes in each animal re- cover from a single burst of exocytosis long before the first sampling, and that this recycling is blocked at the plasmatocyte stage. In L. polyphcmits. the indepen- dence of granulocytes and plasmatocytes is further sup- ported by the detection of coagulogen only in granulo- cytes (Suhr-Jessen et al.. 1989). Large secretory granules The large structured granules seen in the proximity of Golgi complexes in granulocytes are apparently not affected by endotoxin (Figs. 7-10, 13, 14). This supports the interpretation that this class of structured granules is an immature stage leading to the mature uniform secre- Table I iMin nl the ahiindnncc of mitochondria in plasmatocytes (PL) Tachypleus tndentatus Serial section Number of mitochondria in Ratio PL/GR Plasmatocvte Granulocyte #1 #: #3 154 276 138 48 86 51 3.25 3.21 2.71 Total 568 185 = 3 Each serial section number refers to one plasmatocyte and one gran- ulocyte. Eighteen to 23 serial sections were required to completely sec- tion a cell. I'RIDLM'AI'L'S HEMOCYTES 63 Table II .1 comparison o/l he main differences between plasmatocytes ami graiiiilocyte.s in Tachypleus tridcntatus Plasmatocyte Granulocyte Nucleus Euchromatic Heterochromatic RER Flattened and well Distended hut developed poorly developed Free ribosomes Many Few Large secretory Few — if any Many granules Mitochondria Many Few Frequency 3% 97% tory granules, as suggested for L. polyphemus (Copeland and Levin, 1985;Suhr-Jessen eta/.. 1989). Following en- dotoxin stimulation, the content of the mature uniform granules become transitorily structured before exo- cytosis (Fig. 11). This resembles the situation in rat mast cells and human platelets (Bloom, 1974; Morgen- stern et ai, 1987). The different responses to endotoxin suggest that immature and mature secretory granules contain different membrane proteins. Immune defense Granulocytes and plasmatocytes from the Asian T. tri- dentatus are cytologically indistinguishable from those in the American L. polyphemus (Dumont el ai, 1966; Shishikura et ai, 1977; Gupta, 1979; Nemhauser et ai, 1 980; Ornberg and Reese, 1981; Shishikura el ai. 1982; Armstrong, 1985; Copeland and Levin, 1985; Tablin and Levin, 1988; Suhr-Jessen et ai. 1989). This suggests that the cellular part of their immune defense systems has remained unchanged for more than 140 million years (Shishikura et ai. 1982). Do granulocytes also participate in the endocytic part of the immune defense system, as debated by Armstrong and Levin (1979)? Although digestive vacuoles are pres- ent (Fig. 14), we have not observed the formation of large endocytic vacuoles, neither in plasmatocytes nor in gran- ulocytes, but both cells form micropinocytotic (coated) vesicles (Fig. 9). It is tempting to speculate that granulo- cytes and plasmatocytes may operate together, and with the humoral part of the immune defense system, to rec- ognize and destroy invading microorganisms. Although the cellular part of the immune defense sys- tem in horseshoe crabs has been studied extensively, nei- ther the hemocyte stem cell nor its location, regulation of maturation, differentiation, or proliferation is elucidated (Cohen, 1985). The fate of the granulocytes after exo- cytosis is also unknown. The gathering of the organelles in the middle of the granulocyte after exocytosis might be the first step in a recovery process (Fig. 14). The present study describes a hitherto overlooked hemocyte, the plasmatocyte, in the general circulation of T. tridentatiis. It also extends previous studies of the temporal relationship between maturation and struc- ture of the large secretory granules in granulocytes. Both results prompt several questions pertinent to the molecular biology, structure, and function of hemo- cytes in horseshoe crabs in particular, and to the evolu- tion and cell biology of the immune defense system in animals in general. Acknowledgments We thank Tom Mikkelsen for providing the horseshoe crabs and Ulla Hauschildt for technical assistence with the transmission electron microscopy and light micros- copy preparations. Support from Knud H0jgaards Foun- dation (to PPJ) and a student fellowship from the Carls- berg Foundation (to PPJ) is gratefully acknowledged. Literature Cited Armstrong, P. B., and J. Levin. 1979. In vitro phagocytosis by Liinu- lus blood cells. J. Invert. Pathol 34: 145-151. Armstrong, P. B. 1980. Adhesion and spreading of Limulus blood cells on artificial surfaces. J. Cell Sci. 44: 243-262. Armstrong, P. B., and F. R. Rickles. 1982. Endotoxin-induced de- granulation of the L;/m /<«»/. 201: 303-30N. Shishikura, F., and K. Sekiguchi. 1979. Comparative studies on he- mocytes and coagulogen of the asian and the american horseshoe crabs. Prog. Clin. Bwl. Rex. 29: 185-201. Shishikura, F., S. Nakamura, K. Takahashi, and K. Sekiguchi. 1982. Horseshoe crab phylogeny based on amino acid sequences of the fibrino-peptide-hke peptide C. J. E.\p. Zool. 223: 89-91. Shishikura, F., S. Nakamura, K. Takahashi, and K. Sekiguchi. 1983. Coagulogens from four living species of horseshoe crabs (Limulidae): comparison of their biochemical and immunochem- ical properties. ./. Bioeliem. 94: 1279-1287. Suhr-Jessen, P., L. Baek, and P. P. Jakobsen. 1989. Microscopical, biochemical and immunological studiesofthe immune defense sys- tem of the horseshoe crab, Limulus polyphemus. Biol. Bull. 176: 290-300. Tablin, F., and ,). Levin. 1988. The fine structure of the amebocyte in the blood of Limuliix polvpheniits II. The amebocyte cytoskeleton: a morphological analysis of native, activated, and endotoxin-stimu- lated amebocytes. Biol. Bull 175: 4 1 7-429. Watson, S. \V., J. Levin, and 'I . .1. Novitsky, eds. 1987. Delt'clion of Bacterial Endotoxins with the I imulus Amebocyte Lysate Test. Prof;. Clin. Biol Re\. 231, Alan R. Liss. Inc., New York. \\illumsen, N. B. S., F. Siemensma, and P. Suhr-Jessen. 1987. A multinucleute amoeba, Puruelniox zoochlorellae(W\\\umsen, 1982) comb. nov.. and a proposed division of the genus Cluiox into the genera Climn and Parachaos (Gymnamoebia. amoebidae). Arch. Prottxienkd 134:303-313. Reference: Biol. Hull 178: 65-73. (February. 1990) A! Adenosine Receptor Modulation of Adenylyl Cyclase of a Deep-living Teleost Fish, Antimora rostrata JOSEPH F. SIEBENALLER1 AND THOMAS F. MURRAY2 ^Department of Zoology and Physiology, Louisiana State University, Baton Rouge, Louisiana 70803, and 'College oj Pharmacy, Oregon State University, Corvallis, Oregon 97331 Abstract. Low temperatures and high hydrostatic pres- sures are typical of the deep sea. The effects of these pa- rameters on transmembrane signal transduction were determined through a study of the A, adenosine recep- tor-inhibitory guanine nucleotide binding protein-ade- nylyl cyclase system in brain membranes of the bathyal teleost fish. Antimora rostrata (Moridae). The compo- nents of this system were analyzed at 5°C and 1 atm, and the role of the A, receptor in the modulation of adenylyl cyclase was determined. The A, selective radioligand Nh- ['H]cyclohexyladenosine bound saturably, reversibly, and with high affinity. The K^ of N6-['H]cyclohexyladen- osine estimated from kinetic measurements was 1.11 nAf; the Kj determined from equilibrium binding was 4.86 nAf. [32P]ADP-ribosylation of brain membranes by pertussis toxin labeled substrates with apparent molecu- lar masses of 39,000 to 4 1 ,000 Da. Basal adenylyl cyclase activity was inhibited in a concentration-dependent manner by the A! adenosine receptor agonist N6-cyclo- pentyladenosine (IC50 = 5.08 nM). The inhibition of ad- enylyl cyclase activity was dependent upon GTP. Basal adenylyl cyclase activity was unaffected by 272 atm of pressure. The efficacy of 100 /iA/ N6-cyclopentyladeno- sine as an inhibitor of adenylyl cyclase was the same at atmospheric pressure and at 272 atm. The inhibition of adenylyl cyclase by the agonist 5'-N-ethylcarboxami- doadenosine (100 pM) at 272 atm was twice that ob- served at atmospheric pressure. Although consideration Received 1 September 1989; accepted 30 November 1989. Abbreviations: ATP. adenosine triphosphate; cAMP. cyclic adeno- sine monophosphate; [3H]CHA, N6-['H]cyclohexyladenosine; CPA. N''-cyclopentyladenosine; G protein, guanine nucleotide binding pro- tein; GTP, guanosine triphosphate; NAD, nicotinamide adenine dinu- cleotide. NECA, 5'-N-ethylcarboxamidoadenosine; R-PIA. N^-phenyl- isopropyladenosine, R(-) isomer. S-PIA. N6-phenylisopropyladeno- sine, S( + ) isomer. of the effects of low temperature and high hydrostatic pressure on acyl chain order suggest that deep-sea condi- tions will perturb membrane function, signal transduc- tion by the A, receptor system of the bathyal fish A. ros- trata is not disrupted by deep-sea conditions. Introduction The low temperatures and high hydrostatic pressures of the deep ocean may disrupt the biochemical and phys- iological functions of organisms colonizing this habitat (Siebenaller and Somero, 1978, 1989). Membrane-asso- ciated systems are likely to be particularly sensitive be- cause of the ordering effects of these environmental vari- ables on the organization of acyl chains of lipids (Chong and Cossins, 1983; Hochachka and Somero, 1984). Comparisons of homologous cytoplasmic proteins from deep- and shallow-living teleost fishes have established the importance of adaptation to deep-sea temperatures and pressures (Siebenaller and Somero, 1989). Studies of membranes and associated systems in deep-sea organ- isms indicate that these systems also adapt (e.g.. Cossins and Macdonald, 1984, 1986; DeLong and Yayanos, 1985, 1 987; Gibbs and Somero, 1989). To further understand the effects of deep-sea condi- tions, and to identify potential adaptations of transmem- brane signal transduction, we studied the A, adenosine receptor and its associated effector elements in brain tis- sue of a deep-living cold-adapted marine teleost fish. Antimora rostrata. The objectives of this study were: (1) to determine whether the A, receptor of a typical deep- sea species is coupled to adenylyl cyclase, and (2) to as- certain whether this coupling is functional under the conditions of low temperatures (0-6°C) and high hydro- static pressures (85-250 atm) experienced by A. rostrata. To this end, we undertook a molecular dissection of this 65 66 J. F. S1EBENALLER AND T. F. MURRAY signal transduction system, characterized ligand binding to the A, receptor, identified the associated GTP-binding proteins, and determined basal adenylyl cyclase activity and the role of A, receptor agonists and GTP-binding proteins in modulation of cAMP accumulation. A. rostrata is a benthopelagic morid commonly found in the Atlantic and South Pacific Oceans at bathyal depths of 850 to 2500 m (Iwamoto, 1975; Wenner and Musick, 1977). [Pressure increases 1 atm (=101.3 kPa) for every 10 m of depth in the ocean.] A. rostrata is re- placed by the congener. 1. micmlepis in the North Pacific (Small, 1 98 1 ). Many adaptations to hydrostatic pressure and low temperature have been documented for these species (e.g., Hochachka, 1975; Siebenaller and Somero, 1979; Somero and Siebenaller, 1979; Cossins and Mac- donald, 1984, 1986; Avrova, 1984; Yancey and Siebe- naller, 1987; Hennessey and Siebenaller, 1987; Gibbs and Somero, 1989). A, adenosine receptor modulation of adenylyl cyclase was selected for two reasons as a model with which to examine pressure and temperature effects on transmem- brane signal transduction. First, our previous studies had documented the widespread distribution of this receptor among the classes of chordates, including deep-occurring teleosts (Siebenaller and Murray, 1986, 1988), and we had also identified potentially adaptive differences in li- gand binding among species (Murray and Siebenaller, 1987; Siebenaller and Murray, 1988). Agonist occupa- tion of the A, adenosine receptor inhibits cAMP accu- mulation in mammalian central nervous tissue prepara- tions (reviews by Wolff tv a/., 1981; Londos et a/.. 1983; Snyder, 1985; Williams, 1987). The A, adenosine recep- tor is coupled to adenylyl cyclase [ATP pyrophosphate- lyase (cyclizing); EC 4.6.1.1] by an inhibitory guanine nucleotide binding protein (G, protein). Agonist occu- pation of the other subclass of adenosine receptor cou- pled to adenylyl cyclase, the A: receptor, stimulates ad- enylyl cyclase activity. These receptors are further distin- guished on the basis of the rank order potencies of adenosine analogs (Daly, 1983a, b; Stone, 1985; Wil- liams, 1987). At a measurement temperature of 22°C, the binding affinities, specificities, and pharmacological profiles of the A i adenosine receptors in teleost fishes are similar to those of mammals (Siebenaller and Murray, 1986; Mur- ray and Siebenaller, 1987). The binding of agonists to the high affinity state of mammalian A, receptors is dis- rupted by the low temperatures typical of body tempera- tures of many cold-adapted fishes (e.g.. Bruns et a/., 1980; Trost and Schwabe, 1981; Murphy and Snyder, 1982;Lohserfa/.. 1984; Siebenaller and Murray, 1988). However, agonist recognition and binding properties of the A, adenosine receptors are retained in evolutionary adaptation to different body temperatures. For instance. for eight vertebrate species with body temperatures of 1 - 40°C, Kj values for the agonist N6-['H]cyclohexyladeno- sine <[3H]CHA) measured at 5°C varied 30-fold; how- ever, the binding affinities vary only four-fold when com- pared at temperatures similar to the species' body tem- peratures (Siebenaller and Murray, 1988). Materials and Methods Specimens Demersal adult Antimora rostrata (Moridae) were col- lected by otter trawl at their depths of typical abundance (850-2500 m) off the coast of Newfoundland, Canada, on a cruise of the R/V Gyre in May 1986. Brain tissue was dissected, frozen in liquid nitrogen at sea, and trans- ported to the laboratory where tissues were maintained at -80°C until used. For the [3:P]ADP-ribosylation ex- periments described below, brain tissue from the mac- rourids, Macrourus hergla.\, Coryphaenoides rupestris, and C. armatus, taken off the coast of Newfoundland, the scorpaenids, Sebastolobus alascanus and 5. altivelis, taken on a cruise of the R/V H 'ecoma off the coast of Oregon, and the salmonid, Oncorhynclnts mykiss (Salmo gairdneri), raised at the Food Toxicology and Nutrition Laboratory of Oregon State University, were also used. The macrourid species were chosen because they represent a primarily deep-sea family. The Sebasto- lobus species have been employed in a variety of pressure adaptation studies (Siebenaller and Somero, 1989), and O. mykiss is a pelagic freshwater species. Reagents [Adenylate-3:P]-nicotinamide adenine dinucleotide ([3:P]NAD, 31.31 Ci/mmol), [3H]CHA (34.4 Ci/mmol), [«--:P]ATP (800 Ci/mmol) and [3H]cAMP (30.5 Ci/ mol) were from DuPont NEN (Wilmington, Delaware). The R- and S-diastereomers of N6-phenylisopropyladen- osine (PIA), 5'-N-ethylcarboxamidoadenosine (NECA), and papaverine were obtained from Research Biochemi- cals. Inc. (Wayland, Massachusetts). Pertussis toxin was from List Biological Laboratories (Campbell, Califor- nia). Electrophoresis reagents and molecular weight standards were from Bio-Rad (Richmond, California). Adenosine deaminase (Sigma, Type VI), N6-cycIopentyI- adenosine (CPA), 2-chloroadenosine, and all other chemicals used were from Sigma Chemical Co. (St. Louis, Missouri). Water was processed through a four- bowl Milli-Q purification system (Millipore, Bedford, Massachusetts). Preparation of brain membranes Antimora rostrata brain membranes for assays of li- gand binding were prepared following the procedures de- scribed by Murray and Siebenaller ( 1987). ADENYLVL CYCLASE OF A DEEP-SEA FISH 67 For adenylyl cyclase assays, brain tissue was disrupted with a Dounce (pestle A) in 100 volumes of 10 mM HEPES, pH 7.6 at 5°C, and centrifuged at 27,000 X g(0- 4°C) for 10 min. The pellet was resuspended in buffer, centrifuged at 27,000 X g for 10 min, resuspended in buffer, and 7.5 units/ml of adenosine deaminase were added. The homogenate was incubated at 18°C for 30 min, chilled on ice, centrifuged at 27,000 X g, and the pellet resuspended in buffer and 7.5 units/ml adenosine deaminase. Fifty microliters of this homogenate were used in the adenylyl cyclase assays. For ADP-ribosylation experiments, membranes were homogenized with a Dounce (pestle A) in 40 volumes of 50 mM Tris-HCl, pH 7.6 at 5°C. The homogenate was centrifuged at 27,000 X g for 10 min. The pellet was re- suspended in 40 volumes of Tris-HCl buffer. Fifty micro- liters of this were used for the ribosylation experiments. Protein was determined by the method of Lowry et al. (1951) following solubilization of the samples in 0.5 M NaOH. Bovine serum albumin (Sigma Chemical Co.) was used as the standard. Time course of agonist association and dissociation Aliquots of brain membranes were incubated with 2.85 nM [3H]CHA in 50 mM Tris-HCl, pH 7.6 at the incubation temperature of 5°C. Nonspecific binding was determined simultaneously in the presence of 60 nM R- PIA. For the dissociation experiments, samples were first incubated at 5°C with 2.85 nM [3H]CHA for 240 min to allow binding to reach equilibrium. R-PIA (60 nAI) was added in a negligible volume (1% of the total) to initiate the dissociation reaction. Samples were started at timed intervals, and all incubations were terminated simulta- neously by filtration over No. 32 glass fiber filter strips (Schleicher and Schuell Inc., Keene, New Hampshire) using a cell harvester (Brandel Instruments, Gaithers- burg, Maryland). The data were analyzed as described below. Equilibrium binding assay for membrane bound A, adenosine receptors The rapid filtration assay described by Bruns et al. (1980) and Murray and Cheney (1982) was used with minor modifications to determine the specific binding of the A,-selective agonist [3H]CHA to A. rostrata brain membranes. Assays were conducted in Tris-HCl, pH 7.6, at the incubation temperature of 5°C. The procedures de- scribed in Siebenaller and Murray (1988) were followed. Brain membrane protein (0.4- 1 .2 mg) was added to each assay tube. [32F\ADP-ribosylation Pertussis toxin-catalyzed [3:P]ADP-ribosylation of GTP binding proteins followed the procedures described in Ribeiro-Neto et al. ( 1 985 ) and Greenberg et al. (1987). The 100-jul incubation mixture contained 100 mM Tris- HCl, pH 7.5, at the incubation temperature of 5°C, 25 mM dithiothreitol, 2 mM ATP, 0. 1 mM GTP, 5 [32P]-NAD, 1.5 ng soybean trypsin inhibitor, 15 ^ tracin, 2 ^g pertussis toxin, and 37-92 ng membrane pro- tein. After 3 h, the reaction was stopped by adding 50 /ul of stop solution (3% sodium dodecyl sulfate, 42% glyc- erol, 1 5% 2-mercaptoethanol, 200 mM Tris-HCl, pH 6.8, at 20°C) and boiled for 5 min. The denatured sam- ples were subjected to sodium dodecyl sulfate polyacryl- amide electrophoresis in a 1.5-mm thick 12.5% acryl- amidegel following Laemmli(1970). The gel was stained with 0.25% Serva Blue R (Serva Fine Biochemicals, Westbury, New York) in 25% 2-propanol, 10% acetic acid, destained and dried. The dried gels were exposed to Kodak (Rochester, New York) X-Omat AR film. Du- Pont Cronex Lightning Plus intensifying screens were used. Adenylyl cyclase assays The standard adenylyl cyclase assay contained in a to- tal volume of 150 //I, 10 to 20 jtg of A. rostrata brain membrane protein, 50 mM HEPES, pH 7.6 at the assay temperature of 5°C, 50^Af 2-deoxy-ATP, approximately 1 to 1.5 X 10" cpm [«-32P]ATP, 10 juMGTP, 6.25 mM Mg acetate, 100 mM NaCl, 7.5 units creatine kinase, 5 mM phosphocreatine, 1.5 /*g soybean trypsin inhibitor, 1 5 Mg bacitracin, and other constituents as indicated be- low. Assays were conducted in triplicate in a refrigerated water bath for 2 h. The reaction was stopped by adding 250 fi\ of 2% sodium dodecyl sulfate, 45 mM ATP, and 1.3 mA/cAMP. The samples were boiled for 3 min and 600 ^1 of water were added. [3:P]cAMP generated in the assays was determined according to Salomon et al. (1974). For assays of the effects of hydrostatic pressure on ad- enylyl cyclase activity and inhibition, samples were transferred to polyethylene tubing. The tubing was trimmed to exclude air bubbles and sealed using a pipet heat sealer. [3H]cAMP (approximately 20,000 cpm) was used as an internal standard to monitor the recovery of sample through the sealing and incubation, and through the subsequent column chromatography steps isolating the [32P]cAMP from the [3:P]ATP following Salomon et al. (1974). The pKa of HEPES, the buffer used in these experiments, is relatively insensitive to pressure (Bern- hardt et al., 1988). Samples were incubated in high pres- sure vessels maintained at 5°C in a refrigerated circulat- ing water bath. The high pressure apparatus is described in Hennessey and Siebenaller (1985). Samples were incu- bated for 1 20 min. The time required to seal and pressur- ize a group of four samples and the time required to re- 68 J. F. SIEBENALLER AND T. F. MURRAY move the samples was less than 6% of the incubation time at elevated pressure. Samples sealed and incubated at atmospheric pressure have adenylyl cyclase activities identical to samples that are incubated in test tubes. Data analysis The kinetic parameters for the time course of associa- tion and dissociation of specific [3H]CHA binding were estimated using the equations of Weiland and Molinoff (1981). Rate constants were calculated by least squares linear regression. The association data were analyzed as a pseudo-first order reaction described by the equation: In [B,J -[B]) k ,)t = kohst where [BCJ is the amount of [3H]CH A bound at equilib- rium, [B] is the amount bound at time t, [L] is the con- centration of [3H]CHA. The pseudo-first order rate con- stant, kohs is determined from the slope of plots of In [Beq]/([BeJ - [B]) versus time (Weiland and Molinoff, 1981; Kitabgi r/ E \ c 1 o E CL < o CPA IC50 = 5.08 ± 2.65 2.9- 2.7- — i — 25 50 CPA 75 100 Figure 7. Inhibition of Antimora roslrala brain membrane basal adenylyl cyclase activity by the A, adenosine receptor agonist CPA at 5°C. ADENYLYL CYCLASE OF A DEEP-SEA FISH 71 1.0-- _D 0) cr 0.5-- 1 atm 272 atm Pressure Figure 8. The effects of hydrostatic pressure on Anlimora mslrata basal adenylyl cydase activity (open bar) and inhibition of basal ade- nylyl cyclase activity by the adenosine analogs CPA (100 //A/; filled bar) and NECA (100 /i.U.1 hatched bar). Membranes were incubated at atmosphenc pressure or 212 atm pressure for 2 h at 5°C. All values are standardized to the I atm basal adenylyl cyclase activity. The 1 atm and 272 atm basal activities were 3.3 pmol mm ' mg protein' '. The 1 atm data are the mean of three replicates: the 272 atm values are the mean of six replicates. The average standard errors are 1 1.7% of the values of the mean. Discussion Binding of the agonist [3H]CHA to the A, receptor in A. rostrata brain membranes at 5°C is saturable and readily reversible (Figs. 1 . 2. 3). At 5°C, the rate constants determined for A. rostrata from association-dissociation experiments are lower than those reported for two scor- paenid fishes, Sebastolobus alascanus and 5. altivelis, at a measurement temperature of 22°C (Murray and Siebe- naller, 1987). The kobsfortheA rostrata binding reaction is only 2 1 to 25% of the values obtained for the Sebasto- lobus species ai 22°C. Thek_! value for A. rostrata is only 40 to 65% of the 22°C values. At 22°C the binding reac- tion in Sebastolobus membranes is complete in 30 min. In contrast, at 5°C, the reaction in A. rostrata mem- branes takes more than four times as long to reach equi- librium (Fig. 1). At 5°C, which approximates the body temperatures of these three species, the K^ values are similar (Siebenaller and Murray, 1988). At 5°C, the rank order potencies of agonists is compat- ible with that expected for the A, receptor (Fig. 9; Siebe- naller and Murray, 1988). The K, values indicate dis- crimination of the R- and S-diastereomers of PIA (4.5 and 1 1 5.9 nM, respectively). The rank order potency se- ries expected for A, receptors is R-PIA > 2-chloroadeno- sine > NECA > S-P1A. and for A: receptors NECA > 2- chloroadenosine > R-PIA > S-PIA (Daly, 1983 a, b; Stone, 1985; Williams, 1987). The rank order potencies, the discrimination between R-PIA and S-PIA, and the Kj of [3H]CHA values are characteristic of an A, adeno- sine receptor. The substrates specifically ["P]ADP-ribosylated by pertussis toxin in A. rostrata and six other teleost species (Fig. 4) have apparent molecular masses characteristic of the class of alpha subunits from the guanine nucleotide binding regulatory proteins G, and G0 (Oilman, 1987; PfeufTer and Helmreich, 1988). The GTP-dependence of CPA-induced inhibition of cAMP accumulation (Fig. 6) demonstrates a role for these G proteins in the coupling of the A, receptor to negative modulation of adenylyl cyclase activity in A. rostrata brain membranes. In the presence of GTP, CPA inhibited basal adenylyl cyclase activity with an IC5I) of 5 .08 ± 2.65 \iM ( Fig. 7 ). The max- imal inhibition ranged from 7 to 17%. This degree of in- hibition is similar to the maximal inhibition of adenylyl cyclase by CPA in embryonic chick heart membranes ( Blair rt al. 1989). The A, adenosine receptor of the deep-living teleost, Anlimora rostrata, is capable of modulating the activity of adenylyl cyclase under the conditions of low tempera- ture and high hydrostatic pressure, which characterize the bathyal habitat (Fig. 8). Experiments currently un- derway, using brain tissues from other species, indicate that the A, adenosine receptor-G,-adenylate cyclase sys- tem can be markedly perturbed by hydrostatic pressures less than the 272 atm used in the present study. For in- stance, in shallower-occurring fishes, basal adenylyl cy- clase activity is inhibited 1 1 to 25%> by 136 atm pressure (Siebenaller and Murray, work in progress). In contrast, A. rostrata brain tissue adenylyl cyclase is unaffected by 272 atm pressure, the highest pressure tested. The effi- cacy of agonists at the A] adenosine receptor is not les- sened by increased pressure (Fig. 8). CPA-induced inhi- bition of adenylyl cyclase was unaltered, and the efficacy of NECA increased. Thus, basal adenylyl cyclase activ- ity, as well as signal transduction by the A, receptor sys- 100-- o cr i— o o O m -10 -9 -8 -7 -6 -5 -4 LOG DISPLACER CONCENTRATION (M) Figure 9. Inhibition of specific [3H]CHA binding in Aniimora nn- Iraui brain membranes by adenosine analogs: R-PIA (open circle), NECA (open triangle), 2-chloroadenosine (filled circle). S-PIA (filled triangle). Eleven concentrations of each analog were incubated with membranes and 7.9 nA/ [3H]CHA for 1 50 mm at 5°C. 72 J. F. SIEBENALLER AND T. F. MURRAY tern, are functional under the conditions of pressure and temperature at which A. rostrata occurs. Consideration of the effects of low temperature and high hydrostatic pressure on membrane viscosity (Cos- sins and Macdonald, 1989) suggests that any of the com- ponents of the A, receptor-G, protein-adenylyl cyclase complex may be susceptible to perturbation in organ- isms colonizing the deep sea. For A. rostrata, the func- tion of this transmembrane signaling complex is main- tained at low temperature and high hydrostatic pressure. The pressure insensitivity of this membrane-associated system in A. rostrata is analogous to the pressure adapta- tions observed for cytoplasmic proteins (Siebenaller and Somero, 1 989). The Km values of NAD-dependent dehy- drogenases of deep-living species are relatively insensi- tive to perturbation by pressure. In contrast, homologous enzymes from shallow-living, cold-adapted species are perturbed by pressures as low as 68 atm. By having pres- sure-resistant enzymes, function is preserved over the range of depths that may be experienced by an individual during ontogeny or diel vertical migrations, or by a spe- cies maintaining populations over a broad depth gradi- ent (Siebenaller, 1987). Gibbs and Somero (1989) hypothesized, based on their study of Na+/K+-ATPase in teleost gill tissue, that clear adaptations of membrane-associated systems to pressure may only be apparent in species occurring at depths greater than 2000 m. Although our data do not directly test this hypothesis, the pressure insensitivity of the A | receptor and effector system in A. rostrata. which commonly occurs to depths of 2500 m, are compatible with their suggestion. This pressure resistance in A. ros- trata is a standard with which to compare the effects of environmental parameters on transmembrane signal transduction in other deep- and shallow-occurring spe- cies. Acknowledgments This research was supported by NSF grant DCB- 8710155, and ONR contracts N00014-88-K-0426, N0014-88-K.-0432, and ONR grants N00014-89-J-1865 and N00014-89-J-1869. Shiptime on the R/V \Vcconui off the coast of Oregon was supported by NSF grant DCB-8710155. Shiptime on the R/V Gyre off the coast of Newfoundland was supported by NSF grant DMB- 8502857 to Dr. A. F. Riggs. We thank Drs. A. Riggs and R. Noble for their help in obtaining specimens and Drs. P. Franklin and M. Leid for their help with the ADP- ribosylation experiments. Literature Cited Avrova, N. 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To circumvent this problem, we have developed a simple, reliable, enzyme-linked lee- tin sorbent assay (ELLSA) for quantifying discharged spirocysts. With this method, we have shown that the dis- charge of spirocysts, like that of mastigophore nemato- cysts, is chemosensitized in a dose-dependent manner by three classes of low molecular weight substances, typified by N-acetylneuraminic acid (NAN A), glycine, and cer- tain heterocyclic amino compounds, such as proline and histamine. We also show that spirocysts exhibit consider- able agonist-specific variation in the dose-responses of discharge, suggesting the existence of multiple popula- tions of spirocyst-bearing cnidocyte/supporting cell complexes (CSCCs). Our findings call into question commonly held views regarding the respective roles of spirocysts and mastigophore nematocysts in the reten- tion of captured prey. Introduction The cnidom of the feeding tentacles of acontiate sea anemones, including Aiptasia patlida. consists of three types of cnidae: spirocysts; microbasic p-mastigophore nematocysts, and basitrichous isorhiza nematocysts (Hand. 1955) in approximate ratios of 3:1:0.3, respec- tively (Bigger, 1982: Watson and Mariscal, 1983). Cni- dae function primarily in the capture of prey (Ewer, 1947), in aggression (Purcell, 1977; Bigger, 1982), in de- Received 17 July 1989; accepted 30 November 1989. ' To whom all correspondence should be addressed. fense( Francis, 1973), and in the attachment to appropri- ate substrates (Mariscal, 1972). Spirocysts are adherent cnidae found only in zoan- tharian anthozoans (Mariscal et al.. 1978; Mariscal, 1 984). An undischarged spirocyst consists of a single-lay- ered capsule containing a long, spirally coiled, inverted tubule of uniform diameter (Mariscal, 1974). The tubule lacks spines, but bears hollow rods that dissociate upon discharge to form a web of fine, adhesive microfibrillae (Mariscal el al.. 1977). Unlike nematocysts, discharged spirocysts are difficult to see under the light microscope due to their non-refrac- tile, transparent capsules (Weill, 1934). Because the tu- bules of discharged spirocysts entangle extensively (Ste- phenson. 1929; Skaer and Picken, 1965; Picken and Skaer, 1966; Mariscal, 1974; Mariscal et al., 1977), it is difficult to visually distinguish individual tubules. Thus, it is tedious and time-consuming to visually count spiro- cysts discharged onto test probes. To circumvent this difficulty, we developed a simple, sensitive, and reproducible assay to quantify spirocysts discharged onto test probes. The method is based on the recent discovery that the everted tubules of spirocysts have a high affinity for free and conjugated N-acetylated sugars such as occur on mucins, asialomucins, and mu- copolysaccharides ( Watson and Hessinger, in prep. ). The terminal sugars of the unbranched oligosaccharide chains of bovine submaxillary asialomucin are N-acetyl- galactosamine. This saccharide binds specifically to the lectin from I 'icia villosa. Subsequent to binding asialo- mucin to discharged spirocysts, we determine the num- ber of discharged spirocysts adhering to gelatin-coated test probes by measuring the amount of asialomucin bound to probes using a peroxidase conjugate to the I 'icia lectin. We describe a relatively rapid enzyme-linked, lectin 74 CHFMORECEPTORS SENSITIZE CNIDOCVTES TO DISCHARGE SPIROCYSTS 75 sorbent assay (ELLSA) to determine the number of spi- rocysts discharged onto test probes. Using the ELLSA, we show that three classes of agonists sensitize spirocytes to discharge their spirocysts in response to triggering me- chanical stimuli. The dose-response curves of spirocyst discharge to the agonists indicate that multiple popula- tions of discharging spirocysts exist, each characterized by different sensitivities to the agonists. Materials and Methods Sea anemone maintenance Monoclonal sea anemones (Aiplasia pallida, Carolina strain) were fed and maintained individually in glass finger bowls containing natural seawater at 24 ± 1°C as previously described (Thorington and Hessinger, 1988a). Experimental animals and test solutions Prior to each experiment, animals of the same size were starved for 72 h and kept under defined conditions and lighting (Thorington and Hessinger, 1 988a). Test so- lutions of chemosensitizing agonists (N-acetylneur- aminic acid, glycine, proline and histamine; Sigma, St. Louis, Missouri) were prepared in natural, filtered (Type 1, Whatman) seawater adjusted to pH 7.6 with 1 N HC1 or NaOH. Animals were permitted to adapt to changes of medium for 10 min before cnidocyte responsiveness was measured. Assays of cnidocyte responsiveness Three methods were used to measure the discharge of cnidae: (1) cnida-mediated adhesive force; (2) micro- scopic enumeration of discharged microbasic p-mastigo- phores and spirocysts; and (3) an indirect, solid-state en- zyme-linked lectin sorbent assay (ELLSA) of discharged spirocysts. Cnida-mediated adhesive force. Cnida-mediated ad- hesive force was measured as previously described (Thorington and Hessinger, 1988a). In principle, this technique involves using a small, gelatin-coated nylon bead attached to a strain gauge via a stainless steal wire shaft. The gel-coated bead is made to contact the tip of a tentacle on an anemone in a finger bowl containing a solution of chemosensitizing agent in seawater. The dis- charge of cnidae initiated by contact of the probe with the tentacle results in the tubules of the everting cnidae either adhering to or penetrating the gelatin surface. Withdrawing the probe from the tentacle causes the dis- charged cnidae to exert an opposite and downward force on the probe, which is measured from a gravimetrically calibrated force-transducer connected to a strip-chart re- corder. The adhesive force, measured in hybrid units of mg-force (mgf), is the force required to break the cnida- mediated attachment between the probe and the tenta- cle. It is an aggregate measure of several contributions, including the different kinds of discharged cnidae and the inherent "stickiness" of the tentacle surface, and is proportional to the total number of cnidae discharged onto the probe (Geibel el ai. 1988). Enumeration of discharged mastigophores and spiro- cysts. Following the measurement of adhesive force, the same gel-coated probes were used to visually count the number of adhering mastigophore nematocysts by meth- ods previously described (Geibel et ai, 1988). Discharged spirocysts were visually counted by the same procedures used for discharge mastigophores. Even with phase contrast optics, however, fully discharged spirocysts were extremely difficult to see and time-con- suming to count. To expedite counting of discharged spirocysts adhering to test probes, we developed a fast and reliable micro-assay termed an ELLSA. Indirect, solid-state enzyme-linked lectin sorbant assay (ELLSA) This assay for quantifying discharged spirocysts is based upon the observation that the everted tubules of discharged spirocysts bind conjugated N-acetylated sug- ars with high affinity (Watson and Hessinger, in prep.). In brief, the assay involves first dipping the gel-coated tips of spirocyst-bearing probes into a solution of asialo- mucin, then into a solution of I 'icia villosa lectin/peroxi- dase conjugate, followed by colorimetric measurement of bound peroxidase activity. Some of the N-acetylgalac- tosaminyl residues on the asialomucin molecule bind to the adhesive "glue" of the everted tubules while the re- maining terminal sugars bind the lectin/peroxidase. Buffers. The following buffers were prepared: Buffer A (0.69 M NaCl and 0.25 M phosphate, pH 7.6); Buffer B (0. 1 5 M NaCl and 0.0 1 A/ phosphate, pH 6.0 containing 0.02% Tween 20); Buffer C (0.15 M NaCl and 0.01 M phosphate, pH 6.0); and Buffer D (0.5 M sodium citrate- HC1 pH 5.3). Asialomucin solution. Asialomucin (12 //g/ml; A- 0789, Sigma) in filtered seawater was divided into 10 ml aliquots and stored frozen. For assays, a solution of asia- lomucin (10.8 Mg/ml) was prepared by adding nine parts of the stock solution to one part of Buffer D. Leclin/enzyme conjugate. Horseradish peroxidase conjugated to I 'icia villosa lectin (E-Y Laboratories, San Mateo, California) was diluted to a final concentration of 1.5 Mg/m' m Buffer A. Aliquots of lectin/enzyme con- jugate were protected from light and frozen (— 20°C) un- til used. Mannose ( 50 mA/) was added immediately prior to using the lectin conjugate to minimize nonspecific in- teractions between the lectin and the gelatin on test probes. Enzyme substrate. Hydrogen peroxide (30%; Sigma) 76 G. U. THORINGTON AND D. A. HESSINGER was daily diluted to 3% ( v/v) with distilled water and then to 0.3% with Buffer C. The final substrate solution was prepared immediately before use by adding 6 ml of 0.3% HoO: to 0.05 ml of 1% o-dianisidine (Sigma) in meth- anol. Assay procedure. The wells of flat-bottomed, 96-well microtiter plates (Dynatech) were each rinsed with 200 jul of Buffer B, emptied, and then air dried for 30 min. Test probes were secured to a plastic holder that permit- ted individual probes to be immersed in the contents of separate wells without coming into contact with the sides or bottom of the wells. All incubations were performed at room temperature. Probes were incubated in the asia- lomucin solution for 30 min, then rinsed by immersing in individual wells containing Butter C for 2 min, and finally air-dried for 5 min. Mucin-treated probes were incubated in separate wells containing 200 ^1 of lectin/enzyme conjugate for 60 min in the dark. Following a 2-min rinse in Buffer C, they were transferred to wells containing 200 n\ of enzyme substrate where they were incubated for 60 min. Follow- ing the incubation, the probes were removed and 50 ^1 40% sodium azide in Butter C was added to each of the wells to stop peroxidase activity. The absorbance of each well was measured at 492 nm using a microtiter well spectrophotometer (Model EL 308, Biotek Instruments, Cambridge, Massachusetts). The mean values of controls, consisting of gel-coated probes, which had not been touched to sea anemone ten- tacles, were subtracted from the values of individual ex- perimental probes. Probes sputter-coated with gold for 4 min at 1 5 M using a Polaron E 5 100 sputter coaler and then dipped into asialomucin (10.8 Mg/ml) in Buffer D were used as external standards to assess reactivity of re- agents and to normalize data from test probes to the stan- dard curve, when necessary. The "gold" standard gave absorbances of 0.07 (±0.003 S.E.M.) O.D. at 492 nm. For experimental probes, the absorbance at 492 nm is linearly and directly proportional to the number of dis- charged spirocysts. Measurements of absorbance are di- rectly converted to the number of discharged adherent spirocysts on a probe by extrapolation from the standard curve. Results Optimal dilution of ELLSA reagents Checkerboard titrations of asialomucin and of lectin peroxidase were performed in microtiter plate wells. The dilutions ranged from 1:3 to 1:81 for asialomucin and 1: 8 to 1:5832 for lectin peroxidase. Test probes were gold- coated insect pins (See Materials and Methods) with heads of 0.8 mm diameter. Negative controls were probes treated with 0.01 M phosphate buffered saline, pH 6.0. The dilutions chosen were those giving the great- 16 E '2 UJ O m cc 8 m NUMBER OF SPIROCYSTS X 10'2 Figure I. Standard curve for ELLSA determination of discharged spirocysts. The number of discharged spirocysts counted on test probes is plotted against absorbance at 490 nm. Solid circles represent mean values obtained from tentacles chemosensitized by N-acetylneur- aminic acid and open circles from histamine-sensitized animals. Each point is the mean of separate absorbancy readings (n = 24) and direct countings (n = ll)(R = 0.99). est difference between controls and experimentals. The optimal dilution for asialomucin was 1:27, which is equivalent to 10.8 ng/m\; and for the lectin peroxidase it was 1.5 Standard cun-e A standard curve was constructed by plotting visually counted spirocysts per probe as a function of absorbance at 490 nm. Visual counts of spirocysts discharged onto probes from animals that were chemosensitized by vari- ous concentrations of either NANA or histamine were performed under phase contrast optics. Direct counts and absorbancy readings were obtained using replicate probes from the same animals. For absorbancy readings, a total of four separate experiments were performed and averaged. Each experiment consisted of six replicate probes. A linear and direct relationship existed between the absorbance and the visually counted discharged spi- rocysts (Fig. 1). Adhesive force measurements Dose-response curves, expressing the mean adhesive force for all tested chemosensitizers, are biphasic. The curves for glycine, histamine, proline, and NANA ex- hibit a sigmoidal region of sensitization at low concentra- tions of sensitizer, a maximum response or effect (Emax) at higher concentrations (EC100), and a region of appar- ent desensitization occurring at still higher concentra- CHEMORECEPTORS SENSITIZE CNIDOCYTES TO DISCHARGE SPIROCYSTS Table I Dose-responseparameters of agonist-sensitized cmida discharge and adhesive force measurements from MptasiapaSida tentacles 77 Spirocysts Mastigophores Adhesive force Agonists E™ (no.) EC.oo Ko.5 (A r) Emax (I ,0.) EC- UK) 1 M) 5 (" /) Emax (mgf) ECIOO Ko.5 (M) Glvcine 107 ± 15 5 X 10-" 5.4 x 10 l2 ±0 160 ± 24 10" 1.2 x 10 -" ±0.23 8.7 ±0.7 10" 2.0 x 10 8±0.2 Histamine 12.0 ±0.9 2.7X1Q-7 1.4X10 "±0.1 Peak 1 138 ± 18 2.7 X 10 * 9.7 x 10 "' ± 1.0 1 10± 23 10-" 1.6 x 10'° ±0 Peak 2 159 ±23 2.7 X 10" l.Ox 10 ' ±0.3 201 ± 40 io-7 1.9 X 10 * ±0.3 Proline 10.7 ±1.0 10-" 3.6x10 "±0.4 Peak 1 128 ± 6 2.7 x 10 * 3.2 x 10 " ±0.2 70 ± 15 10 " 5.4 x 10 " ±0.3 Peak 2 93 ± 7 2.7 x 10" 1.7 X IO-7 ±0.3 86 ± 4 10" 5.0 x io-7 ±0 NANA 157± 9 10 5 8.1 x 10" ±0.5 14.0+1.0 l.SxlO5 3.6xlO~7±0.5 Peak 1 233 ± 15 io-8 5.0 x IO-9 ±0 Peak 2 396 ± 9 io-7 8.0 x to-8 ±0.4 Peak 3 172 ±46 10~s 3.2 x 10" ± 1.0 Emax (no.) represents the maximal number of cnidae discharged onto single test probes at optimal sensitization. ECioo is the molar concentration of agonist producing a maximal effect. This value was obtained by visual inspection of dose-response curves. K<, 5 (M) represents the molar concen- tration of agonist producing the half-maximal effects. Emax (mgf) represents the maximal cnida-mediated adhesive force at optimal sensitization. Both E,^ and Ko5 values are determined from least-square double reciprocal plot of the sensitized region of the dose-response curve. Values represent the response to agonists alone (/ c . controls subtracted) and are means ± standard error of the mean. tions (Figs. 2A, 3 A, 4A, and 5 A, respectively). The dose- response curves differ with regard to the specific dose- response parameters (Table I): Emax, the maximum effect; Ko.5, the dose at which a half-maximum effect oc- curs; and ECiou. the dose at which the maximum effect occurs. Dose-responses of mastigophore and spirocyst discharge Glvcine. The dose-response curves representing the discharge of mastigophores (Fig. 2B) and spirocysts ( Fig. 2C) to glycine are biphasic. The dose-response of the dis- charge of spirocysts to glycine consists of a single modal dose-response similar to that obtained from adhesive force measurements and from the discharge of mastigo- phores (Fig. 2A, B). However, there are significant differences in the dose-responses of these two types of cnidae. The response of spirocysts sensitized by glycine is shifted significantly to the left of the glycine-sensitized mastigophore response, indicating that responding spiro- cytes are approximately 10,000 times more sensitive to glycine than are the responding mastigophore-bearing cnidocytes (Table I). Before chemosensitization, the mean number of dis- charged spirocysts on control probes was 116; after sensi- tization, the number rose to 2 1 4. This is equivalent to an average increase of 86%. Because insignificant spirocyst discharge occurs at higher concentrations, and because the dose-response for adhesive force and for discharged mastigophores coincide, it appears that the discharged mastigophores are the major contributors to glycine-in- duced adhesive force. Histamine. The dose-responses of discharging spiro- cysts and mastigophores sensitized by histamine are bi- modal, each displaying two biphasic peaks (Fig. 3) that are complementary and non-overlapping. The two peaks of discharging mastigophores each appear to be about ten times more sensitive to histamine than the corre- sponding two peaks of discharging spirocysts. Proline. The dose-response curves of cnida discharge to proline (Fig. 4) are similar to those obtained for hista- mine. Both the mastigophore and spirocyst response pro- files are bimodal, but unlike histamine, they are comple- mentary and coincidental, rather than non-overlapping. The discharge of spirocysts is less sensitive to proline than to histamine (Table I). N-acetylneuraminic acid (NANA). The pattern of dis- charge elicited by NANA for spirocysts is trimodal, but for mastigophores it is modal. This is in contrast to the responses elicited by the tested "amino" agonists in which agonist-induced patterns were similar for both spirocysts and mastigophores. Each of the three biphasic spirocyst responses is fairly narrow (Fig. 5C), in compari- son to the mastigophore response (Fig. 5B), which spans a range of NANA concentrations of five to six orders of magnitude. Effect of target hardness on retention of cnidae To determine whether the hardness of the target con- tributes to the number of cnidae retained on target probes, we varied the concentrations of the gelatin used (5-50%; w/v) to coat target probes. We sensitized all anemones at !0~5 M NANA to assure that the number of discharging cnidae remained constant. Thus, the number of discharged cnidae retained on probes mea- 78 G. LI. THORINGTON AND D. A. HESSINGER log Glycine Cone. (M) Figure 2. Dose-responses of glycine on discharge of cnidae. A. Effect of glycine on cnida-mediated adhesive force. Values express the mean of four separate experiments. Each experiment consists of eight replicate probes for each concentration: each probe and each tentacle is used only once (n = 32). B. Effect of glycine on the number of dis- charged mastigophores (n = 8). C. Effect of glycine on the number of discharged spirocysts (n = 24). The number of spirocysts was deter- mined by the ELLSA assay. Vertical bars represent the standard error of the mean at 95% confidence limit. showing maxima at 40% and steep declines at 50%. The adhesion curve for spirocysts (Fig. 6B), on the other hand, is sigmoidal, reaching a maximum at 30% and then plateauing at harder coatings of gelatin. At concentrations of gelatin below 20% the numbers of retained mastigophores predominated by as much as 2.5-fold (Fig. 6A, D). Approximately equal numbers of mastigophores and spirocysts were retained on probes o £1 0. g 2.0 * o 1 E sured the adhesion of the discharging cnidae to target surfaces of differing degrees of hardness. We find that the retention of discharged mastigo- phores and spirocysts onto test probes of differing de- grees of hardness is minimal at soft gelatin coatings of 5% (Fig. 6A, B). The adhesion curves with respect to gelatin concentration for retained mastigophores (Fig. 6 A) and for adhesive force measurements (Fig. 6C) are biphasic. log Histamine Cone. (M) Figure 3. Dose-responses of histamine on discharge of cnidae. A. Effect of histamine on cnida-mediated adhesive force. Values express the mean of four experiments. Each experiment consists of eight repli- cate probes for each concentration; each probe and each tentacle is used only once (n = 32). B. Effect of histamine on the number of discharged mastigophores (n = 8). C. Effect of histamine on the number of dis- charged spirocysts (n = 24). The number of spirocysts was determined by the ELLSA assay on Figure 2C. Vertical bars represent the standard error of the mean at 95% confidence limit. CHFMORECEPTORS SENSITIZE CNIDOCYTES TO DISCHARGE SPIROCYSTS 79 45 2.0 o 01 0 2.5 log Proline Cone. (M) Figure 4. Dose-responses of proline on discharge of cnidae. A. Effect of proline on cnida-medialed adhesive force. Values express the mean of four experiments. Each experiment consists of eight replicate probes for each concentration; each probe and each tentacle is used only once (n = 32). B. Effect of proline on the number of discharged mastigophoresfn = 7).C. Effect of proline on the number of discharged spirocysts (n = 24). The number ot spirocysts was determined as in preceding figures. Vertical bars represent the standard error of the mean at 95% confidence limit. dae occur: the spirocysts, the microbasic p-mastigo- phores, and the basitrichous isorhizas ( Hand, 1955). Re- cently, using cnida-mediated measurements of adhesive force in A. pallida. three different classes of chemorecep- tors were identified that sensitize cnidocytes to discharge their cnidae in response to triggering mechanical stimuli (Thorington and Hessinger, 1988a, b). Although the dis- charge of the microbasic p-mastigophores is under the t 3 E z o I o I coated with 20, 30 and 40% gelatin (Fig. 6D). However, the spirocysts predominated by about 3-fold at 50% gela- tin (Fig. 6B, D). Discussion In the feeding tentacles of the sea anemone Aiptasia pallida, as in all acontiate anemones, three types of cni- loq NANA Cone. (M) Figure 5. Dose-responses of N-acetylneuraminic acid (NANA) on discharge of cnidae. A. Effect of NANA on cnida-mediated adhesive force. Values express the mean of four experiments. Each experiment consists of eight replicate probes for each concentration; each probe and each tentacle is used only once (n = 32). B. Effect of NANA on the number of discharged mastigophores (n = 1 1 ). C. Effect of NANA on the number of discharged spirocysts (n = 24). The number of spirocysts was determined as in preceding figures. Vertical bars represent the stan- dard error of the mean at 95%> confidence limit. 80 G. U. THORINGTON AND D A. HESSINGER 03 LLJ CC O Q. o g co o o X o o CC Q. CO O Z 3.5 2.5 1.5 0.5 3.5 2.5 0.5 • 10'5 M NANA D CONTROL A 1CT5 M NANA A CONTROL • 10 5 M NANA O CONTROL 10'5 M NANA 0 CONTROL 80 70 60 D> o CC o LL LLJ CO LLJ I O 50 40 CD i a 2.0 1.0 10 30 50 10 30 50 % GELATIN (wt/vol) % GELATIN (wt/vol) Figure 6. Dose-responses of retained discharged cnidae and of measured adhesive force using targets coated with varying concentrations of gelatin. A. Effect of target hardness on the number of mastigophores retained onto probes (n = 5). B. Effect of target hardness on the number of spirocysts retained onto probes (n = 5). C. Effect of target hardness on the measured adhesive force (n = 5). D. Ratio of retained mastigo- phores to retained spirocysts. All experiments were carried out either in 10 " M N-acetylneuraminic acid or in seawater (controls). Data points are the mean ± standard error of the mean. influence of at least two classes of sensitizing agonists, namely glycine and N-acetylated sugars (Geibel et a/., 1988), it is unknown whether such chemosensitizers. along with a third class of sensitizers, typified by hetero- cyclic amino compounds, also elicit similar responses from spirocytes. Spirocysts have been described ultrastructurally (Mariscal and McLean, 1976; Mariscal et al., 1976, 1977), but few experimental studies have been per- formed on spirocytes. The qualitative effects of remote mechanical stimuli (Conklin and Mariscal, 1 976) and of food extracts (Williams, 1968) on the discharge of spiro- CHEMORECEPTORS SENSITIZE CNIDOCYTES TO DISCHARGE SPIROCYSTS 81 cysts have been reported. Until now, the local, chemical control of spirocyst discharge and the purported primary role of spirocysts in retaining captured prey has not been quantitatively or experimentally verified. This lack of in- formation is due in large part to the difficulty of detecting discharged spirocysts because they possess a highly trans- parent and non-refractile capsule. The counting of dis- charged spirocysts by optical methods is further compli- cated by the fact that the everted tubules entangle exten- sively. While the visibility of the capsules of discharged spirocysts is enhanced with phase contrast optics, the counting of these cnida is, nonetheless, tedious and time- consuming. A rapid and sensitive assay of discharged spirocysts To circumvent these problems, we developed a sensi- tive indirect, solid-state, enzyme-linked lectin sorbant assay (ELLSA) to detect discharged spirocysts. The assay is highly reproducible and is significantly faster than vi- sually counting discharged spirocysts using phase con- trast optics. The potential applications of this procedure include enumerating discharged spirocysts on experi- mental targets as well as detecting and characterizing the adhesive substance of spirocysts. In the present report we use this assay to study the effects on spirocyst discharge of two classes of substances known to sensitize the dis- charge of mastigophores (Geibel el al., 1988), in addition to a third class of sensitizer known to sensitize cnida-me- diated adhesive force (Thorington and Hessinger, 1988b). Sensitiiation ofspirocytes to discharge spirocysts We have found that the three known classes of sensitiz- ers as typified by glycine, NANA, and the heterocyclic amino compounds, histamine and proline, all sensitize spirocyst- and mastigophore-bearing CSCCs, albeit in very different and specific ways. In spite of the variability in sensitivity, magnitude, and pattern of spirocyte re- sponsiveness induced by these agonists, each of the dose- response profiles consists of one or more biphasic peaks. Each biphasic peak reveals a region of sensitization reaching a maximal effect (Emax), followed by a region of desensitization at higher concentrations. The dose-re- sponse parameters (Table I) indicate that the discharge of spirocysts is most sensitive to glycine, followed by his- tamine, proline, and then NANA, while the discharge of mastigophores is most sensitive to histamine, followed by proline, NANA, and glycine. The differences in the sensitivity ofspirocytes and nematocytes to glycine were the most pronounced. In addition to differences in sensitivity to agonists, the dose-response patterns also exhibited differences. In con- trast to the modal (i.e. biphasic) dose-responses exhibited by measurements of adhesive force (Thorington and Hessinger, 1988a, b; Geibel el al.. 1988; Figs. 2A, 3 A, 4A, 5A), we observe that dose-responses of the discharge of spirocysts to glycine is modal, while the dose-re- sponses to proline and histamine are both bimodal, and the response to NANA is trimodal. These contrast to the dose-responses of discharging mastigophores, which for glycine and NANA are modal, while for proline and his- tamine are bimodal. Although the dose-responses of mastigophore and spirocyst discharge are not coinciden- tal for any of the tested agonists, except possibly proline, the fact that all of the adhesive force dose-response curves are coincidental with those of the mastigophores implies that discharging mastigophores contribute sig- nificantly more to adhesive force than do discharging spirocysts. Are all of the receptors effecting multimodal responses (i.e.. NANA) associated directly with the cnidocytes or possibly located on remote sites where they exert indirect control over cnidocyte responsiveness, such as via the nervous system or by initiating changes in behavior that affect the availability of cnidae to discharge? By using mucin-labelled colloidal gold, we find that 99.4% of the labelled gold binds to supporting cells adjacent to spiro- cytes and nematocytes (Watson and Hessinger, 1988), while no label binds to tentacle sensory cells. We con- clude that the receptors to the multimodal agonist, NANA, are entirely located on supporting cells of CSCCs and not on remote sensory sites. A salient feature of modal dose-responses is that the response is "turned off" at concentrations of agonist ex- ceeding those needed to evoke a maximum response. Where multimodal responses are exhibited, high concen- trations of agonist turn off the response of CSCCs having dose-response maxima below that concentration. The existence of bimodal and, particularly, trimodal dose-re- sponses provides for discharge of cnidae over a wide range of agonist concentrations while ensuring that only a portion of the available CSCCs are sensitized at any one time and dose. Thus, the total number of discharging cnidae never reaches the total number present. This effectively conserves cnidae by preventing both excessive discharge against living prey and nonproductive dis- charge against killed prey. Multiple populations ofcnidocyte/supporting cell complexes (CSCCs) The display of bimodal and trimodal dose-responses implies the existence of multiple populations of spiro- cytes distinguished by different sensitivities (i.e., KO 5 val- ues) to a given agonist. That multiple populations of CSCCs exist is indicated by the fact that there are CSCCs, termed type C CSCCs, that discharge their cnidae in re- sponse to tactile stimuli in the absence of added agonist (Figs. 2, 3, 4, 5), in addition to CSCCs, termed type B 82 G. U. THORINGTON AND D. A. HESSINGER CSCCs, that require chemosensitization by agonists be- fore they can be triggered to discharge by static (i.e., non- vibrating) targets. Furthermore, mastigophore-bearing CSCCs triggered by targets vibrating at specific frequen- cies (Watson and Hessinger, 1989) are termed type A CSCCs. Although we do not yet know if vibration-sensi- tive, spirocyst-bearing type A CSCCs exist, there obvi- ously exist different populations of spirocyst- and nema- tocyst-containing CSCCs distinguished by differences in their sensitivities and specificities to agonists and by the ways they are triggered by mechanical stimuli to dis- charge their cnidae. the primary contributors to measured adhesive force. On the other hand, when targets are too hard for the dis- charging mastigophores to penetrate, then the spirocysts predominate as the retained cnida and, collectively, they provide the major contribution to adhesive force. Thus, the correlation between measured adhesive force and the number of discharging mastigophores on both dose-re- sponsive curves and on adhesion curves suggests that dis- charged mastigophores contribute significantly more to adhesive force than do discharged spirocysts under con- ditions in which the target is penetrable to discharging mastigophores. Roles of discharged mastigophores and spirocysts in the capture of prey In the light of our current findings, the commonly ac- cepted roles of the spirocysts and the mastigophores in the capture and adherence of prey must be re-evaluated and modified. We consider these matters from the per- spectives of two questions addressed by this report: (i) which of the two kinds of discharged cnida contribute most to cnida-mediated adhesive force; and (ii) which physical types of target retain the two kinds of cnida. To adequately address the first question, we must rec- ognize that the cnida-mediated components of adhesive force measurements reflect both the number and the kinds of cnidae discharging onto targets. A quantitative analysis of the contributions and magnitudes of these in- dividual factors to adhesive force is beyond the scope of the present discussion, but we can make preliminary qualitative assessments based upon the findings pre- sented here. We have seen for several agonists that the dose-responses for adhesive force measurements and for the number of discharged mastigophores coincide and more closely resemble each other than do the dose-re- sponses for discharging spirocysts ( Figs. 2-5 ). This is also seen by comparing EC100 values for the discharge of cni- dae with those for adhesive force (Table I). To assess the second question, we performed measurements of adhe- sive force and cnidae discharge in which the hardness of the gelatin-coating on the target probes was varied. With gelatin coatings below 20%, the number of discharged mastigophores retained on the target probes predomi- nated over spirocysts (Fig. 6A, D), presumably because proportionally fewer discharging spirocysts can adhere to the "softer" targets. Equal and maximal numbers of mastigophores and spirocysts are retained on probes coated with 20, 30, and 40% gelatin (Fig. 6D). Above 40% gelatin, however, the spirocysts predominate (Fig. 6B. D), presumably because mastigophores are incapa- ble of penetrating these "harder" targets. Thus, when the targets are too soft for the discharging spirocysts to ad- here, the penetrant nematocysts predominate as the kind of cnida retained on the target and, collectively, they are Conclusions In this paper, we show that the discharge of spirocysts is chemosensitized by the same agonists that sensitize the discharge of mastigophores. That is not to say that there may not also exist agonists that sensitize only the dis- charge of mastigophores or of spirocysts. However, the dose-responses of these two kinds of cnidae differ both qualitatively and quantitatively. The dose-responses of discharging mastigophores are either modal (e.g.. to glycine and NANA) or bimodal (e.g.. to proline and histamine), and are coincidental to dose-responses obtained from measuring adhesive force under the same conditions. We have presented strong ev- idence that the similarity between the dose-response curves of adhesive force measurements and the discharge of mastigophores is due to the discharged mastigophores contributing significantly more to cnida-mediated ad- herence onto 30% gelatin-coated targets than the dis- charged spirocysts. It seems appropriate, therefore, to modify the pur- ported roles of penetrant microbasic p-mastigophores and adhesive spirocysts in the capture of prey. Spirocysts have been generally regarded as the primary means by which adhesion of prey to the tentacle occurs (Williams, 1968; Doumenc, 1971; McFarlane and Shelton, 1975; Mariscal, 1984). Mastigophores have been regarded ^ primarily penetrating and envenomating prey while, by implication, not contributing significantly to prey adhe- sion unless the tubules wrap around bristles or projec- tions on prey (Mariscal, 1984). Our findings, however, indicate that mastigophores play a significant, and some- times primary, role in the adhesion of prey, depending most likely upon the hardness of the prey surface. In- deed, it appears that mastigophores and spirocysts may be complimentary in their relative contributions to prey adhesion so that the contribution of mastigophores to adhesive force predominates with soft-surfaced targets, which they can penetrate. The contribution of spiroi , ^ to adhesive force predominates when the target surface is hard enough for the spirocysts to adhere and too hard for the mastigophores to penetrate. Thus, in addition to CHEMORECEPTORS SENSITIZE CNIDOCYTES TO DISCHARGE SPIROCYSTS 83 penetrating and immobilizing prey, discharging mastigo- phores contribute significantly, even predominantly, to the adhesion of prey, provided they are able to penetrate the surface of the prey. Acknowledgments Funded in part by NSF grant DCB-8609859 to D.A.H. Literature Cited Bigger, C. H. 1982. The cellular basis of the aggressive acrorhagial response of sea anemones. . / Mor/>liol. 173:259-278. Conklin. E. J., and R. N. Mariscal. 1976. Increase in nematocyst and spirocyst discharge in a sea anemone in response to mechanical stimulation. Pp. 549-558 in Coelentrate Ecology and Behaviour. G. O. Mackie, ed. Plenum Press, New York. Doumenc, D. 1971. Aspects morphologiques de la devagination du spirocyste chezAclinia et/uina L J Microsc. 12: 263-270. Ewer, R. F. 1947. On the functions and mode of action of the nema- tocysts of hydra. Prot: Zooi Sot: Land- 117: 365-376. Francis, L. 1973. Intraspecific aggression and its effect on the distri- bution of Anthopleura elegantissima and some related sea anemo- nes. Biol. Bull. 144: 73-92. Geibcl, G., G. Thorington, R. Y. Lim, and D. A. Hessinger. 1988. Control of cnida discharge: II. Microbasic p-mastigophore nematocysts are regulated by two classes of chemoreceptors. Biol. Bull. 175: 132-136. Hand, C. 1955. The sea anemones of central California. Part III. The acontianan anemones. WasmannJ. Biol. 13: 189-251. Mariscal, R. N. 1972. The nature of adhesion to shells of the symbi- otic sea anemone Calliaclis tricolor. (Leseur). ./ E.\f>. Mar Bio. /fee/. 8:217-224. Mariscal, R. N. 1974. Nematocysts. Pp. 1 29- 1 78 in Coelenterate Bi- ology: Reviews and New Perspectives. L. Muscatineand H. M. Len- hoff, eds. Academic Press, New York. Mariscal, R. N. 1984. Cnidaria: Cnidae. Pp. 57-68 in Biology of the Integument. Vol. I. Invertebrates, J. Bereiter-Hahn. A. G. Maltolsy, and K. S. Richards, eds. Springer- Verlag, Berlin. Mariscal, R. N., and R. B. McLean. 1976. The form and function of cnidarian spirocysts. II. Ultrastructure of the tip and wall and mechanism of discharge. Cell Tissue Rex. 169: 3 1 3-32 1 . Mariscal, R. N., C. II. Bigger, and R. B. McLean. 1976. The form and function of cnidarian spirocysts. I. Ultrastructure of the capsule exterior and relationship to the tentacles sensory surface. ( '<•// / n sue Res. 168:465-474. Mariscal, R. N., R. B. McLean, and C. Hand. 1977. The form and function of cnidarian spirocysts. III. Ultrastructure of the thread and the function of spirocysts. Cell Tissue Res 178: 427-433. Mariscal, R. N., E. J. Conklin, and C. II. Bigger. 1978. The putative sensory receptors associated with the cnidae of cnidarians. Scanning Electron Microsc. 2: 959-966. McFarlane, I. D.,and G. A. B. Shellon. 1975. The nature of the adhe- sion of tentacles to shells during shell-climbing in the sea anemone Calliaclis parasitica (Couch). J E\p. Mar. Biol. Ecol. 17: 177-186. Ficken, L. E. R., and R. J. Skaer. 1966. A review of researches on nematocysts. In The Cnidarians and Their Evolution. W. J. Rees, ed. Symp. /.ool Soc Lond 16: 15-50. Purcell, J. E. 1977. Aggressive function and induced development of catch tentacles in the sea anemone Metridium senile (Coelenterata. Actiniana). Biol. Bull. 153: 355-368. Skaer, R. J., and L. E. R. Picken. 1965. The structure of the nemato- cyst thread and the geometry of discharge in Corynactis virulis. (All- man). Phil. Trans. Roy. Soc Land. 250: 131-164. Stephenson, T. A. 1929. On the nematocysts of sea anemones. J. Mar Biol.Assoc. U.K. 16: 173-200. Thorington, G. V., and D. A. Ilessinger. 1988a. Control of cnida dis- charge: I. Evidence for two classes of chemoreceptors. Biol. Bull 174: 163-171. Thorington, G., and D. A. Hessinger. 1988b. Control of discharge: factors affecting discharge of cnidae. Pp. 233-253 in Biology oj Ne- nnitocysls. D. A. Hessinger and H. M. Lenhoff. eds. Academic Press, San Diego. Watson, G., and R. Mariscal. 1983. The development of a sea anem- one tentacle specialized for aggression: morphogenesis and regres- sion of the catch tentacle of Haliplantila luciae (Cnidaria, Antho- zoa). Biol. Bull 164: 507-5 1 7. Watson, G., and D. A. Hessinger. 1988. Localization of a purported chemoreceptor involved in triggering cnida discharge in sea anemo- nes. Pp. 255-272 in Biology of Nematocysts, D. A. Hessinger and H. M. Lenhoff, eds. Academic Press, San Diego. Watson, G., and D. A. Hessinger. 1989. Cnidocyte mechanorecep- tors are tuned to the movements of swimming prey by chemorecep- tors. Scienee 243: 1589-1591. Weill, R. 1934. Contribution a I'etudedescnidairesetdeleurs nemato- cysts. I. Recherches sur les nematocystes. Trav. Sta. Zool. tt'imer- tttt-10: 1-347. Williams, R. B. 1968. Control of the discharge of cnidae in Diadu- i>H'iic/ucu:e(Ven\\).Natiire2\9:959. CONTENTS DEVELOPMENT AND REPRODUCTION Bentley, M. G., S. Clark, and A. A. Pacey The role of arachidonic acid and eicosatrienoic acids in the activation of spermatozoa in Arenicola manna L. (Annelida: Polychaeta) . Martin, VickiJ. Development of nerve cells in hydrozoan planulae: III. Some interstitial cells traverse the ganglionic pathway in the endoderm Sicard, Raymond E., and Mary F. Lombard Putative immunological influence upon amphibian forelimb regeneration. II. Effectsof x-irradiation on regeneration and allograft rejection ECOLOGY AND EVOLUTION Smith, David A., and W. D. Russell-Hunter Correlation of abnormal radular secretion with tis- sue degrowth during stress periods in Helisoma tn- volvis (Pulmonata, Basommatophora) .... 25 GENERAL BIOLOGY Hose, Jo Ellen, Gary G. Martin, and Alison Sue Gerard A decapod hemocyte classification scheme integra- ing morphology, cytochemistry, and function PHYSIOLOGY 33 deFur, Peter L., Charlotte P. Mangum, and John E. Reese Respiratory responses of the blue crab Callinectes sapidus to long-term hypoxia 46 Jakobsen, Per Ploug, and Peter Suhr-Jessen The horseshoe crab Tachypleus tridentatus has two kinds of hemocytes: granulocytes and plasmatocytes Siebenaller, Joseph F., and Thomas F. Murray A, adenosine receptor modulation of adenylyl cy- clase of a deep-living teleost fish, Antimora rostrata . . Thorington, Glyne U., and David A. Hessinger Control of cnida discharge: III. Spirocysts are regu- lated by three classes of chemoreceptors 74 55 65 Volume 178 THE Number 2 BIOLOGICAL BULLETIN APR 2 5 1990 •• APRIL, 1990 Published by the Marine Biological Laboratory THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board Marine Biological Laboratory LIBRARY APR 2 5 1990 Woods Hole, Mass. GEORGE J. AUGUSTINE, University of Southern JOHN E. HOBBIE, Marine Biological Laboratory California GEORGE M. LANGFORD, University of RUSSELL F. DOOLITTLE, University of California North Carolina at Chapel Hill at San Diego WU.LIAM R. ECKBERG, Howard University Louis LEIBOVITZ- Manne Biol°Sical Laboratory ROBERT D. GOLDMAN, Northwestern University RUDOLF A. 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Addi- tional reprints may be ordered at time of publication and nor- mally will be delivered about two to three months after the is- sue date. Authors (or delegates for foreign authors) will receive page proofs of articles shortly before publication. They will be charged the current cost of printers' time for corrections to these (other than corrections of printers' or editors' errors). Other than these charges for authors' alterations. The Biologi- cal Bulletin does not have page charges. Reference: Rial. Hull 178: 85-93. (April, 1990) The Sperm Transfer System in Kinbergonuphis simoni (Polychaeta:Onuphidae) HWEY-LIAN HSIEH1 AND JOSEPH L. SIMON Department of Biology, University of South Florida, Tampa. Florida 33620 Abstract. Tube dwelling Kinbergonuphis simoni (San- tos, Day and Rice) achieves a 98.9% fertilization effi- ciency by means of a sperm transfer system involving spermatophores and seminal receptacles. The spermato- phores are mushroom-shaped structures released as clumps. The seminal receptacles are paired sac-like or- gans embedded in the dorsal epidermis of female genital segments. Males release spermatophores into the envi- ronment, and females pick them up with their ventral palps and first pair of parapodia. Stored sperm remain viable for fertilization for at least one month. Spermato- phore release and egg laying are independent of the pres- ence of the opposite sex. Advantages associated with this system are discussed, and include asynchronous repro- duction, a long breeding season, reduced sperm loss, and reduced exposure to risks. This sperm transfer mode is the first reported in the family Onuphidae and is pro- posed for other small, tube-dwelling onuphids. Introduction Sperm transfer in polychaetes occurs in two main modes: non-aggregate transfer, in which sperm are free swimming and not packed together before reaching eggs; and aggregate transfer, in which sperm are packed to- gether by varying complex structures before reaching eggs. Of the non-aggregate transfer modes, three different types have been recorded: broadcast spawning (Clark, 1961; Schroeder and Hermans, 1975), copulation (Just. 1914; Gray, 1969; Schroeder and Hermans, 1975; West- heide, 1984), and pseudocopulation (Reish, 1957; Petti- bone, 1963; Daly, 1973). Three types of aggregate trans- Received 17 October 1989: accepted 29 January 1990. ' Present address: Institute of Zoology, Academia Sinica, Taipei, Tai- wan, 11529R. O. C. fer have been recognized: indirect hypodermic impreg- nation, free transfer of spermatophores, and free transfer of spermatozeugmata. Among these three types, sperma- tozeugmata transfer (Austin, 1963; Eckelbarger, 1974) has not been elucidated with certainty, and will not be discussed further here. In hypodermic impregnation, males actively place spermatophores on the body surface of females. Sperm may then be collected into seminal receptacles, or may penetrate through the epidermis into the coelom of fe- males (Ax, 1968;Jouin, 1970; Westheide, 1984). In free transfer, the spermatophores are released into the envi- ronment and later picked up by females. Seminal recep- tacles are often noted. Free spermatophore transfer has been well demonstrated in members of the spionid genus Polydora (Rice, 1978a, 1987a), and has been strongly suggested to occur in serpulids and sabellids (Daly and Golding, 1977; Picard, 1980). The members of these three Families are tube dwellers. Life history characteristics and habitat choice have been considered strong selective forces for the mode of sperm transfer (Rice, 1978a; Clark, 1981; Mann, 1984; Westheide, 1984). For example, sessile or tube dwelling life styles limit direct bodily contact, or decrease the mo- bility of individuals so that encounters between sexes are infrequent or impossible; thus, neither copulation, pseudocopulation, nor indirect hypodermic impregna- tion would be favored. Broadcast spawning or free trans- fer of spermatophores may be the only alternative for such species. However, broadcast spawning requires large numbers of gametes and synchronous reproduction in the population. The disadvantages of broadcast spawning have been reported (e.g., in corals, Harrison et ai. 1984;Shlesingerand Loya, 1985; and in sea urchins, Pennington, 1985). In contrast, free spermatophore transfer with sperm storage, as found in the spionid Poly- 85 86 H. HSIEH AND J. L. SIMON tiora. has been proposed as an efficient low risk mode of sexual reproduction (Rice, 1978a). Liberation of sper- matophores into the sea also has been considered as an adaptive character in sessile tubicolous pogonophorans (Fliigel, 1977) and vermetid gastropods (Hadneld and Hopper, 1980). Recently, a high efficiency of fertilization has been recorded in bivalves with similar free spermato- phore transfer ( 6 Foighil, 1985). Reproduction in the Onuphidae has been reviewed in general, and developmental patterns have been studied in a few species (Blake, 1975; Fauchald, 1983; Paxton, 1986; Hsieh and Simon, 1987). However, no studies have been done on sperm transfer modes in this group. The characteristics of life style and life history of Kinhcr- gonnpliis simoni are similar to those of many Po/ydora species. Both are dioecious tube dwellers and are small in size. Females produce few, large yolky eggs, brood their young in the tubes, and have an extended breeding sea- son (Rice, 1978a, b; Hsieh and Simon, 1987; Hsieh and Simon, unpub. data). The goals of this study are to address: ( 1 ) the mode of sperm transfer in Kinbergonuphis simoni: (2) the fertil- ization efficiency of this mode; and (3) the possibility that the mode represents convergent evolution between spio- nids and onuphids. Materials and Methods Seminal receptacles Worms were collected from an intertidal sandy flat in Upper Tampa Bay, Florida, and brought alive into the laboratory in January 1985. The presence of seminal re- ceptacles was determined as follows: Two treatments — control and isolation — were set up. Five replicates were used as controls. In each replicate, a pair of male and female worms were reared in a plastic dish surrounded by mesh cloth to keep adults and juveniles from escap- ing. In the isolation experiment, seven females were sep- arately incubated in the same way. Three of the seven females were brooding when experiments began. The fe- males were tapped out of their tubes and thus separated from their young. These young were at embryonic or seg- mented stages. Seawater, which had been sealed in jars for four months, was filtered through Whatman No. 1 filter paper before being added to the aquaria. Salinity and temperature were maintained at 22%o and 20°C, re- spectively. The presence of larvae and juveniles was noted at one- or two-week intervals. This study was con- ducted for three months. Spermatophores Mature worms were collected in March 1988 to deter- mine the occurrence of spermatophores. In the labora- tory, males were reared in mesh-enclosed dishes with and without females. Salinity was kept at 22-24%», and tem- perature at 20-22°C. Observations on behavior and sper- matophore production were made at intervals of 2 to 3 h during daylight hours for three weeks. In all laboratory experiments, the worms were fed ground alfafa. Seminal receptacles and spermatophores: morphology Mature females collected in May 1985, were prepared for paraffin sections after being fixed in Bouin's fixative. Subsequently, they were cut into 7- to 10-jjm sections and stained in Ehrlich's hematoxylin and eosin (Knud- sen, 1966). Spermatophores were prepared for SEM studies following the procedures of Hsieh and Simon (1987). Fertilization efficiency Worms were collected at the study site monthly in 1982, and from June to October in 1985, to determine the fertilization efficiency. Worms were relaxed in 0. 1 5% propylene phenoxytol and fixed in 10% formalin in the field. Broods were examined in the laboratory. Unfertil- ized eggs could be recognized by a white coloration and a clear space appearing at one end (Fig. 1). Only the broods at early developmental stages (blastula to 5-set- iger stages) were used to avoid underestimating the num- ber of unfertilized eggs due to disintegration. Fertiliza- tion efficiency was expressed as the percentage of eggs fertilized of all eggs spawned. Results Seminal receptacles Table I shows that, over three months, paired females produced one to three normally developing broods. Iso- lated females also laid eggs, suggesting that spawning was not induced by the presence of males. In some isolated females (No. 3, 4, and 6), eggs were present in maternal tubes, but no development was observed. In four of the seven isolated females, each produced only one viable brood, indicating that females did store sperm, but that the amount was insufficient for subsequent broods. In isolated females, 0 to 7 juveniles were produced, while in control pairs 9 to 64 offspring were produced (Table I). Spermatophores Spermatophores were released from male tube open- ings as clumps, which stuck to the bottom of the culture dishes or to pieces of debris. Occasionally, in clean dishes where no food particles were present, spermatophores were trapped in the water surface film. Freshly released spermatophores were white, almost transparent, very POLYCHAETE SPERM TRANSFER Table I ( 'out/wrist »i ol breeding sum's* between isolated /enuile.\ anil paired male* and females of Kinbergonuphis simoni reareil in the laboratory from January to March, 87 Date (1985) Number of Number of viable broods juveniles Treatment Jan 27 Feb 3 10 17 24 Mar 3 9 produced produced Control pairs 1 J. 3 * * * J.6. * 2 9 2 J.27 * * J.4 * * J, 28 3 64 3 * * J, 17 1 17 4 J. 18 * * J, 16.* * * J.8 3 42 5 J. 5 * * * J.7 2 12 Isolated females 1 J.2 1 2 2 * * J,4 1 4 3 * * * 0 0 4 * * * * 0 0 5 brooder J. 3 1 3 6 brooder * * * 0 0 7 brooder J. 7 1 7 Larvae or eggs present in maternal tubes; J = juveniles present in dishes: numerals following J = brood sizes. sticky, and easily broken when handled. Motile sperm were observed inside the intact spermatophores. The thin mucous sheets to which the spermatophores were attached were quickly broken down by ciliates or rotifers; however, the spermatophores themselves remained in- tact for more than 48 h. Spermatophores produced at one time by individual males could form more than one clump. The number of spermatophores in each clump varied, ranging from 33 to 160 (mean ± 1 S.E. = 84.90 ± 12.66, n = 10). The average number of spermatophores produced by an indi- vidual male at one time was 80.25 ± 1 8.26 (n = 4). Sper- matophores were present in all of the culture dishes, with or without females, indicating that production of sper- matophores was not influenced by the presence of fe- males or other males. Seminal receptacles and spermatophores: morphology Seminal receptacles are found in the genital segments of females, which run roughly from the 80th segment to the 100th segment. They are located dorsal and posterior to the nephridiopores, near the intersegmental junctions (Figs. 2, 3). Seminal receptacles are paired, blind, sac-like organs embedded in the body wall (Fig. 4). Each sac is about 40 ^m long, and possesses a single 6 /urn wide open- ing to the exterior. The wall of the sac is composed of columnar cells, except at the blind end where cuboidal cells predominate (Fig. 5). Some sacs are branched into two to four lobes (Fig. 6), and the number of lobes varies among and within females. Each spermatophore is mushroom shaped, with a stalk and a spherical portion (Figs. 7, 8) containing sperm. Heads of individual spermatophores are about 40 ^m in diameter, with the stalk about 135 ^m in length. The spherical heads are covered by two layers, the outer char- acterized by a granular appearance, and the inner one with symmetrically arranged bands (Fig. 9a-c). Not ev- ery spermatophore produced is equipped with both lay- ers, some occasionally lacking the outer layer (see arrows in Fig. 7). The sperm from broken spermatophores are morphologically identical to mature sperm seen in the coeloms of males (Fig. 10; also see Fig. 20c in Hsieh, 1984). Transfer of spermatophores Although the direct release of spermatophores from male gonoducts was not observed, expulsion of sper- matophores from the tube openings of males was ob- served several times. When spermatophores were placed around the tube openings of female worms, these fe- males— usually within one minute — would extend their anterior body portions out of the tubes, searching. Upon locating the spermatophores, they would pick them up with their first pair of parapodia and ventral palps, and then immediately withdraw to their tubes. In one in- stance, some spermatophores were literally carried out of a female's tube by larvae when the female and larvae were disturbed by routine observations. Upon examina- tion under SEM, these spermatophores did not contain the outer granular layer (see Fig. 9c), suggesting that fe- 88 H. HS1EH AND J. L. SIMON .' A •*"* ifc/ Figure 1. Unfertilized eggs and a developing embryo within a tube of Kinbergonuphis sinioni Dl = developing larva with 5 setigers; Ue = unfertilized eggs; Sg = sand grams. Figure 2. Dorsal view (SEM) of a female A'm/vn,><>ml. Bull 124: 115-124. Austin, C'. R. l%5. Feriili:alion Prentice-Hall Inc.. Englewood Cliffs, New Jersey. 59 pp. Ax, P. 1968. Das fortptlanzungs verhalten von Tnlnbndnlus ( Archi- annelida. Dinophilidae). Mar. Binl 1: 330-335. Blake, .1. A. 1975. The larval development of polychaeta from the northern California coast. II. Nnlhna elegans (F&mUy Onuphidae). Ophelia 13:43-61. Clark, R. B. 1961 . The origin and formation of the heteronereis. Binl. Rev 36: 199-236. Clark, \V. C. 1981. Sperm transfer mechanisms: some correlates and consequences. A'. Z J Zool. 8: 49-65. Daly, J. M. 1973. Some relationships between the process of pair for- mation and gamete maturation in Harmothoe imhricaia (L.) (An- nelida: Polychaeta). Mar. Behav Phyxiol. 1:277-284. Daly, J. M. 1978. Growth and fecundity in a Northumberland popu- lation of Spimrhis spirorbis (Polychaeta: Serpulidae). J. Mar Binl Assoc. L'. K 58: 177-190. Daly, J. M. and D. W. Golding. 1977. A description of the sperma- theca of Spirorbis spirorhis (L.) (Polychaeta: Serpulidae) and evi- dence for a novel mode of sperm transmission. J. Mar. Biol. Assoc. f K 57:219-227. Eckelbarger, K. J. 1974. Population biology and larval development of the terebellid polychaete Nicnlca instencola. Mar Biol. 27: 101- 113. Eckelbarger, K. J., and J. P. Grassle. 1987. Spermatogenesis, sperm storage and comparative sperm morphology in nine species of Capi- u-lla. Capitomaxtus and Capilellidcs (Polychaeta: Capitellidae). Mar Binl 95:415-429. Fauchald, K. 1983. Life diagram patterns in benthic polychaetes. Prnc. Binl Snc Wash. 96( 1 ): 1 60- 177. Kliigel, II. 1977. infrastructure of the spermatophores ofSihoxlinum cknuini Jagersten (Pogonophora). Nature 269: 800-KO 1 . Goodrich, E. S. 1930. On a new hermaphrodite syllid. Q J Microsc. Sti. 73(4): 65 1-666. Gray, J. S. 1969. A new species of Saccocirrus ( Archiannelida) from the west coast of North America. Pac. Sci. 23: 238-25 1 . Greve, VV. 1974. Planktonic spermatophores found in a culture de- vice with spionid polychaetes. Helgolander. HY.v.v. Meeresunters. 26: 370-374. lladfield, M. G., and C. IN. Hopper. 1980. Ecological and evolution- ary significance ol pelagic spermatophores of a vermetid gastropod. Mar. Binl 57:315-325. Harrison, P. L., R. C. Babcock, G. D. Bull, J. K. Oliver, C. C. Wallace, and B. L. Willis. 1984. Mass spawning in tropical reef corals. Sci- ence 223: I 186-1 184. I hut MI. ni. 0. 1947. Polychaetous Annelids. Part 7 Capiicllidae. Allan Hancock Pacific Expeditions vol. 10. 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Spnnger-Verlag, Berlin. 217 pp. Myohara, M. 1980. Reproduction and development of Pseudopolv- dnra paucibranchiala (Polychaeta: Spionidae) under laboratory conditions, with special regard to the polar lobe formation. J. Fac. Sci Hokkaido Univ. (Scr f>). 22(2): 145-155. O Foighil, D. 1985. Sperm transfer and storage in the brooding bi- valve Myxella linmda. Binl Bull 169:602-614. Okuda, S. 1946. Studies on the development of Annelida Polychaeta 1.7 Fac. Sci Hokkaulnlmp Univ. (Ser. 6)9: I 15-219. Paxton, H. 1986. Generic revision and relationships of the family Onuphidae (Annelida: Polychaeta). Rec Auxl. Mux. 38: 1-74. Pennington, J. T. 1985. The ecology of fertilization of echinoid eggs: the consequences of sperm dilution, adult aggregation, and syn- chronous spawning. Bin! Bull 169:417-430. Pettibone, M. H. 1963. Marine Polychaete ll'ormx nt the New En- gland Region. Museum of Natural History, Smithsonian Institu- tion, Washington. 356 pp. Picard, A. 1980. 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Riot Sue H'axh. Hull 7: 1 14- 127. Rice, S. A., and K. J. Eckelbarger. 1989. An ultrastructural investiga- tion of spermatogenesis in the holopelagic polychaetes I 'anadisfor- mosa and Krohnia Icpidota (Polychaeta:Alciopidae). Hiol Hull 176: 123-134. Richards, S. L. 1970. Spawmngand reproductive morphology o(Sco- lelepi v \i/nannila (Spionidae, Polychaeta). Can .1 '/.ool. 48: 1369- 1374. Schroedcr, P. O., and C. O. Hermans. 1975. Annelida: Polychaeta. Pp. I -2 1 3 in Reproduction of Marine Invertebrate!;, vol. 3: A nnelids and Echiurans, A. C. Giese and J. S. Pearse, eds. Academic Press, New York. Shlesinger, Y., and Y. l.oya. 1985. Coral community reproductive patterns: Red Sea versus the Great Barrier Reef. Science 228: 1 333- 1335. Simon, J. L. 1967. Reproduction and larval development ofSpio se- tosa (Spionidae: Polychaeta). Bull. Mar Sci. 17: 398-431. Soderstrom, A. 1920. Studien uber die polychaetenfamilie Spioni- dae. Inaug. Dis.\. Uppsala. Almqvist anil H'iksell. 286 pp. Stearns, S. C. 1976. Life-history tactics: a review of the ideas. Q Rev Bioi 51(1): 3-47. Weslheide, \V. 1984. The concept of reproduction in polychaetes with small body size: adaptations in interstitial species. In Poly- chaete Reproduction. A. Fischer and H-D. Pfannenstiel, eds. Forl- schr. Zooi 29: 267-287. Westheide, W. 1988. The ultrastructure of Polychaeta. Pp. 263-279 in Micro/anna Marina, vol. 4, W. Westheide and C. O. Hermans, eds. Gustav Fischer Verlag, Stuttgart. New York. Reference: Biol. Bull 178: 94-100. (April. 1990) Structure and Function of a Special Tissue in the Female Genital Ducts of the Chinese Freshwater Crab Eriocheir sinensis TAI-HUNG LEE* AND FUMIO YAMAZAKI Laboratory of Embryology and Genetics, Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido 041, Japan Abstract. The histological anatomy of the genital ducts of adult females of Eriocheir sinensis was studied before and after copulation, and during and after egg-laying. A strongly basophilic valve-like tissue was discovered at the junction of the spermatheca and the oviduct. This tissue prevents communication between the spermatheca and the oviduct except during oviposition. At this time, it functions as a valve, allowing ripe eggs out of the oviduct and preventing sperm from entering the oviduct during and after egg-laying. These findings suggest that the ac- tual site of gamete contact in E. sinensis is within the spermatheca, instead of in the lumen of the ovary or in the oviduct. The presence of the valve-like tissue assures that the ripe eggs collected from the ovary during egg- laying are unfertilized. This observation is of great im- portance for obtaining unfertilized ripe eggs in studies of artificial fertilization (//; vitro) and hybridization. The valve-like tissue has not been described in other brachy- urans, and this genital duct should be classified as new for the Brachyura. Introduction The goal of this study was to define the actual site of fertilization in the Chinese freshwater crab. Eriocheir si- nensis, in preparation for artificial fertilization (//; vitro). This crab is widely distributed in fresh and brackish wa- ters in southeastern China and has great economic value in the country. The female reproductive system of the Brachyura, with the exception of two superfamilies, consists of a se- ries of ducts leading from the ovary to the exterior of the Received 15 May 1989; accepted 19 January 1990. * To whom all correspondence should he addressed. animal. These ducts are composed of four regions: ovi- duct, spermatheca, vagina, and vulva (Hartnoll, 1968). During copulation, the male transfers its spermato- phores into the spermatheca of the female; therefore, fer- tilization in the Brachyura is generally accepted as being internal. But what is the actual site of this fertilization? And what is meant by "internal fertilization" in the Bra- chyura? These two questions have not yet been answered conclusively. Early reports were contradictory. Binford (1913) sug- gested that in Menippe mercenaria, fertilization occurred in the lumen of the ovary, because spermatozoa were found on the surface of the ripe eggs, and many could develop into embryos. Spalding (1942), Cheung (1966), and Goudeau ( 1982) reported that fertilization of Card- mix maenas occurs in the lumen of the ovary or within the oviduct. On the other hand, studies ofPortunus san- giiinolentus (by Ryan, 1 967 ) and Libinia emarginata (by Hinsch, 1971) suggested that the spermatozoan contacts the membrane of the ripe egg internally, and that the re- maining processes in fertilization occur outside the body of the female. This suggestion agrees with that of Yonge ( 1 937). In E. sinensis, we found a valve-like tissue within the spermathecal wall which is connected to the oviduct. Ex- cept during oviposition, this valve-like tissue prevents communication between the oviduct and the sperma- theca. During egg-laying, the tissue functions as a valve, freeing eggs and preventing spermatozoa from entering the oviduct. Thus, we will comment on the expression "internal fertilization" as it pertains to E. sinensis and other brachyurans. Materials and Methods Specimens of the Chinese freshwater crab, Eriocheir sinensis. were obtained from Yang Qin Lake in Jiangsu 94 GENITAL DUCT TISSUE IN E SINENSIS 95 mu op- Fif>urv I. Schematic illustration of the longitudinal section of the genital ducts of the female Eriocheir sinensis. Abbreviations: (co) co- lumnar epithelium; (en) endocuticle; (ex) exocuticle; (ep) epicuticle; (In) hinge; (in) inner wall; (mu) muscles; (op) operculum; (ou) outer wall; (ova) ovary; (ovi) oviduct; (spa) spermatheca; (vag) vagina; (val) valve-like tissue; (vu) vulva. Bar = 1 mm. Province, China, in November 1986 during the crab's spawning migration season. The maximum carapace widths of the specimens used in this study were 7-8 cm. Some specimens were used immediately after they ar- rived at the laboratory (Faculty of Fisheries, Hokkaido University, Hakodate, Japan); these were at the germinal vesicle stage. The remaining specimens were maintained for about two months in individual compartments of a well-aerated, closed circulating seawater system (28%o S, 20°C). Female genital ducts, in various stages — before and after copulation, during egg-laying, and one day and four days after egg-laying — were excised carefully and fixed in Bouin's solution for histological observations. Sections (8-10 jmi) were made by the standard paraffin method and stained with Delafield's haematoxylin and eosin. Results Structure of the female genital ducts and the valve-like tix\ne In adults of Eriocheir sinensis, we found that the fe- male genital ducts have four regions: oviduct, sperma- theca, vagina, and vulva (Fig. 1 ). The oviduct, a short, tube-like passage connecting the ovary and the spermatheca, is about 4. 1 mm long. The opening of the oviduct leading to the spermatheca is on the epithelium of the spermatheca, just above the outer wall of the vagina. The undulant wall of the oviduct is composed of a columnar epithelium. The cavity of the oviduct is full of basophilic material in colloidal form. The spermatheca is ovoid, about 19 mm high and 8 mm wide. It consists of a single crumpled layer of colum- nar epithelium; its cavity is filled with a basophilic colloi- dal substance. The portion of the spermathecal cavity nearest the vagina sharply tapers downward, its narrow end continuous with the cavity of the vagina. The vagina, about 3 mm long, is formed by two cuticular walls; one face (inner wall) is invaginated into a concavity of the other (outer wall) (Fig. 2). Muscles run diagonally from the inner wall to the sternum. The opening of the vagina (i.e., the vulva) is on the sternite of the sixth thoracic seg- ment. The vulva is characterized by the presence of an operculum — the continuation of the inner wall of the va- gina. The operculum can be opened and closed by con- tracting and relaxing the muscles of the inner wall during copulation or egg-laying. A strongly basophilic tissue (hereafter referred to as the valve-like tissue) can be found at the opening of the sper- matheca leading to the oviduct (Fig. 1 ; Fig. 3A); it is about 1 . 1 mm high and 0.4 mm wide. Because the border of the valve-like tissue is connected to the epithelium of the open- ing, it prevents communication between the oviduct and the spermatheca. The tissue is composed of a mass of cells in which no nuclear division is observed; thus, it appears to originate from epithelial cells somewhere nearby. Under light microscopy, the tissue appeared to be a syncytium because no cell membranes were observed. Most of the nuclei are nearly oval, with a long axis of about 5.6 ^m and a short axis of about 3.5 urn; the direc- tions of the long axes are random (Fig. 3B). In contrast, the nuclei in the middle of this tissue are somewhat con- densed and spindle-shaped, with a long axis of about 7.3 jum and a short axis of about 2.0 ^m; all the long axes of these nuclei point toward the cavity of the spermatheca (Fig. 3C). The nuclei within the middle part of that sur- face of the tissue facing the spermathecal cavity appeared to be pycnotic (Fig. 3D). This observation suggests that old nuclei are displaced through the middle part of the tissue into the cavity of the spermatheca. ou en vag Figure 2. Schematic illustration of a transverse section of the va- gina showing its concave shape. Abbreviations: (en) endocuticle; (ep) epicuticle; (in) inner wall; (mu) muscles; (ou) outer wall; (vag) vagina. 96 T. LEE AND F. YAMAZAKI 'B ^^•HHHH| :>" «^ Figure 3. Transverse sections from the middle of the valve-like tissue and oviduct before copulation. A: Whole figure of the valve-like tissue. B: Most of the nuclei of the tissue are oval and the directions of long axes are at random. C: The nuclei in the middle of the tissue are somewhat condensed and spindle-shaped, all the long axes of these nuclei point toward the cavity of the spermatheca. D: The nuclei within the middle part of that surface of the tissue facing the spermathecal cavity appear to he pycnotic. Abbreviations: (co) columnar epithelium; (ovi) oviduct; (spa) spermatheca: (val) valve-like tissue. Bar (A) = 200 pm. Bar (B, CD) = 20 fim. Structural changes in the valve-like tissue and the oviduct Five hours after copulation. Spermatophores and free spermatozoa introduced during copulation swell the spermatheca to about three times its pre-copulatory size. The valve-like tissue is a bit flattened due to the pressure of the seminal fluid (Fig. 4A). Besides these, no other changes in the oviduct were observed. During egg-laying. Changes occur in both the oviduct and the valve-like tissue. The undulant surface of the ovi- duct straightens, and its circumference expands to some degree. The valve-like tissue is perforated by the ex- truded eggs in the middle with its broken parts prolonged toward the cavity of the spermatheca (Fig. 4B, C, Fig. 5A). Figures 4D and E and 5B show that near the end of egg-laying, eggs are extruded continually. The split parts of the tissue are closely attached to each other when there are no eggs passing through. One day after egg-laying. A new thin layer of valve- like tissue appears on the border that is connected to the epithelium of the spermatheca. The split parts of the valve-like tissue have already fused together. However, no nuclear-division is observed in this tissue. The old tis- $$:$&'&•:. GENITAL DUCT TISSUE IN E. SINENSIS • •iiiiiiiiiiiiii ' mmam 97 piSii-y. ^^Mi^-^^^ii f^t fW •"^s v:M vffW'j'v:^ m«?' ;,.•• : vv^^:^3^^!*^ r/f«l^^^:?i L'7Js» >«» t&ix-SftfX , ^,-? &.\ if'1 \ -- . W' . nt- of. ;**--' "— -':: '; v ..• •;, •• :. -• :^,' JBSU ' Figure 4. Transverse sections from the valve-like tissue and oviduct showing the structural changes in different stages. A: Five hours after copulation. B and C: During egg-laying (from the same spermatheca). D and E: Near the end of egg-laying (from the same spermatheca). F: One day after egg-laying. G: Four days after egg-laying. Abbreviations: (eg) egg; (mu) muscles; (nt) new valve-like tissue; (ot) old valve-like tissue; (ovi) oviduct; (sp) spermatozoa; (spa) spermatheca; (val) valve-like tissue. Bar (A, C, E, F, G) = 200 Mm. Bar(B, D) 98 T. LEE AND F. YAMAZAKI OVI CO sp -Fig.4B ovi -Fig.4C Fig.4D Fig.4E Figure 5. Schematic illustration of the longitudinal sections of the valve-like tissue. A: Based on obser- vation of continuous transverse sections of the same spermatheca as shown in Figure 4B and C. B: Based on observation of continuous transverse sections of the same spermatheca as shown in Figure 4D and E. Abbreviations: (co) columnar epithelium; (eg) egg; (ovi) oviduct; (sp) spermatozoa; (spa) spermatheca; (val) valve-like tissue. sue is being discharged into the cavity of the sperma- theca. The surface of the oviduct changes again from be- ing straight to undulant (Fig. 4F). Four days after egg-laying. The old valve-like tissue is almost discharged and a new valve-like tissue is formed (Fig. 4G). No spermatozoa were found inside the oviduct or the ovary during the different stages discussed above. More- over, none of the ripe eggs removed from the egg-laying ovary developed into embryos. Discussion In the present study, no spermatozoa were found in either the oviduct or the ovary in Erioclieir sinensis be- fore, during, or after egg-laying. Moreover, the ripe eggs removed from the egg-laying ovary did not develop into embryos. Therefore, the spermatozoa in the sperma- theca never entered the oviduct or ovary. This phenome- non can be explained by the presence of the valve-like tissue. This tissue not only prevents the sperm from en- tering the oviduct or the ovary before egg-laying, but it also functions as a valve. It allows ripe eggs out of the oviduct during egg-laying and prevents the sperm from entering the oviduct, both near the end of egg-laying and after egg-laying, by closing once the positive pressure of the seminal fluid acts upon it ( Fig. 6 ). Therefore, the only site where the eggs and sperm come into contact is in the spermatheca. In E. sinensis, egg-laying continues for approximately 1 5-30 min. About 300,000-500,000 eggs or more can be found in one brood. The capacity of the spermatheca is no more than 100 eggs, and the opening of the vagina will only allow the passage of two or three eggs at one time. Thus, we estimate that the time an egg takes from entering the spermatheca to release from the vagina is no more than 1 s (unpub. data). Accordingly, there is only enough time for the sperm to attach to or penetrate the surface of the outer membrane of the ripe egg in the sper- matheca, so the remaining events of fertilization must then occur externally. This conclusion is similar to the suggestions made by Yonge (1937), Ryan (1967), and Hinsch(l971). Because fertilization is a series of phenomena that gen- erally involves the contact of sperm and egg, penetration, and karyogamy, the term "internal fertilization" is ap- parently not appropriate forE. sinensis. However, in this study, we could not determine whether any interaction (e.g., acrosome reaction) occurred between the sperm and the egg within the spermatheca. If such an interac- tion does occur, then fertilization in this crab cannot be external. On the other hand, if the sperm simply attaches to the egg membrane and has no further interaction with it within the spermatheca, then the term "external fertil- ization" is applicable. Hence, further research is required to determine whether this crab performs "external fertil- ization." In early studies of the brachyurans by Binford (1913; Menippe nicrcencina), Ryan (1967; Port nuns sanguino- lentns), and Hartnoll (1968; Carcinus maenas, Ifyas coarctatus and Hyas araneus), the structure of the ovi- duct and its opening into the spermatheca were de- scribed as being similar to one another. Unlike E. si- nensis. the oviducts of these crabs do not function as a passage from the ovary into the cavity of the sperma- theca; rather, they are simply a convoluted cord of cells with a blind end extended toward the stratified epithe- lium of the spermatheca, except during ovulation (Fig. 7A). Some time before either ovulation or egg-laying, this cord of cells forms a passage between the ovary and the cavity of the spermatheca; but no valve-like tissue preventing the sperm from entering the oviduct was ob- served (Fig. 7B). GENITAL DUCT TISSUE IN E SINENS1S 99 OVI CO val spa spa A B Figure 6. Schematic illustration of the transverse sections of the valve-like tissue showing the function of freeing eggs and preventing sperm from entering the oviduct. A: Valve-like tissue opens when the eggs are extruded. B: Valve-like tissue closes when the positive pressure of the seminal fluid acts upon it. Abbre- viations: (co) columnar epithelium; (eg) egg; (ovi) oviduct; (spa) spermatheca; (val) valve-like tissue. Arrows indicate the directions of positive pressure. Our view of the relation between the structural changes in the oviduct and the site of fertilization differs from those of previous investigators. Ryan ( 1967) found a small amount of sperm in the open oviduct of P. san- guinolentus. With no further explanation, he concluded that the spermatheca was the site of sperm-egg contact, and that the rest of the fertilization process occurred ex- ternally. He thought that there was insufficient time for the sperm to penetrate the egg within the body of the female crab. Diesel (1989) also reported sperm-egg con- tact within the spermatheca of /. phalangium, but did not report whether any sperm were present in the oviduct or ovary immediately before or after spawning. In studies of M. mercenaria (by Binford, 1913), and C. macnas (by Spalding, 1942; Cheung, 1966; Goudeau, 1982), the au- thors believed that fertilization occurred in the lumen of the ovary or within the oviduct because: ( 1 ) sperm were found in the lumen of the ovary; and (2) some of the eggs removed from that ovary could develop into embryos. We cannot deny that sperm might be naturally pressed into the oviduct and the lumen of the ovary when the blind-ended oviduct opens. However, the evidence cited ovi se Figure 7. Schematic illustration of the transverse sections of the oviduct and spermatheca described in early studies by Binford (1913), Ryan (1967). and Hartnoll (1968). A: Non-spawning stage. B: Some time before or during ovulation and egg-laying. Abbreviations: (ovi) oviduct: (se) stratified epithelium; (spa) spermatheca. by Binford (1913) and other investigators is too weak to support their conclusions. During their dissection and removal, the genital organs may have experienced nega- tive pressure inside the ovary, drawing the sperm into the oviduct or the ovary. This artificial phenomenon might have misled investigators. This may also account for the presence of the sperm in the oviduct of P. sanguinolentus (by Ryan, 1967). Further experiments are needed to clar- ify the site of fertilization in the crabs that have no appa- ratus to prevent sperm from entering the oviduct. There are two known types of oviducal openings into spermatheca: one is that reported by Binford (1913), Ryan (1967), and Hartnoll (1968), and the other is the one we describe in the present study. Besides E. sinensis, we also found the same valve-like tissue and patent ovi- duct in Eriocheir japonicus and Hemigrapsus sangui- neus (unpub. data). In Pachygrapsus crassipes, Chiba and Honma (1971) discovered an oviduct of the same structure; unfortunately, they did not mention whether there was a valve-like tissue. What does the distribution of these two types of openings in the Brachyura mean? What is their taxonomic significance? Does the valve-like tissue have functions other than preventing sperm from entering the oviduct? From where are the cells that re- build the split valve-like tissue? The presence of the valve-like tissue in crabs helps en- sure that the ripe eggs removed from the egg-laying ovary are all unfertilized. This finding is of importance for ob- taining unfertilized ripe eggs in studies of artificial fertil- ization (in vitro) (Lee and Yamazaki, 1989) in E. si- nensis. Furthermore, crabs with this valve-like tissue would be good laboratory animals for studies on fertiliza- tion, hybridization, and embryology in the Brachyura. Acknowledgments The authors thank Dr. Akira Goto of Laboratory of Embryology and Genetics, Faculty of Fisheries at Hok- 100 T. LEE AND F. YAMAZAK1 kaido University, for his constructive criticism, and John Goodier for reading the manuscript. We also thank our friends Chun-Min Liu and Yaichiro Kamataki for their assistance in collecting the specimens, and Paul Endo for his helpful suggestions. Finally we wish to express our hearty thanks to Mrs. Enid Mok Lee for her continuous assistance. Literature Cited Binford, R. 1913. The germ-cells and the process of fertilization in the crab, Menippe mercenaria. J Morpli 24: 147-201. Cheung, T. S. 1966. The development of egg-membranes and egg at- tachment in the shore crab, Carciiiu.^ luacnas. and some related decapods./ Mar. Biol .-l.vvoc. V K 46: 373-400. Chiba, A., and Y. llonma. 1971. Studies on gonad maturity in some marine invertebrates — II. Structure of the reproductive organ of the lined shore crab. A'/'/)/'"" .VH/MW Gakkaishi. 37: 699-706. (in Japa- nese, with English abstract) Diesel, R. 1989. Structure and function of the reproductive system of the symbiotic spider crab Imitinis plialanxiwn (Decapoda: Maji- dae): observations on sperm transfer, sperm storage, and spawning. J. Crust. Biol. 9:266-277. Goudeau, M. 1982. Fertilization in a crab: I. Early event in the ovary, and cytological aspects of the acrosome reaction and gamete con- tacts. 7V.v.vwCV//14:97-ll 1. Hartnoll, R. G. 1968. Morphology of the genital ducts in female crabs. / Limn. Soc. (Zool). 47: 279-300. Hinsch.G. \V. 1971. Penetration of the oocyte envelope by spermato- zoa in the spider crab. J L'llrastrucl. Res 35: 86-97. Lee, T. II.. and F. Yamazaki. 1989. Cytological observations on the fertilization in the Chinese freshwater crab Erioclieir .v/wmv.v by artificial insemination (in vitro) and incubation. Aquaculture 76: 347_36(). Ryan, K. P. 1967. The structure and function of the reproductive sys- tem of the crab. Purtunus saiiguinolentu.i(Hert>sl) (Brachyura: Por- tunidae). II. The female system. Pmc. Symp. Crustacea, Mar liiol ,-l.v.sw , India. Jan. 12-15. 1965. Ernakulam. Pt II: 522-544. Spalding, J. F. 1942. The nature and formation of the spermatophore and sperm plug in Carcumt nuicnas. Q J Microsc. Sci. 83: 399- 422. e, C . M. 1937. The nature and significance of the membranes surrounding the developing eggs Homarus vulgaris and other Deca- poda. Pruc. tool. Sue. Loud. A. 107: 499-5 1 7. Reference: Biol. Bui! 178: 101-1 10. (April, I WO) Sperm Attachment and Acrosome Reaction on the Egg Surface of the Polychaete, Tylorrhynchus heterochaetus* MASANORI SATO2 AND KENZI OSANAI Marine Biological Station, Toliokit University, Asarnitshi, Aomori, 039-34, Japan Abstract. Sperm binding to the egg envelope (chorion) was examined in fixed eggs and isolated chorions of the polychaete, Tylorrhynchus heterochaetus. Sperm bind- ing included two successive steps: attachment (acroso- mal outer surface-chorion binding) before the acrosome reaction and adhesion (acrosomal process-chorion bind- ing) after the acrosome reaction. The attachment be- tween sperm head-tip and the outermost layer of the cho- rion was observed in Ca-free seawater, in which the acro- some reaction did not occur. The surface of the chorion was stained with phosphotungstic acid (PTA). Sperm did not attach to pronase-treated eggs, in which the PTA- positive layer disappeared. When isolated chorions were soaked in distilled water for several hours, they lost the capacity for sperm attachment, and the PTA-positive layer thinned. The acrosome reaction was induced by material that was dissolved from the chorions into dis- tilled water. This suggests that both the receptor for sperm attachment and the inducer of the acrosome reac- tion are involved in the PTA-positive layer. Introduction In many animals, ripe unfertilized eggs have one or more extracellular coats (envelopes). During fertiliza- tion, egg envelopes play a key role in sperm binding, in the induction of the sperm acrosome reaction, and in the exclusion of supernumerary sperm (see Epel and Vac- Received 16 December 1988; accepted 18 December 1989. 1 Contribution No. 558 from the Marine Biological Station. Tohoku University. 2 Present address: Department of Biology. Faculty of Science, Kago- shima University, Korimoto, Kagoshima 890, Japan. quier, 1978;Lopo, 1983;MonroyandRosati, 1983;Jaffe and Gould, 1985). Previous studies on sperm-egg binding have suggested that two types of binding exist (see Epel and Vacquier, 1 978): ( 1 ) binding between the outer surface of unreacted sperm heads and egg envelopes before the acrosome re- action (referred to as attachment in the present paper) in mice (Saling and Storey, 1979; Bleil and Wassarman, 1983; Wassarman et a/., 1985; Soldani and Rosati, 1987), ascidians (DeSantis et a/., 1980; Rosati, 1985), a horseshoe crab (Brown, 1976; Barnum and Brown, 1983), polychaetes (Anderson and Eckberg, 1983; Osa- nai, 1983; Sato and Osanai, 1983, 1986), an abalone (Lewis et ai, 1982) and a sea urchin (Aketa, 1973, 1975); and (2) binding between acrosomal processes of reacted sperm and egg envelopes after the acrosome reaction (re- ferred to as adhesion in the present paper) in ascidians (DeSantis et ai, 1980; Rosati, 1985), a horseshoe crab (Brown, 1976), polychaetes (Osanai, 1983; Sato and Osa- nai, 1983, 1986), sea urchins (Summers and Hylander, 1975; Vacquier, 1980), a sand dollar (Summers and Hy- lander, 1974), bivalves (Hylander and Summers, 1977; Brandriff et a!., 1978), and a crustacean (Clark et ai, 1981). Studying sperm-egg binding can be difficult be- cause, during normal fertilization in most organisms, the acrosome reaction usually follows sperm attachment too quickly to be examined. Using phase-contrast micros- copy, Osanai (1983) observed that sperm remained at- tached to isolated egg envelopes (chorions) without the acrosome reaction in Ca-free solution, and that sperm adhered to the chorion with its acrosome reacted in Ca- containing solution in the polychaete Tylorrhynchus het- erochaetus. In the present study, we use electron microscopy to 101 102 M. SATO AND K. OSANAI b . c Figure I. Sperm binding to the fixed Tylorrhynchus eggs. Two hours after insemination. • 260. (a) Tylorrliynchu.i sperm were bound to the egg in artificial ordinary seawater. (b) Tylurrliym'lius sperm were bound to the egg in artificial Ca-free seawater. (c) Sperm of the sea star I \icniM peel 1 11 1 tern were not bound to the egg in artificial ordinary sea- water. confirm these sperm bindings, and demonstrate that fac- tors for the reception of initial sperm attachment and for the induction of sperm acrosome reaction are distributed in the outermost layer of the egg envelope all over the egg surface. Materials and Methods Preparation oj gametes Mature worms of the nereidid polychaete Tylorrhyn- c/ius heterochaetus were collected in Natori, Miyagi Pre- fecture, Japan. They were placed in 30% seawater (salin- ity: about 10), and refrigerated at 0-5°C. Gametes were obtained by compressing or cutting the body with for- ceps. Unfertilized eggs were washed several times in 30% seawater; sperm were diluted in ordinary seawater (cf. Osanai, 1978). Experimental media Natural seawater filtered with a paper filter (Toyo- roshi No. 2) or Herbst's artificial seawater (ordinary and Ca-free seawater) modified by Motomura (1938) were used. Ca-free seawater was prepared by substituting NaClforCaCl2. Isolation ofchorion from eggs Egg envelopes (chorions) were isolated from unfertil- ized eggs as described by Osanai ( 1 976, 1 983). The unfer- tilized eggs were suspended in 30% seawater and then gently homogenized with a teflon homogenizer. The ho- mogenate was centrifuged at 300 X g for 5 min. After removing the supernatant, the sedimented chorions were resuspended in fresh 30% seawater and centrifuged again. Transparent chorions were obtained by repeating this procedure several times. Preparation of fixed eggs The sperm-egg binding process was examined using fixed unfertilized eggs as described in sea urchins by Kato and Sugiyama (1978). The eggs were prefixed in 1%. glu- taraldehyde in 30% seawater for 0.5-2 h. After rinsing in 30%' seawater several times, the eggs were inseminated. In other cases, unfertilized eggs were pretreated with 0.1% pronase ( Kaken Chemical Co.) in 30% seawater for 20 min prior to prefixation. Insemination Tylorrhynchus eggs are fertilizable in media over a wide range of salinity. In this study, sperm were added to isolated chorions and fixed eggs in 30%- or 100%> seawa- ter. When insemination occurred in Ca-free seawater, the chorions and eggs had been rinsed several times in Ca-free seawater, so that the concentration of contami- nating Ca at insemination might be less than 1/1000 of that in ordinary seawater. Egg or chorion suspensions ( 1 - 5 X 102/ml) were inseminated with sperm suspension (final concentration: 10 4 dilution of dry sperm, 5 X 106- 1 X 107/ml) at room temperature (10-20°C). SPERM-EGG BINDING IN TYLORRHYNCHUS 103 AV Figure 2. Attachment ot'unreacted sperm to the fixed eggs in artificial Ca-free seawater. One minute after insemination. X31.500. (a) A spermatozoon attached to the first layer ( I ) of chorion by its head-tip without any acrosomal change. (2, 3. 4) The second, third, and fourth layer of the chorion, respectively. AV: acrosomal vesicle, (b) A spermatozoon with its acrosomal vesicle open at the head-tip. The outer membrane of sperm head was attached to the first layer of chorion. Test ofacrosome reaction-inducing activity in material dissolving from cliorions Isolated chorions were placed in distilled water (DW) (2% V/V) for 0.5-5 h. The chorion suspension was fil- tered through filter paper. The filtrate was diluted with ordinary seawater to 30% seawater and used as chorion extract. Sperm suspension was added to the chorion extract (final sperm concentration: 1-5 X 107/ml). The test solu- tion was fixed with 1-2% glutaraldehyde 15-40 min later. Sperm acrosome reaction was checked by phase- contrast microscopy (X 1000). Electron microscopy Specimens were fixed in 2% glutaraldehyde in 70-90% seawater for several days at 0-4°C. After rinsing, they were postfixed in 1% OsO4 in 70-90% seawater for 1 h at 0-4°C. The specimens were dehydrated in ethanol and embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate or with 1 0% phosphotung- 104 M. SATO AND K. OSANAI I Figure 3. Adhesion of reacted sperm to the fixed eggs in artificial ordinary seawater (a, b, c). One minute after insemination. The acrosomal process (AP) penetrated the first and second layers of chorion and adhered to the third layer. The outer membrane of acrosome was in contact with the fibrous compo- nent of the first laver of chorion (arrows). • 35,000. stic acid (PTA), and then examined with a transmission electron microscope. Results Sperm binding to fixed eggs In both ordinary seawater and Ca-free seawater, Tylor- rhyncliux sperm were bound to the surface of fixed eggs at their head-tip (Fig. la, b). When the fixed eggs were inseminated with sperm of the sea star Asterina pectini- fera and the sea urchin Strongylocentrotus nuclus, the sperm were not bound to the eggs (Fig. Ic). The chorion (1-1.5 ^m thick) consists of four layers (Fig. 2; see also Sato and Osanai, 1 983). The first (outer- most) layer is composed of a row of small packed spheres wrapped in fibrous matter. The second layer is composed of less electron-dense material. The third layer is a thin electron-dense layer. The fourth layer (innermost and thickest) is composed of densely packed material with many cavities opening toward the inner surface. The ultrastructure of the sperm-egg binding was exam- ined with specimens fixed 1 min after insemination. In Ca-free seawater, most sperm were attached to the first layer of chorion by their head-tip without any acrosomal change, though the acrosomal vesicle had opened in a few sperm (Fig. 2). The opening of the acrosomal vesicle was sometimes observed in free sperm suspended in sea- water, and the usual morphological change associated with a true acrosome reaction did not occur. Thus, open- ing of the vesicle may be either a spontaneous phenome- non or an artifact of fixation. In any case, the outer sur- face of the sperm head-tip made contact with the fibrous component of the first layer. When live eggs were inseminated in Ca-free seawater, sperm temporarily attached to the egg surface, but soon detached. The sperm-egg attachment without a sperm acrosome reaction seems to be less stable in live eggs than in fixed eggs. In ordinary seawater, most sperm bound to the cho- rion underwent the acrosome reaction and formed a lob- ular acrosomal process (Fig. 3). The acrosomal process penetrated the first and second layers of the chorion and adhered to the third layer. At the same time, the outer membrane of the acrosome (the lateral side of the opened acrosomal vesicle) continued to contact the fibrous com- ponent of the first layer of the chorion. Some sperm that had not undergone the acrosome reaction were attached to the first layer of the chorion. Sperm binding to isolated chorions In intact unfertilized eggs, the first layer of the chorion was stained by PTA (Fig. 4a, see also. Sato and Osanai, 1983). The outer surface of the isolated chorion (mor- phologically similar to the third layer) was stained with PTA (Fig. 4b), suggesting that the first and second layers had been deformed or had collapsed onto the third layer. SPERM-KGG BINDING IN TYLORRHYNCHVS 105 1234 Figure 4. (a) Ultrastructure of the surface of an intact unfertilized egg. A section stained with phosphotungustic acid (PTA). The first layer (1) of the chorion was stained intensively. (2, 3, 4) The second, third, and fourth layer of the chorion, respectively. > 40.800. (b) Ultrastruc- ture of the isolated chorion. A section stained with PTA. The outer surface (arrow), which was morphologically similar to the third layer, was stained. X45.600. (c) Sperm binding to the isolated chorions in artificial ordinary seawater. Two minutes after insemination. X140. Sperm were bound to the isolated chorion in both or- dinary and Ca-free seawater, and the bindings of sperm were kept for a long time (Fig. 4c). Because the isolated Table I Percentage of occurrence of acrosome reaction in \pcnn hound to isolated chorion in presence or absence of Co?* Percentage of acrosome reaction Expt. No. Ordinary seawater Ca-free seawater 1 83.0 0.4 2 64.4 0 3 61.3 0.2 4 11.6 0 Isolated chonons were fixed 1-10 min after insemination. Occur- rence of acrosome reaction was checked by phase contrast microscopy in 200-300 spermatozoa on 3-6 chorions. chorion was transparent, the acrosome reaction of the attached sperm could be checked by phase-contrast mi- croscopy. In ordinary seawater, many sperm underwent the acrosome reaction (Table I, Fig. 5). These sperm were also examined by electron microscopy. The lobular acro- somal process adhered to the outer surface of the chorion and did not penetrate it (Fig. 6a). In Ca-free seawater, the KigureS. Phase contrast micrographs of sperm binding to the trans- parent isolated chorions. x 1 900. (a) Control. An intact free-swimming spermatozoon, (b, c) Spermatozoa undergoing acrosome reaction and adhering to the chorion in artificial ordinary' seawater. Ten minutes after insemination. Arrows indicate the development of acrosomal pro- cess, (d) Spermatozoa attached to the chorion without acrosome reac- tion in artificial Ca-free seawater. An arrow indicates an intact acroso- mal vesicle. Ten minutes after insemination. 106 M. SATO AND K. OSANAI Figure 6. Fleet ron mierographs of sperm binding to the isolated chorion. One minute after insemination. • 24.800. (a) A spermatozoon adhering to the outer surface of chorion with the spread acrosomal pro- cess (AP) in artificial ordinary seawater. (h) A spermatozoon attached to the outer surface of the chorion without acrosome reaction in artifi- cial Ca-free seawater. AV: Acrosomal vesicle. sperm bound to the isolated chorion did not undergo the acrosome reaction (Table I. Fig. 6b). The outer acroso- mal membrane of sperm head-tip was attached to the chorion surface. No spermatozoon was bound to the in- ner surface of the chorion (the fourth layer) in both ordi- nary and Ca-free seawater. Effect of pronase treatment of eggs on sperm-egg binding We tried to remove the egg-surface component that binds sperm and induces the acrosome reaction. Unfer- tilized eggs were pretreated with pronase and then fixed. After rinsing, the eggs were inseminated in 30% seawater. Sperm attachment was blocked or greatly reduced (Table II, Fig. 7). The pronase-treated eggs were examined by electron mi- croscopy. The first and the second layers were removed from the chorion in the pronase-treated eggs (Fig. 8). The outer surface of the chorion did not stain with PTA. Acrosome reaction-inducing activity in chorion extract Chorion extracts were prepared by soaking chorions in DW. Sperm were added to the chorion extract diluted with natural seawater. Many of the free swimming sperm underwent acrosome reaction (Fig. 9, Table III). The sperm did not undergo acrosome reactions in control media (30% seawater). After isolated chorions were treated with DW, they were mixed with sperm in 30% seawater. Few sperm were bound to the chorions (Fig. 10). These chorions were ex- amined by electron microscopy. The PTA-positive layer at the outer surface of the chorions became thinner after the DW-treatment (Fig. 1 1). No morphological change was observed in other parts of chorion. Discussion Osanai (1983) used light microscopy to examine sperm binding to the isolated chorion in Tylorrhyncluts heteroclmetm. He showed that sperm binding includes two steps: sperm attachment before the acrosome reac- tion and sperm adhesion after the acrosome reaction. He also showed that the progression from sperm attachment to adhesion requires external calcium ions. We used elec- tron and light microscopy to observe sperm-chorion binding using fixed eggs and isolated chorions. Our re- sults confirm the validity of Osanai's (1983) report. Figure 7. Inhibition of sperm binding by pronase-pretreatment of eggs. The eggs were inseminated in 30% natural seawater after glutaraldehyde-fixation, and observed 10 min after insemination. X 140. (a) The eggs pretreated with pronase for 20 min. Sperm did not bind to the eggs, (b) The eggs without pronase- pretreatment. Many sperm hound to the eggs. SPERM-EGG BINDING IN TYLORRHYNC/ll'S Table 1 1 i 'I v/xvm binding by pronase-prelreatrnenl o/eggx 107 Eggs No. of sperm hound on the egg contour* Pronase-treated Untreated 3.2±0.7(n = 20) 66.5 ± 3.6 (n = 12) ! Average ± SD (No. of eggs examined). Sperm attachment and adhesion were demonstrated ul- trastructually in Ca-free seawater and ordinary seawater, respectively. Both were also photographed 1 min after insemination during normal fertilization (Sato and Osa- nai. 1983). However, in normal fertilization, the sperm attachment step is rather inseparable, because it is fol- lowed quickly by the acrosome reaction. We could sepa- rate the sperm attachment step in Ca-free medium, in which the acrosome reaction was prevented. Sperm attachment occurred at only the head-tip of Figure 9. Induction of acrosome reaction by the chorion extract. *2, 100. (a) Unreacted sperm in control medium (30% seawater). (b. c) Sperm undergoing acrosome reaction in the chonon extract. conspecific sperm. This appears to be a species-specific and site-specific reaction for the first sperm-egg recogni- tion. Specific attachment between sperm and egg enve- lope before the acrosome reaction is also known in an ascidian (Rosati and De Santis, 1978) and in mammals (Wassarman et al. 1985). Sperm attachment was inde- Figure 8. infrastructure of the pronase-treated eggs. The eggs were treated with pronase for 20 min. X24.000. (a) A section stained with uranyl acetate and lead citrate. Most parts of the first and second layers of the chorion disappeared with a few spherical components of the first layer remaining on the surface (arrow), (b) A section stained with phos- photungstic acid. The outer surface of the chorion was not stained as compared with the untreated egg and the isolated chorion (Fig. 4). Table III osonii' reaction-inducing activity of the chorion extract Percentage of acrosome Duration of reaction* incubation in Expt. No. distilled water (h) Extract Control** 1 0.5 82.4 0 2 1.5 3.7 2.7 3 5 77.4 1.9 4 5 19.1 7.6 * Occurrence of acrosome reaction was checked in 100- 1 50 sperma- tozoa by phase contrast microscopy. ** 30% natural seawater. 108 M. SATO AND K. OSANAI Figure 10. Decrease of sperm binding by soaking of the isolated chorions in distilled water. The isolated chorions were inseminated in 30/ a/.. 1 978; Saling and Storey. 1979; Soldani and Rosati, 1987). Why sperm attachment to live Tylorr- hynchus eggs is less stable than to fixed eggs or isolated chorions, in Ca-free seawater, is unknown. The outer- most layer of the chorion of live eggs may be less tightly fastened to the chorion proper. Alternatively, attached sperm may be detached by a factor secreted from live eggs, as in normally fertilized eggs (Osanai, 1976). How- ever, it is unknown whether unfertilized eggs secrete the sperm-detaching factor in Ca-free solution. The acrosome reaction in Tylorrhynchus heterochae- tus evidently requires external calcium ions as in sea ur- chins (Dan, 1954; Collins and Epel, 1977). The jelly, the outermost layer of the sea urchin egg envelope, induces the acrosome reaction and sperm aggregation behavior Figure 1 1. Ultrastructural alteration of the isolated chorion by the distilled-water treatment. Sections were stained with phosphotungstic acid(PTA). -36.700. (a) An isolated chorion just after preparation, (b) An isolated chorion after distilled-water treatment for 60 mm. The PTA-positive layer at the outer surface of the chorion became thinner. SPERM-EGG BINDING IN TYLORRHYM'I/LS 109 showed that a sugar or glycoprotein on an egg envelope plays an important role in sperm attachment or induc- tion of the acrosome reaction in the eggs of a mouse (Shur and Hall, 1982a, b). an ascidian (Rosati and De- Santis, 1980; Pinto ct a/.. 1981), a horseshoe crab (Bar- num and Brown, 1983), a sea urchin (Segall and Len- narz, 1979; Yoshida and Aketa, 1983), a seastar (Uno and Hoshi. 1978), and a bivalve (Tumboh-Oeri and Koide. 1982). The acrosomal process of the fertilizing spermatozoon fuses with a microvillus projecting from the egg through the chorion in T. heterochaetus (Sato and Osanai, 1983). However, no morphological difference of the PTA-posi- tive layer was observed between regions around micro- villi and the other regions. In contrast with T. hetero- chaetus. the acrosome reaction-inducing activity was lo- calized to a limited number of specialized sites above egg microvilli in the nereidid polychaete Ncanlhes japonica (Sato and Osanai, 1986). Sperm seem to initiate the acrosome reaction just after attaching to the chorion in the presence of Ca. Their acrosomal processes usually penetrate the first and sec- ond layers of the chorion and adhere to the third layer. Such sperm can fuse with an egg microvillus to fertilize the egg (Sato and Osanai, 1983). The initial attachment between the sperm head-tip and the outermost layer of chorion may be important for keeping the sperm ori- ented for successful fertilization during the acrosome re- action. Holding the sperm erect on the chorion surface should lead to proper penetration and adhesion of the acrosomal process. Literature Cited Akcta. K. 1973. 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Greve. 1985. Recep- tor-mediated sperm-egg interactions in mammals. Pp. 341-360 in Biologv ot Fertilization, Vol. 2, C. B. Metz and A. Monroy, eds. Academic Press, New York. Yoshida. M., and K. Aketa. 1983. A 225 K dalton glycoprotein is the active core structure of the sperm-binding factor of the sea urchin. Anthocidarii crut\i^pina. E.\p. Cell Re\. 148: 243-248. Reference: Biol. Hull 178: 111-117. (April. I WO) A Photoperiod Determined Life-Cycle in an Oligochaete Worm BERND SCHIERWATER1 AND CARL HAUENSCHILD Zoologisches Institut der Technischen Universilaet, Pockelsstr. Wa, 3300 Braunschweig, West Germany Abstract. For one common cosmopolitan naidid worm, Stylaria lacustris, we studied the effects of differ- ent environmental factors upon ( 1 ) the alternation of re- productive modes, (2) the rates of population increase, and (3) the combination of each of ( 1 ) and (2 ). While age, temperature, population density, or rate of feeding did not affect the mode of reproduction, photoperiod had a dominant effect. Under long-day conditions (LD > 12: 12), all worms reproduced exclusively by paratomic fis- sion, theoretically ad infinitum. When transferred to sh- ort-day conditions (LD < 12:12) the worms ceased vege- tative reproduction, and within 2 to 4 weeks developed the hermaphroditic genital apparatus and a clitellum. Af- ter an additional two weeks, the first cocoons were pro- duced. The switch to the bisexual mode of reproduction was cum grano sails irreversible. These findings are con- sistent with observations of field samplings, and allow one to predict the annual life-cycle strategy of S. lacus- tris. This is the first example of a photoperiod deter- mined life-cycle within the oligochaete worms. The vegetative mode of reproduction led to extremely high rates of population increase, whereas with the bisex- ual mode of reproduction the number of individuals was roughly stable. However, because 5. lacustris could not withstand temperatures of 5°C or lower, the switch to sexual reproduction and the formation of diapausing co- coons appear to be the only mechanism of overwinter- ing. Nevertheless, some 'asexual' clones never switch to sexual reproduction, whereas a loss of the asexual vegeta- tive mode of reproduction did not occur. In contrast to some general predictions from life-history theories, the reproductive strategy of 5. lacustris is highly prepro- Received 13 November 1989; accepted 2 January 1990. 1 Present address: Yale University. Department of Biology. P.O. Box 6666, New Haven, CT 065 1 1 . grammed and cannot respond to sudden and unexpected environmental changes. Introduction With the pollution of our environment, present day ecology demands investigation of the biological mecha- nisms that regulate the distribution and dynamics of populations in a given environment (e.g., Brinkhurst and Jamieson, 1971; McElhone, 1978; Brinkhurst and Cook, 1980; Tauber et al., 1986; Zaslwaski, 1988; Klerks and Levington, 1989). The rapid increase in theories on the evolution of life histories demands extended experimen- tal work and empirical data (cf. Stearns, 1976, 1980; Rez- nick, 1985; Hoekstra, 1987; Michod and Levin, 1988; Hauenschild, 1989;Nunney, 1989). The study of particular oligochaete worms can be highly fruitful to our understanding of both the ecologi- cal and the evolutionary implications of animal life-cy- cles for two reasons: ( 1 ) the oligochaete worms in general are regarded as perhaps the most important group con- cerned with the retrieval of organic matter in freshwaters (e.g., Brinkhurst and Jamieson, 1971; Dumnicka and Pasternak, 1978; Brinkhurst and Cook, 1980). (2) Those oligochaete worms that are capable of reproducing both by a bisexual and by a vegetative mode of reproduction, in particular the Aeolosomatidae and Naididae, allow in- traspecific comparisons of the consequences of sexual vs. asexual life-history tactics; such systems allowing experi- mental work are badly needed but are difficult to find (cf. Bell, 1980; Townsend and Calow, 1981; Calow, 1983; Reznick, 1985; Hoekstra, 1987; Abugov, 1988; Schier- water, 1989; Hadrys et al., 1990). Unfortunately, the number of well understood life-cycles that include an 'al- ternation of reproductive modes,' is surprisingly low (cf., Giese, 1959; Giese and Pearse, 1959; Kinne, 1970; Brinkhurst and Cook, 1980; Townsend and Calow, 1981; Holm, 1988). in 112 B. SCHIERWATER AND C. HAUENSCHILD Records of the reproductive ecology of most oligo- chaetes are limited to notes on the presence of sexually mature specimens in field populations, but almost no conclusions can be drawn from these scattered notes; thus, little is known about the mechanisms affecting the mode of reproduction and hence their annual life-cycles (e.g., Vershinin and Semernoi, 1977; McElhone, 1978, 1982; Mill, 1978; Brinkhurst and Cook, 1980; Pascar- Gluzman, 1981; Wetzel, 1982). In this study we will present the annual life-cycle model as well as quantitative data on the consequences of sexual vs. asexual reproduction for the cosmopolitan naidid Stylaria lacustris Linnaeus 1 758. Its life-cycle has not been described, though growth rates of vegetative worms have been studied (Streit, 1978; McElhone, 1982; Finogenova, 1984) and several brief notes about sexual worms are available (Kamlyuck and Kovaltchuk, 1972; McElhone, 1978, 1982; Wetzel, 1982). McElhone (1982) suggested that food supply, food quality, and water qual- ity may affect the 'alternation of reproductive modes' in S. lacustris. In this study we will demonstrate that the life cycle of S. lacustris is strictly and exclusively determined by the photoperiod (day-length). We shall discuss this first finding of a photoperiod-determined life-cycle in an oligochaete worm in an ecological context regarding the evolution of the life-history strategy. Materials and Methods Animals Stylaria lacustris is one of the most common and widely distributed oligochaete species, found in Europe, Asia, Africa, and North America (e.g., Brinkhurst and Jamieson, 1971; Vershinin and Semernoi, 1977; McEl- hone, 1978;Pascar-Gluzman, 1981; Wetzel, 1982). In 5. lacustris, the vegetative mode of reproduction follows the type of paratomic fission of animal chains of between two to three individuals (Stephenson, 1 930). The biology of sexual reproduction has not been described. Worms were counted as 'sexual' if either gonads and/ or a clitellum were visible. All other worms were called 'vegetative' independent of the formation of tomits. Field samples All animal material of Stylaria lacustris was collected from the field at different times and transferred into the laboratory for culturing under defined laboratory condi- tions. Worms were collected in W. Germany from a pond at Weddel. Braunschweig, in July 1985, Sept. and Oct. 1987, June and July 1988, and from the river II- menau at Uelzen in August 1986, '87, '88. Right after sampling, as many worms as possible were isolated and checked within 24 h for their reproductive status (i.e., presence or absence of a clitellum, gonads, tomits) by means of a dissection microscope at 20X. Laboratory studies Under defined laboratory conditions, we investigated whether the following environmental factors influence the 'alternation of reproductive modes': temperature, feeding, population density, and photoperiod. Culturing. Culture dishes (400 cm3 'deep freeze' plastic containers) were kept in thermoregulated rooms or in chambers providing temperature constancy to ±1°C, as controlled by mini-max-thermometers; normal photo- period setting was LD = 16:8, unless otherwise stated. S. lacustris was cultured either in filtered and heated (2 h at 80°C) water of its natural environment, or in carbonic- acid-free natural mineral water ('VitteF or 'Volvic'). Worms were fed on the green algae Haematococcus la- custris and Goniuin sociale ad lib. The air bubbled cul- ture dishes were washed, and water and food were re- newed twice a week. Acclimation time to any new experimental condition was 24 h. The highest changes in temperature were 5°C per day. Temperature changes of 10°C were done step- wise within 4 days. For long-period observations on the reproductive activity under different feeding, tempera- ture, and photoperiod conditions, acclimation time was 14 days, unless otherwise stated. Animals were observed through a binocular micro- scope ('Zeiss' 475052-9901 ) with variable magnifications from 8x to 50x. One ocular was equipped with a //m- scale for in vivo measurements of one-dimensional dis- tances. Photoperiod settings. Experiments on the effects of photoperiod on the mode of reproduction were run at 20 ± 1 °C, unless otherwise stated. The following LD settings were used: LD = 6: 1 8, 1 2: 1 2, 1 6:8, 1 8:6, and 24:0. Light intensities were 500-2000 Ix during light periods and = 0.05 Ix in the dark, respectively. The light intensities were measured with a lightmeter (Gossen, Mavolux 6C 18493), and because of the use of fluorescent lights (Os- ram L40W/22-1), the light intensity values have to be taken with care. During all experiments and observa- tions on the effects of temperature, feeding rate, popula- tion density and age. light-dark rhythm was held con- stant at LD = 16:8. Mean doubling times. For observations on mean dou- bling times (mdt), 30 worms each were placed in plastic chambers of =400 cm3, and the number of worms per chamber was counted once a week. After each counting, the total number of worms was reduced to a maximum of 50 worms per chamber. Two populations (one from Weddel pond and one from the river Ilmenau) were fol- lowed over 8 weeks (2 weeks acclimation plus 6 weeks of registration) at 10, 15, 20, and 25°C. The mdt's were PHOTOPERIOD1SM IN AN OL1GOCHAETE 113 u T> O CL 03 L X (D to 100 50 A) LD = 6ilB T=20 t .« B) LD = 6,18 T=20 °C A) LO = 12,12 T=20 t * B) LO = 12,12 T=20 °C 14 21 28 t I me (d) 35 42 Figure 1 . Examples for the time course of switching from the vege- tative to the bisexual mode of reproduction induced by the photoperiod in Sty/aria lacusiris. Vegetatively reproducing worms from the field were exposed to short-day conditions of either LD = 1 2: 1 2 or LD = 6: 18 at day 0 in the figure; A = Weddel 1985, B = Ilmenau 1986 popula- tion; the points for LD = 16:8 are not shown, for all of them would lie along the abscissa (= 0% sexual reproduction); N > 1000 for each population. calculated from the initial population size (N 1 ), the final population size (N2) and the time (t) in days between the countings: mdt = log 2 t/(log N2 - log N 1 ). Statistics. The non-parametric Mann-Whitney-U test (two-tailed) was used to compare the means of two inde- pendent samples, and the Jonckheere test was used to look for monotonous trends in three or more indepen- dent samples (Lienert, 1976). The number of statistical replicates (i.e.. number of cultures tested under the same experimental conditions) is given as n in the text, whereas N means the total number of worms checked for their reproductive mode during one experiment. The alternation of reproductive mode worked only in one direction, i.e., only vegetative worms switched to sexual reproduction. Once this switch had occurred, it was cum grano salis irreversible. Ninety-one clitellate worms had been transferred back from LD = 6: 1 8 to LD = 16:8 (T = 20°C) and watched for 42 continuous days. Only three worms reverted to vegetative asexual repro- duction by forming tomits; in these three worms the cli- tellum of the parent individual was retained, whereas the daughter individuals showed normal asexual shape. Bisexual reproduction excluded fission, i.e., clitellate worms never formed tomits. Therefore, once a popula- tion became sexual, the number of worms per chamber became constant or even declined slightly, because some worms always died after changing the photoperiod. Un- der laboratory conditions, the number of cocoons pro- duced per sexual worm was low at all tested tempera- tures. Rates of cocoon production ranged from 0.25 to 2.0 cocoons per sexual worm until death. The highest rates of cocoons produced per sexual worm, within 3 weeks after the clitellum became visible, were 0.5 (T = 15°C), 0.7 (T = 20°C), and 0.8 (T = 25°C). Generally the first cocoons were produced at 16.3 ± 5.61 days (range 7-3 1 days; N = 650, n = 13) after clitellum forma- tion. Bisexual reproduction thus led to a very limited rate of production of reproductive units. Cocoons were lem- on-shaped and preferably placed in the corners of the culture dishes. Mean ± SD cocoon length was 0.8 ± 0.07 mm (n = 78), and each cocoon contained a maximum of three embryos. Asexual clones. Two culture populations of 5. lacustris that came from the Weddel pond and had been cultured exclusively vegetatively (T = 20°, LD = 16:8) over 14 or 23 months, respectively, failed to show a photoperiodic Results Photoperiod The mode of reproduction was strictly determined by the photoperiod. Under long-day conditions (LD > 12: 12) the worms reproduced exclusively vegetatively by paratomic fission. Exposure to short-day conditions ( LD < 12) induced a quantitative switch from paratomic fis- sion to sexual reproduction within 10 to 30 days (Fig. 1), i.e.. the hermaphroditic genital apparatus has been developed. The formation of the clitellum always oc- curred after the gonads became visible (in some cases up to 25 days later). The reaction time to the short-day con- dition, i.e.. for the switch in the mode of reproduction, was affected by the temperature. One population was di- vided into three portions and each portion cultured at either 15, 20, or 25°C. With increasing temperature the time for switching from the vegetative to the sexual mode of reproduction was significantly shortened (P < 0.001, Jonckheere, N = 3 X 38; see Fig. 2). c O O L D. O L 100 50 OOP D LO = 6,18 T=15 °C »-.«-.« C) LO = 6.18 T=20 °C - C) LO = 6.18 T=25 °C 4 21 28 t I ma (d) 35 42 Figure 2. Example of the effect of temperature on the time course of switching from the vegetative to the bisexual mode of reproduction in Sly/aria lacusiris; one vegetative population (C = Weddel 1988) of 1 14 worms was divided into 3 portions, and each group of 38 worms was exposed to one of three different temperatures under short-day con- ditions (LD = 6: 1 8) at day 0. 114 B. SCHIERWATER AND C. HAUENSCHILD Table I The mean doubling limes (nidi) ofvciu'lativcly reproducing Stylaria lacustns at different experimental temperatures range T mdt ra N n [d] min max 10 277 7 11.1 ±4.04 9.3 16.9 15 657 12 6.9 ±2.47 4.8 10.9 20 733 12 5.1 ±2.83 2.7 13.2 25 854 12 5.0 ± 3.49 2.5 14.4 The means ± SD and the ranges over a 6-week observation period of each two cultures are given. At 10°C, one culture died after the first week of observation period, hence here n = 7. reaction. Samples of each population were tested at both LD = 1 2: 1 2 and LD = 6: 1 8 each at 1 5°C and 20"C over 13 weeks. From 238 worms, only 8 worms, i.e., =3%, became sexually mature. Mean doubling times for vegetative populations. The differences in mdt between the Weddel and the Ilmenau populations were not significant (U-test) and hence the groups were pooled in Table I. The mdt decreased sig- nificantly with increasing temperature (P < 0.05, Jonck- heere). However, the differences between 20°C and 25°C were not significant (U-test). Other environmental factors No S. lacustris worm reproduced sexually when the light period was > 12 h per day. Thus, the mode of repro- duction was independent of temperature, feeding and population density, and age (see Table II). Temperature. Different populations of 5. lacustris were exposed to temperatures between 5 and 30°C for 14 weeks. Temperatures of 5°C and 30°C were not tolerated, and all experimental animals died within 6 days (N = 2 X 63, n = 2 X 3). In the zone of thermal-tolerance, not one sexually reproducing individual was found (N > 1000) during 14 weeks of observation. One culture at 10°C was cultured for another 9 weeks and checked 3 times per week. On 10 October 1988 (after 21 weeks in controlled conditions), two worms were found that had a well developed genital apparatus and a clitellum. Neither worm was found after two weeks; their fate is unknown. Cocoons were not found. Feeding. Feeding rates reduced to 3 days feeding per week over 10 weeks never led to a sexual worm. All worms kept reproducing vegetatively (N > 600, n = 3). Starvation experiments, resulting in LD5(I values of 18 ± 3.75 days (range for total population extinction: 20- 31 days), also never led to sexually reproducing worms (N = 100, n = 3; T = 20°C, LD = 16:8). Population density. Different population densities of 0.1-1.5 worms cm 2 bottom area of culture dish were tested over 10 weeks at T = 20°C. No sexual worms were found (N> 1000, n = 3). Field samples Field samples of S. lacustris were taken at different times of the year. Only in one sample, collected in Octo- ber 1987, were both vegetative as well as sexual worms found. In all other samples collected between June and September exclusively aclitellate worms were found (see Table III). The only exception was observed on 19 Au- gust 1988. Two clitellate worms were detected in a field sample from the Weddel pond collected on 1 7 July 1988. The sample (including plant material) had been stored in a plastic beaker in our laboratory for four weeks at natural daylight. Other worms found in the sample were aclitellate (N = 108) on 19-21 August. Discussion Although studies of the seasonal development of or- ganisms have always occupied an important place in ex- perimental biology, the leading role of the signaling fac- tors in determining seasonal phenomena have been largely unknown. For 5". lacustris, the results of this study unmistakably demonstrate that the life-cycle is strictly determined by the photoperiod as the relevant external signaling factor. Since the outstanding discoveries of Garner and Allard (1920) and Rowan (1926) on photo- periodic phenomena in plants and animals, many im- portant contributions have derived from studies in par- ticular on polychaetes and insects (for overview see Giese, 1959; Kinne. 1970; Segal, 1970; Hauenschild. 1975; Tauber et a/., 1986; Zaslawski, 1988), but none from the phylogenetically closely related oligochaetes. The alternation of reproductive modes Stylaria lacustris apparently measures the day-length (proximate factor) to prepare for the sharp temperature decline (ultimate factor) during winter. Under long-day (summer) conditions, the worms reproduced exclusively asexually by paratomic fission, theoretically ad infini- tum. In the 1960's, Hauenschild (unpubl.) cultured a population of 5. lacustris for more than six continuous years in the laboratory at LD = 16:8 and T = 20°C, and he did not find a single sexually mature worm during this period. In the short-day (autumn conditions), S. lacustris reproduces only once and then the worms die. Hence, S. lacustris can best be termed as a 'continuous asexual and monotelic bisexual breeder' (using the terminology as re- viewed by Mill, 1978). The two findings of sexual worms of unknown origin under long-day conditions can hardly weaken the results. However, the two observed asexual populations that al- PHOTOPER1ODISM IN AN OLIGOCHAETE 115 Table 1 1 The effects of age and different environmental factors on the switch from vegetative to bisexual reproduction in Stylaria lacustris Age Temperature Pop. dens. Feeding rate Short -day N^(n) >1000(5) > 1000 (5) > 1000 (3) >600(3) >1000(5) time[d] 98 98 70 70 21-36 sexual p ] 0 0 0 0 >95 Nveg = initial number of vegetative worms exposed to the conditions listed. (n) = Number of different populations tested. time = Observation time. Short-day = LD < 12:12. The photopenod (short-day) is the only factor found to determine the switch from the vegetative to the bisexual mode of reproduction. Under long-day conditions the worms never became sexually mature (as followed continuously over more than 20 generations), independent of temperature, population density (pop. dens.) and feeding rate. most did not switch to sexual reproduction under short- day conditions are noteworthy. It is unknown whether an irreversible genetically based loss of sexuality or some kind of 'permanent modification' had occurred in these populations. The latter was first observed by Hauen- schild (1956, 1957) in the anthomedusae of Eleutheria dichotoma from the Mediterranean. Here, a small per- centage of primary medusae directly budded off from the polyp was regularly found to be asexual. In the field a loss of the sexual mode of reproduction is known from the sedentary polychaete Ctenodrilus serratus. In the North Sea, C. serratus reproduces exclusively asexually by par- atomic fission, whereas in the Mediterranean Sea sexu- ally mature (hermaphroditic) worms are known. In S. lacustris, asexual clones were only found in populations that had been cultured vegetatively for a long time in the laboratory (here, more than 14 or 23 months, respec- tively). Whether field samples also include a small per- centage of asexual clones cannot be answered, because some worms always died when changing the photope- nod from long- to short-day. In the field, the asexual clones would go extinct whenever the temperature dropped below 5°C, i.e.. normally during the winter in the Palaearctic area. Only the 'normal' clones showing the photoperiodic reaction can survive. However, in bio- topes showing annual temperature fluctuations between only 10°C and 25°C, a loss of the bisexual mode of repro- duction in field populations of S. lacustris seems likely, analogous to the polychaete C. serratus. If those habitats are found this can be easily tested by exposing popula- tion samples to short-day conditions. The annual life-cycle From the findings of this study, the life-cycle of S. la- custris can be roughly described as shown in Figure 3. The prediction is rough in the sense that the tested LD scalings were broad and the within-population genetic variation is unknown. The predicted life-cycle from this study allows 5. lacustris to start sexual reproduction and hence production of diapausing cocoons prior to. and in anticipation of, the cold winter period, which is critical for the worms' survival (cf. Denlinger et al.. 1978; In- grisch, 1984; Zaslawski, 1988). At 52°N. lat. this would be from October to November, corresponding to the nat- ural habitats in north Germany, where ponds usually do Table III Proportions of reproductive modes infield samples of Stylana lacustris Date Place N Sex [N] Veg [N] { } [N] cut [%] Aclit [%] 14July 1985 pond Weddel 26 0 11 15 0 100 10 Aug. 1986 river Ilmenau 161 0 68 93 0 100 30 Aug. 1987 river Ilmenau 107 0 57 50 0 100 20 Sept. 1987 pond Weddel 4 0 1 3 0 100 16 Oct. 1987 pond Weddel 48 41 0 7 85 15 12 June 1988 pond Weddel 96 0 53 43 0 100 Samples were taken from different plants (e.g.. of the genera Ceratophyllum, Cham, Elodea. Nasturtium) from a pond at Weddel (near Braun- schweig) or from the river Ilmenau near Uelzen (Niedersachsen. W. Germany); Oct. 16. 1987 is the only sample collected under natural short-day conditions: { | refers to individuals in which no kind of actual reproduction was obvious; Clit and Aclit refer to clitellate and aclitellate individuals, respectively. 116 B. SCHIERWATER AND C. HAUENSCHILD O) JB X o> T3 23456789 month 10 Figure 3. The annual lite-cycle of Sly/aria lacuxtri.i. The external signaling factor daylength is given for 52°N. lat. The laboratory studies and field samples suggest that the switch from asexual paratomic fission to bisexual reproduction occures in mid September at this latitude. About three weeks later the first worms become sexually mature, and the first diapausing cocoons are produced during October. Further ex- planations are given in the text. not freeze before December. The genetic variation within and between populations would be the predicted basis for adapting 5". lacustris to different annual cycles with respect to temperature and photoperiod (e.g.. Sauer, 1977; Sauer el a/.. 1986; Groeters and Dingle, 1987). This could be tested by collecting 5. lacustris at different latitudes with similar annual temperature cycles, or at different altitudes with similar annual photoperiod cy- cles, and measuring the threshold for the photoperiodic reaction (c;/. Hairston and Olds, 1984, 1986). 'All or nothing' life-history strategy By using the vegetative mode of reproduction, S. la- custris can double its number at least every 5 days at 20 to 25°C (Table I). Data on the rates of population in- crease by vegetatively reproducing populations of S. la- custris given in the literature range from mean doubling times of 3.6 to 12 days between 15°C and 20°C (Streit, 1978; McElhone, 1982; Finogenova, 1984). The data agree with Streit (1978), who calculated mdt of 3.6 (T = 19°C) and McElhone (1982), who estimated values of 4-6 days (T == 20°C). In the latter case it is not clear whether these are mean values or maximal values. Dur- ing summer all efforts are invested in vegetative repro- duction, leading to the most rapid population increases, regardless of the actual physical environmental condi- tions (r-strategy); during one season (April through Sep- tember) one single worm can theoretically give rise to a population of 3.4 billion worms. During autumn all effort is invested in sexual reproduction, i.e., the number of cocoons produced for overwintering is maximized, re- gardless of whether the winter temperatures go below 5°C or not. An unexpected and abnormal temperature de- cline to <5°C during summer or early autumn would lead to the total extinction of populations. Furthermore, the reproductive strategy cannot respond to unexpected changes in other environmental factors, like food supply and population density. Therefore, the life-cycle strategy of S. lacustris is an 'all or nothing' strategy maximizing reproductive output as far as possible (cf. Hirshfield and Tinkle, 1975; Pianka, 1976; Stearns, 1976). This is con- sistent with a high degree of adaptation to predictable annual cycles of environmental conditions; thus the life history strategy of S. lacustris does not fulfill some pre- dictions from life history theories. In a fluctuating and unpredictable environment (such as small freshwater ponds), we would expect S. lacustris (a) to reach early sexual maturity, instead of postponing it as far as possible to the end of the season or (b) to show reproductive flex- ibility in order to minimize the risk of total failure in a bad year (cf. Stearns, 1976, 1980; Glesener and Tilman, 1978; Mill, 1978; Sauer, 1984; Groetersand Dingle, 1987). The high abundances and the wide distribution of 5. lacustris, however, indicate that the photoperiodic life- cycle strategy can also be very successful in oligochaetes. 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Christophersen, H. Hensel, and W. Larcher. 1973. Temperature and Life. Springer, New York. 779 pp. Reznick, D. 1985. Cost of reproduction: an evaluation of the empiri- cal evidence. Oikos 44: 251-261. Rowan, W. 1926. On photoperiodism, reproductive periodicity and the annual migration of birds and certain fishes. Proc. Boston Sac. Nat. Hist. 3S: 141-189. Sauer,K.P. 1977. Die adaptive Bedeutung der genetischen Variabili- taet der photoperiodischen Reaktion von Panorpa vulgaris (Mec- optera, Panorpidae). Zool. Jahrb. Syst. 104: 489-538. Sauer, K. P. 1984. The evolution of reproductive strategies as an ad- aptation to fluctuating environments. Adv. Inv. Reprod. 3: 3 1 7-326. Sauer, K. P., H. Speith, and C. Gruener. 1986. Adaptive significance of genetic variability of photoperiodism in Mecoptera and Lepidop- tera. Pp. 153-172 in The Evolution oj Insect Life Cycles. F. Taylor and R. Karban, eds. Springer, New York. Schierwater, B. 1989. Allometric changes during growth and repro- duction in Eleutheria dichotoma (Athecata, Hydrozoa) and the problem of estimating body size in a microscopic animal. / Afor- phol. 200: 255-267. Segal, E. 1970. Light. Animals. Invertebrates. Pp. 159-211 Marine Ecology, Vol I, part I, O. Kinne, ed. Wiley, New York. Stearns, S. C. 1976. Life-history tactics: a review of ideas. Q. Rev Biol. 51:3-47. Stearns, S. C. 1980. A new view of life-history evolution. Oikos 35: 266-281. Stephenson, J. 1930. The Oligochaeta. Oxford University Press, Ox- ford. Streit, B. 1978. A note on the nutrition of Sly/aria lacuslris (Naidi- dae, Ohgichaeta). Hydrobiol. 61: 273-276. Tauber, M. J., C. A. Tauber, and S. Masaki. 1986. Seasonal Adapta- tion in Insects. Oxford University Press, Oxford. 4 1 1 pp. Townsend, C. R., and P. Calow, eds. 1981 . Physiological Ecology. An Evolutionary Approach to Resource Use. Blackwell Scientific, Ox- ford. 393 pp. Vershinin, N. V., and V. P. Semernoi. 1977. Qualitative and quantita- tive characteristics of oligochaetes of the Krasnoyarsk reservoir. Ekologiyal: 105-107. Wetzel, M. J. 1982. Aquatic oligochaeta in Kansas, USA, with notes on their distribution and ecology. Tech. Puhl. Slate. Biol. Surv. Kans. 12: 112-130. Zaslawski, V. A. 1988. Insect Development. Photoperiodic and Tem- perature Control. Springer, Berlin. 187 pp. Reference: Biol. Bull 178: 1 18-125. (April, 1990) Visualization of the Transparent, Gelatinous House of the Pelagic Tunicate Oikopleura vanhoeffeni Using Sepia Ink PER R. FLOOD1. DON DEIBEL2, AND CLAUDE C. MORRIS: 1 Institute oj 'Anatomy, University of Bergen, Arstadveien 19, 5009 Bergen, Norway and ^'Marine Science Research Laboratory, Ocean Sciences Centre, Memorial University of Newfoundland, St. John's, Newfoundland, Canada, A1C 5S7 Abstract. Appendicularian tunicates of the genus Oi- kopleura feed using an external, acellular, transparent structure known as the house. Previously, dilute particu- late dyes have been used to visualize the internal struc- ture of this house. However, because of toxicity, large particle size, and flocculation, many of these dyes have been of limited practical and scientific use. We report on a new marker, the ink from the cephalopod Sepia offi- cinal is. that solves many of these problems. Specimens of Oikopleura vanhoeffeni relished Sepia ink, having dark black stomachs and producing many dark fecal pellets over several days. When O. vanhoeffeni expanded houses in dilute ink, the internal walls, septae, and filters were shown in great detail, whereas high con- centrations of ink showed delicate patterns of lines on the internal walls. We present documentary photographs of previously unillustrated or undescribed morphologies: the escape slot; the incurrent funnels; two dimples caused by inser- tion of suspensory filaments on the upper wall of the pos- terior chamber, a large, posterior keel; both the open and closed positions of the exit valve; and the complex pat- tern of lines on the inner walls. However, the external walls of the house had no affinity for the dye and could only be seen by dark field illumination. We believe that Sepia ink can be used to visualize functionally important transparent structures of other gelatinous zooplankton and can be a colloidal marker in feeding experiments of a wide range of filter feeders. Received 25 September 1989; accepted 30 January 1990. Introduction Oikopleurid appendicularians are suspension feeding zooplankters that are surrounded by a transparent, acel- lular, gelatinous "house," which they secrete. The house contains a complex system of fine filters that are used by the animals to concentrate and remove food particles from suspension. Using its muscular tail as a pump, the animal draws water into the house through a pair of coarse, bilateral, incurrent filters. The water is then pumped through the tail chamber into bilateral passage- ways leading to the lateral edge of expansive food-con- centrating filters. Here much of the water is pushed through a mesh with 0.22 nm pore size (Deibel et a/.. 1985). Particles are retained between the food concen- trating filter screens, resulting in a concentrated food sus- pension that is drawn into a medial food-collecting tube leading to the animal's mouth. This food suspen- sion is 100 to 1000 times more concentrated than are particles in the environment surrounding the animal (Jor- gensen, 1984; Flood, in prep.). A third filter inside the pharynx of the animal traps the food particles for inges- tion. The filtered water exiting the food concentrating filter leaves the house through a narrow exit spout and valve, producing a jet that propels the house and animal slowly through the sea. The existence of the house has been known since the work of Fol (1872) and Lohmann (1899). However, many details of its structure remained unknown until re- cent improvements in microscopical techniques and spe- cial staining procedures made further progress possible. Dilute particulate dyes have been used to visualize the internal walls, chambers, and filters of the house (All- dredge. 1977; Flood, 1978, 1983; Deibel et a/.. 1985; 118 VISUALIZATION OF OIKOPLEURA HOUSES USING SEPIA INK 119 Deihel, 1986; Fenaux, 1986). When added to seawater, dye particles are retained by the filters within the house in the same way as are naturally occurring particles. Loh- mann (1899) and Alldredge (1977) used dilute suspen- sions of carmine particles to visualize both the incurrent and food-concentrating filters of many oikopleurids. However, the animals may not feed normally when car- mine is present (Alldredge, 1977). We have found that freshly prepared dilutions of carmine and extreme care are required to prevent the animals from leaving their houses. In addition, carmine particles settle rapidly and stain only the incurrent and food-concentrating filters. Fenaux ( 1986) used dilute India ink to stain the incur- rent and food concentrating filters, and the internal walls and septae of houses of Oikopleura dioica. One of us (P.R.F.) has used a similar technique since 1978. If India ink is added to seawater before the animal expands a new house, all internal walls and septae of the house are stained (Flood, in prep.). However, if the ink is added after the house has been expanded, only the food-con- centrating filter is stained. This approach requires freshly prepared ink solutions and great care to prevent the ani- mal from leaving its house. The carbon particles that make up India ink tend to aggregate in seawater and set- tle rapidly as do particles in carmine suspensions. Deibel ( 1986) used several types of particles to mark specific parts of the house of Oikopleura vanhoeffeni differentially. These particles included finely ground charcoal, starch, latex beads, and the unicellular green alga I\ochrysis galbana. Charcoal particles adhered spe- cifically to an intermediate, coarse screen between the two walls of the food-concentrating filter. The alga, on the other hand, stuck to the upper and lower walls of the food-concentrating filter. Starch granules did not adhere to any of the filters of the house, but stained the pharyn- geal filter within the trunk of the animal. This suggests that physical and chemical properties of both the marker and the house structures in question affect the staining result. We recently found another marker that may be used to visualize structural details of oikopleurid houses that, in the past, have been difficult or impossible to docu- ment. Information about these structures is needed to understand the behavioral and functional details of the feeding process on which the ecology of these animals depends. The new marker may be used not only to visu- alize feeding structures and quantify particle clearance rates of pelagic tunicates, but also to observe transparent structures of other marine plankton. Materials and Methods Individuals of Oikopleura vanhoeffeni. in their houses, were collected in 500-ml glass jars by SCUBA diving in Logy Bay, Newfoundland, during May and June 1989. Animals were maintained in these jars for up to 10 days in laboratory tanks containing circulating seawater at 1 to 5°C, about 1°C above ambient sea surface tempera- ture. Ink was collected from freshly dead specimens of the cuttlefish Sepia officinal/sal the Plymouth Marine Labo- ratory, Devon, U.K. The ink duct of each animal was clamped with a hemostat while the ink sac was removed. Once excised, a loop of the duct was placed in a collec- tion vial and the duct cut to allow the ink to drain into the vial. This ink was diluted immediately with 10 parts of distilled water and 1 0.000 units of pennicillin G added per ml of solution to help prevent bacterial decomposi- tion. Diluted ink remains liquid for several years, but un- diluted ink coagulates after several weeks at room tem- perature. The ink was dispersed with gentle agitation in seawater to a dilution of cu. 10 ? just before use. Solid Sepia ink is available commercially, but must be ground before use. It also contains phenol or other chemical preservatives that may be noxious or toxic to marine animals, and therefore it was not used in these experiments. Houses were examined using a Wild M420 macro- scope with bright or dark-field illumination. The light source used for routine observation was a 100W halogen lamp, whereas two modified Sunpak GX14 electronic flashes and a Wild MRS 5S/-5 1 photoautomat were used for still photography. We used Kodak Ektachrome 100 and 400 ASA film for slides, and Kodak Tmax at 100 to 3200 ASA for prints. By using 0.5- and 2.0-times acces- sory lenses on the macroscope, final magnification of the photographs ranged from 1 .25- to 25-times. Results and Comments Liquid Sepia ink was easily miscible in seawater, and, contrary to other dyes that have been used to visualize the house of Appendicularia. it stayed evenly dispersed in solution for up to 14 days. Transmission electron microscopy (TEM) revealed that this ink consists of uniformly spherical melanin granules with diameters ranging between 56 and 161 nm (arithmetric mean = 102 nm. Standard deviation = 21 nm. Flood, pers. obs. by TEM). In spite of this low particle size, some of the ink parti- cles were easily concentrated and ingested by Oi- koploeura vanhoeffeni. In fact, these animals seemed to relish the Sepia ink, having full stomachs and producing abundant opaque, black fecal pellets (Fig. 1) that ap- peared to be composed entirely of ink. However, much of the ink passed through the house of Oikopleura vanhoeffeni, without being witheld by the food-concentrating filters. Some of these particles ad- 120 P. R. FLOOD ET AL EW : ExV Figure I . Lateral view of a live Oikupleura vanhoeffeni heating its tail inside a house faintly stained by Sepia ink. Bright field macrograph at seven times magnification. [The nomenclature used is adopted from Flood ( 1 983) and is largely a direct translation of Lohmann's German names (Lohmann. 1956).] In addition to numerous details of the inside walls (/»") of the house, like the prominent exit spout (ExS) and valve (E\ "I "), a keel (A ), cushion chambers lateral (l.CC) and antero-medial (aCC) to the inlet openings, inlet funnels (//•"»). roof dimples (rd), and a roof hump (rli), numerous internal details can be seen. The animals trunk ( 7>), tail ( Ta), and escape chamber (EsCh) as well as the trunk chamber ( TrCh), tail chamber (TaCh). supply passage (SP). and suspension of the food-concentrating niters (FCF) in the posterior chamber (PCh) are faintly outlined. Numerous fecal pellets (/•"/*) stained completely black by Sepia ink are seen along the floor of the posterior chamber. The external wall (/:'H') is only visible above the hump in the inside roof. 1mm hered to the internal walls and septae of the house and made them easily visible. By varying the concentration of Sepia ink from experi- ment to experiment, the intensity of staining could be controlled to reveal different features of the house. When a specimen of Oikopleura vanhoeffeni expanded its house in seawater containing very dilute Sepia ink, the internal walls, septae, and niters were shown in great de- tail (Fig. 1 ), whereas heavy staining made the house less transparent and revealed delicate patterns of lines and fields on the internal walls and septae throughout the house (Figs. 2, 3). The outer wall of the house, however, had no affinity for the ink and was rarely seen at all in bright field illumi- nation (Fig. 1). However, in most cases its presence was revealed by adhering detritus particles. This was particu- larly true for the prominent bow of the house (Fig. 2). In dark field illumination, on the other hand, the external walls and their variable thickness in distinct parts of the house became more evident (Fig. 3). The difference in volume between the internal water- filled spaces and the total house could be estimated from such pictures. If the internal transverse diameter of the house was considered to be unity, the external transverse diameter was generally close to 1.2, the internal longitu- dinal diameter about 1.3. and the outside longitudinal diameter about 1 .7. Considering the house to be an ellip- tical rotatory body, this makes the total volume approxi- mately 1.5 times as large as the internal water-filled spaces. We do not know if the spaces between the inside and outside walls are filled with a compact (gel-like) sub- stance or if they are water-filled chambers unaccessible to the Sepia particles. By varying the staining intensity of the house, we dis- VISUALIZATION OF O1KOPLEURA HOUSES USING SEPIA INK 121 ExS EsP . / 4 1mm Figure 2. Top view of live Oikupk'iira vanhoeffeni inside its house. Bright field macrograph at seven times magnification after strong staining with Sepia ink. Intricate patterns of Sepia ink are seen on many walls, as for example near the escape passage (EsP) and supply passages (SP). Note also the attachment (arrows) of the animal trunk (7>) to the walls separating the trunk chamber (TrCli) from the escape chamber (EsC'h). The inlet openings (1O), inlet niters (//•'/), and the inlet funnels (IFu) are visible on both sides of the house. Note the prominent bow (B) made visible only by adhering detritus particles. Otherwise, same labeling as in Figure I. covered many structural details of which we were pre- viously unaware or had insufficient knowledge. Here we will only describe some of the most prominent features and comment briefly on their functional significance. 1 I ) The escape port in the anterior chamber (Figs. 2, 5B). The animal forces its way through this preformed weak part when it leaves the house, thereby tearing it open to a wide escape slot. This escape port is covered by the massive bow of the house (Fig. 2), and somehow a preformed channel must exist through this bow material towards the external house wall. Otherwise the animal could not force its way out of the house as easily, fre- quently, and uniformly as it does (cp. Fenaux, 1985). (2) The incurrent funnels leading into the house (Figs. 1-3). In the only existing description of the house ofOi- koplewa vanhoeffeni (Deibel, 1986. Fig. 1), these have been given quite a different shape from what we have been able to photograph. (3) The attachment of the anterior walls of the incur- rent funnels to the lateral part of the trunk (Fig. 2). These walls seem to meet the trunk exactly where the Langer- hans bristle is located. Through this sensory organ the animal may monitor accordingly the inflation and con- dition of the house (Bone and Ryan, 1979). Perhaps the entire house in this context may be regarded as a tactile sensory structure. A rather rigid suspension of the trunk of the animal within the house is needed for the tail to perform its pumping action. This prevents the "tail from wagging the dog" as may be observed just before the animal leaves its house, when the trunk has detached from some of its anchoring points. Stimulation of the Langerhans recep- tor may then initiate the vigorous jerk and swimming movement that enables the animal to detach completely from the house and force its way through the escape slot. (4) The shape of prominent chambers and lateral flaps 122 P. R. FLOOD ET AL Figure .V Inhabitated house of Oikopleura vanhocffeni as seen in dark held illumination from a point above, behind, and to the side of the house. (The axes of the house as it normally moves through the sea are indicated in the lower right hand corner.) Magnified seven times. Note the prominent patterning of the internal walls (/IT) corresponding to the attachment sites of the filter ridges of the food-concentrating filters (arrows) along the periphery of the supply passages (SP), and the presence of a prominent semitransparent jelly-like substance (G') covering the posterior side and the anterior bow-like pole (/J) of the house. The outer limit of this jelly-like substance represent the true exter- nal walls (All) of the house. The orientation of the house as it moves through the water is indicated by axes in the lower right hand corner of the figure. For other abbreviations refer to Figure 1 . in the anterior part of the house, medial and lateral to the incnrrent openings (Fis>.s. 1-3). These chambers are probably rilled by water flowing from the tail chamber through a hole in its distal floor (Flood, in prep.). It is also possible that the anterior chambers communicate with the upper compartment of the posterior chamber and may be tilled by water via this route, as suggested by Fenaux (1986). A positive pressure in the anterior cham- bers surrounding the incurrent funnels is needed to resist the collapsing force generated by the negative pressure within these passageways as water is drawn into the house. The lateral flaps may serve as vertical stabilizers to control the orientation of the house as it moves through the sea or as flaps to prevent the immediate re- clogging of the incurrent niters after they have been back- washed (Flood, in press). (5) Two large dimples in the inner house wall of the upper compartment of the posterior chamber (Figs. 1. 3). These probably represent the anchoring sites of suspen- sory filaments originating somewhere along the anterior edge of the food-concentrating filters. (6) A medial hump in the inner house wall above the trunk oj the animal (Fig. I). The external house wall had its highest optical density and could be faintly seen even in bright field illumination above this hump. Although of unknown functional significance, this hump is also found in houses of Oikopleura dioica and O. labrado- riensis( Flood, pers. obs.). VISUALIZATION OF OIKOPLEURA HOUSES USING SMI. -I INK 123 (7) A large "keel" at the hack of the house just above the exit valve (Figs. 1. 3). This keel may serve as a rudder to inhibit rolling and to facilitate looping motions as the house is propelled through the water. A looping motion, which has been described for other oikopleurans by All- dredge (1976), allows the animal to stay within and ex- ploit a patch of nanoplankton more efficiently than by a linear motion. This keel was discovered by Deibel (1986), but due to poor visibility, even in dark field illu- mination, his description is incomplete (Compare his Fig. 1 to our Fig. 1 ). (8) A posterior exit spout and valve below the longitu- dinal midline of the house (Figs. I, 3). Strong staining by Sepia ink allowed us to observe the opening and closing action of this pressure sensitive valve. In its closed posi- tion its upper and lower lips were inverted (Fig. 4A). One to five seconds after the pumping action of the tail started and increased the pressure inside the posterior chamber and exit spout, the lips everted and exposed a medial oval opening with a strongly birefringent and elastic rim (Fig. 4B). This central exit opening was evident even when the animal pumped slowly. However, when the tail pumped at maximum efficiency, the exit spout became much longer, and four additional exit openings were exposed peripheral to the central one. The tissue surrounding the exit valves was then stretched to such a degree that it left very little contrast in our photographs (Fig. 4C). Fenaux (1986), studying Oikopleura dioica houses, found the four peripheral openings to open before the central one. The propulsive thrust generated by the jet of water leaving the house was directed somewhat below the cen- ter of the house, resulting in a tendency to turn the front of the house upward. When combined with the slightly upward-pointing bow and the directional control of the keel and lateral flaps (see above), this thrust will result in a slow upward movement of the house, or even a vertical looping motion as sometimes seen in the field (cp. All- dredge, 1976). The more intense staining resulting from higher con- centrations of Sepia ink revealed delicate patterns of lines and fields on most internal walls of the house (Figs. 2, 5). In some areas, complex patterns of straight or curved lines were visible (Fig. 5A). These may corre- spond to decorated filaments, corrugated surfaces, or small pockets. In other areas, faint patterns of polygonal fields were apparent (Fig. 5B). Although each polygon was quite large, their pattern reminded us of the oi- koplast cell pattern on the trunk of the animal. These cells are responsible for the production of the house (Lohmann, 1933/1956); perhaps Sepia ink might be used to map the areas of the house made by individual cells. This represents a major problem yet to be properly elucidated for all appendicularians. Discussion The usefulness of the Sepia ink for visualizing distinct parts of Oikopleura houses probably depends on three or four factors: ( 1 ) Sepia ink forms stable solutions in seawater and does not aggregate and sediment like most other particulate dyes. Such flocculent particles seem to interfere with the house expansion process of animals kept in captivity. (2) The particle size of Sepia ink is small enough to allow a significant proportion of parti- cles to pass the food-concentrating filters to stain the walls of the posterior chamber, the exit spout, and possi- bly the anterior chambers of the house. (3) The animals seem to relish the Sepia ink as a food source and do not find it noxious or toxic like many other dyes. (4) The physico-chemical properties of the Sepia ink particles may be particularly favorable to stain the internal walls and septae of the house. These excellent properties of Sepia ink may make it useful in the study of other gelatinous zooplankters. The reason why Sepia ink, like all other particulate dyes we have used, failed to stain the external walls of the house remains obscure. It may depend on special physi- co-chemical properties of this layer, but a more likely ex- planation may be that the dye particles are prevented from having direct physical contact with it. The walls surrounding the water-filled spaces inside the house are probably not entirely waterproof. Due to the higher hy- drostatic pressure inside the house, water will seep slowly out through the walls, leaving its particles behind as a decoration on the internal walls, and producing a thin halo of particle-free water just outside the house. Such a halo may be enough to prevent the proper staining of the external walls. The pore size of the food-concentrating filters of Oi- kopleura vanhoeffeni — 1.0 X 0.22 ^m according to Deibel et al. (1985) — was significantly larger than was the particle size of Sepia ink (0. 1 ± 0.02 ^m according to Flood, unpub. res.). In spite of this, the animals used in this study easily concentrated and ingested the dye, and incorporated it into fecal pellets. This may depend on a selection of the largest particles in the ink, on a selection of aggregated particles, or on an ability to retain smaller particles than hitherto believed. In fact, the carbon bud- gets of oikopleurans seem to be such that ingested parti- cles > 0.2 ^m in diameter rarely account for more than 30% of the energy expenditure for growth, respiration, and house production (Paffenhofer, 1976;Gorsky, 1980; King, 198 1 ). It seems likely that the animals may obtain much of their nurishment from particles < 0.2 ^m in diameter, or from dissolved organic matter. We foresee the use of monodisperse Sepia ink particles in future feeding experiments on appendicularians and other filter feeding marine animals. 124 P. R. FLOOD ET AL PExO CExO 4C PExO EW Figure 4. Bright field macrographic details of the exit spout and valve of a heavily Sepia-ink stained house of Oikopleura vanhoeffeni at 20 times magnification. (A) In its closed state. (B) in its half open state, and (C) in its full open position. Unfortunately the exit openings themselves [one central (CExO) and four peripheral ( PE\O)\ didn't give sufficient contrast to be seen in picture C. The external wall (EM ") of the house is seen next to the exit spout. Figure 5. Bright field macrographic details of an Oikopleura vanhoeffeni house heavily stained by Sepia ink at 23 times magnification. In (A), parallel ruffles ( TChR) and numerous pockets ( TCliP) are seen in the roof of the tail chamber. In ( B), polygonal fields ( /)/) resembling cell outlines are seen next to the escape passage ( EsP) of the house. VISUALIZATION OF OIKOPLEURA HOUSES USING SEPIA INK 125 Acknowledgments Many thanks to the members of the Diving Unit (G. Chaisson, Divemaster) of the Ocean Sciences Centre, Memorial University, for assisting D.D. with the collec- tion of Oikoplcwa vankocffcni. and to Mr. Edward Downton for designing and fabricating laboratory equip- ment. This work is a result of a Bergen-Memorial Uni- versities Exchange Fellowship to P.R.F., and we thank Drs. Bodil Larsen and R. L. Haedrich, Memorial Uni- versity, for making this visit possible. This work was sup- ported by a grant from the Norwegian Research Council for Science and the Humanities to P.R.F., and by Oper- ating and Equipment Grants from the Natural Sciences and Engineering Research Council of Canada to D.D. This is Ocean Sciences Centre contribution number 6 1 . Note added in proof: We have used commercial ink from Sepia recently available from Sigma Chemical Co. (St. Louis, Missouri). Oikoplcwa vanhoeffeni took up this ink similarly to that we collected from Sepia ourselves. Literature Cited Alldredge, A. L. 1976. Field behavior and adaptive strategies of ap- pendicularians (Chordata: Tunicata). Mar. Binl. 38: 29-39. Alldredge, A. I.. 1977. House morphology and mechanisms of feed- ing in the Oikopleuridae (Tunicata. Appendicularia). J. /.. 1986. Feeding mechanism and house of the appendicular- ian Oikoplcwa vanhocllcni. Mar. Biol. 93: 429-436. Deibel, D., M.-L. Dickson, and C . V. I.. Powell. 1985. Ultrastructure ot the mucous feeding tiller of the house of the appendicularian Oikoplcwa vanhocltcni. Mar. i.col I'rogr. Ser 27: 79-86. Kenaux, R. 1985. Rythm of secretion of oikopleurid's houses. Bull Mar Sci. 37: 498-503. Fenaux, R. 1986. The house ofOikopleura dioica (Tunicata. Appen- dicularia): structure and functions. Zoomorphology 106: 224-23 1 . Flood, P. R. 1978. Filter characteristics of appendiculanan food catching nets. Expericntia 34: 173-1 75. Flood, P. R. 1983. The gelationous house ofOikopleura dioica (Ap- pendicularia, Tunicata); its architecture and water filtration mecha- nism. Ann. Meet. Western Soc. Naturalists. Burnaby. B. C. Canada. Dec. 1983 (Abstract). Fol, H. 1872. Etudes sur des Appendiculaires de Detroit de Messine. Mem. Soc. Phys. Hist. Nat. Geneve 21(2): 445-499. Gorsky, G. 1980. Optimisation des cultures d'appendiculaires. Ap- proche du metabolisme de O dioica. Ph. D. thesis, Univ. P. & M Curie, Paris VI. 110pp. Jorgensen, C. B. 1984. Effect of grazing: metazoan suspension feed- ers. Pp. 445-464 in Heterotrophic Activity in the Sea. }. E. Hobbie and P. J. leB. Williams, eds. New York, Plenum Press. King, K. R. 1981. The quantitative natural history of Oikoplcwa di- oica (Urochordata. Larvacea) in the laboratory and in enclosed wa- ter collumns. Ph. D. thesis. Univ. Washington, Seattle. Lohmann, H. 1899. Das Gehause der Appendicularien. sein Bau. seine Funktion und seine Entstehung. Schr. Nantrwiss. I 'er. Schles- H-ix-Holstein 11:347-407. Lohmann, H. 1933/1956. Appendiculariae. Handb. Zool. 5,11: 15- 202. PafTenhofer, G. A. 1976. On the biology of the Appendiculana of the southeastern North Sea. lO/li Enr. Symp. Afar. Biol.. Oslcml, Bel- gium, Sept. 1975. 2:437-455. Reference: Biol. Bull. 178: 126-136. (April. 1490) The Morphology and Mechanics of Octopus Suckers WILLIAM M. KIER AND ANDREW M. SMITH* Department of Biology, Coker Hall, CB# 3280, The University of North Carolina, Chapel Hill. North Carolina 27599-3280 Abstract. The functional morphology of the suckers of several benthic octopus species was studied using histol- ogy and cinematography. The suckers consist of a tightly packed three-dimensional array of musculature. Three major muscle orientations are found in the wall of the sucker: ( 1 ) radial muscles that traverse the wall; (2) circu- lar muscles that are oriented circumferentially around the sucker, including a major and minor sphincter mus- cle; and (3) meridional muscles that are oriented perpen- dicular to the circular and radial muscles. The connec- tive tissue of the sucker includes inner and outer fibrous connective tissue layers and an array of crossed connec- tive tissue fibers embedded in the musculature of the sucker. Attachment is achieved by reducing the pressure in- side the sucker cavity. We propose the following mecha- nism to explain this pressure reduction. Contraction of the radial muscles thins the wall and thus increases the enclosed volume of the sucker. If the sucker is sealed to the substratum, however, the cohesiveness of water re- sists this expansion. Thus, contractile activity of the ra- dial muscles reduces the pressure of the enclosed water. The radial muscles are antagonized by the circular and meridional muscles so that the three-dimensional array of muscle functions as a muscular-hydrostat. The crossed connective tissue fibers of the sucker may store elastic energy, providing a mechanism for maintaining attachment over extended periods. Introduction Octopus suckers perform a remarkable variety of func- tions. Packard (1988) listed six distinct roles of the suck- ers of benthic octopuses including: (1) locomotion; (2) anchoring the body and holding prey; (3) sampling, col- Received 16 October 1989; accepted 30 January 1990. * Order of authorship is merely alphabetical. lecting, and manipulating small objects; (4) chemotactile recognition; (5) displays; and (6) cleaning maneuvers. These diverse roles demand that the suckers be flexible and dexterous yet capable of generating large forces (see Dilly c/ ai, 1964). Previous research has focussed on the chemotactile ability of the suckers (see Wells, 1978), on the sensory receptors of the suckers (Graziadei, 1962; Graziadei and Gagne, 1976a, b). and on their morphol- ogy (see below). Our understanding of how the sucker generates the movements that allow it to manipulate and forcefully grip objects is incomplete. The morphology of octopus suckers has been de- scribed previously. Nixon and Dilly (1977) described the surface features of octopus and squid suckers from different genera. The sucker musculature has been de- scribed by Girod (1884), Guerin (1908), Nachtigall (1974), Niemiec (1885), and Tittel (1961, 1964), but the proposed mechanisms of action are incorrect both in their analysis of the function of the musculature and in understanding the ability of water to sustain sub-ambi- ent pressures. Previous studies also overlooked impor- tant features of the connective tissue. The suckers are muscular-hydrostats as defined by Kier and Smith (1985) (see also Smith and Kier, 1989). The musculature is arranged in a tightly packed, three- dimensional array that provides the skeletal support and the force for movement. This type of system produces movements that are localized and remarkably complex, allowing precise changes in shape by bending, contracting, or stretching at any point. In this paper we describe the muscle arrangements in the suckers of several octopus spe- cies and discuss the function of these arrangements. Materials and Methods Experimental animals Specimens of Eledone cirrosa were supplied by The Laboratory of the Marine Biological Association of the 126 SUCKER FUNCTIONAL MORPHOLOGY 127 United Kingdom, Plymouth. Specimens of Octopus jou- bini and Octopus maya were supplied by The Marine Biomedical Institute of the University of Texas Medical Branch at Galveston, Texas. Specimens of the Octopus bimaculoides/bimaculatus complex (see Pickford and McConnaughey, 1949) were supplied by Pacific Bio-Ma- rine, Venice, California, and Chuck Winkler Enterprises, San Pedro, California. Observations of sucker behavior and kinematics were made primarily on O. bimacu- loides/bimaculatus and O. maya. A detailed morpholog- ical analysis of the suckers was performed on specimens of E. cirmsa, O. joubini, and O. bimaeulatus/bimacu- loides. Histology Blocks of arm tissue that included several suckers were obtained from freshly killed animals that were anesthe- tized in 1% ethanol in seawater. The tissue was fixed in Bouin-Dubosq fixative (Humason, 1979) or in 10% for- malin in seawater for 24-48 h. In some cases, blocks of tissue were obtained from specimens that had been fixed whole in 10% formalin in seawater after anesthesia. The tissue was dehydrated in ethanol and embedded in par- affin (MP 56°C). The blocks were sectioned serially at 5- 10 /urn on a rotary microtome. Serial sections were made in three mutually perpendicular planes. The sections were stained using one of the following techniques: ( 1 ) Mallory's triple stain as outlined by Pantin (1946); (2) Milligan trichrome stain; (3) Picro-Ponceau with iron hematoxylin; or (4) Mowry's colloidal iron method. The procedures followed for stains 2-4 above are outlined by Humason (1979). Sections were examined with bright- field, phase contrast, and polarized light microscopy. Computer-assisted three-dimensional reconstruction The extrinsic musculature of the suckers of one speci- men of E. cirrosa was examined using a computer program for three-dimensional reconstruction (PC3D Three-Dimensional Reconstruction Software, Jandel Scientific, Corte Madera, California). Serial frontal sec- tions (see description of section planes below) 10 nm thick were used for the reconstructions. The outlines of the major muscle groups of every fourth section were traced using a camera lucida on a compound micro- scope. Alignment of the series of tracings was performed according to the visual best-fit method (Gaunt and Gaunt, 1978; Young etai, 1985). The tracings were then digitized with a Numonics 2210 digitizing tablet. The PC3D software, running on a CompuAdd 286/12 AT microcomputer, stacked the outlines of specified muscle bundles from each section, producing a three-dimen- sional representation of the muscular morphology that could be viewed in any orientation. The reconstructions shown in Figure 8 were plotted on a Hewlett-Packard HP 7475A plotter. Cinematography A specimen of 0. maya was filmed walking on a glass aquarium wall with a Canon Scoopic 1 6mm movie cam- era filming at 48 frames/s using Eastman Ektachrome Video News Film. The film was viewed frame by frame on an L-W International film analyzer, and calipers were used to measure the diameter of the sucker and the diam- eter of the opening to the acetabulum. The measurement error was <5%. Measurements were made from one 100- ft roll of film, choosing every sucker (total of 26 suckers) that attached or released and whose outlines were dis- tinct enough to measure. Suckers attached to the glass could be distinguished because they remained stationary relative to the movement of the arm. Results Gross morphology of the suckers The gross morphology of the suckers of different octo- pus species has been described previously (Girod, 1 884; Guerin, 1908; Niemiec, 1885; Nixon and Dilly, 1977; Packard, 1988), and a brief summary of observations on the species we examined is provided here. The sucker consists of two general regions: the acetabulum and in- fundibulum (Girod, 1884) (Fig. 1). The infundibulum is the exposed portion of the sucker that is applied to the substratum during attachment. The acetabulum is a more or less spherical cavity that opens to the infundibu- lum through a constricted orifice (Fig. 1). The surface of the infundibulum bears a series of radial grooves and ridges while the surface of the acetabulum is smooth. The sucker is covered by a chitinous cuticle or sucker lining (see below) that is particularly well-developed on the in- fundibulum. The sucker lining is shed periodically and renewed continuously (Girod, 1884; Naef, 1921-1923; Nixon and Dilly, 1977; Packard, 1988). The infundibu- lum is encircled by a rim covered with a deeply folded, loose epithelium. The suckers are attached to the arms by a short muscular base that is covered by a continua- tion of the dermis and epidermis of the arms. A single row of suckers is present on the arms of E. cirrosa and two rows of suckers are present on the arms of the Octo- pus species. Sucker microanatomy For the purposes of this discussion, we refer to trans- verse and frontal sectional planes. Transverse sectional planes are defined as sections perpendicular to the long axis of the arm. Frontal sections are parallel to the plane defined by the opening of the sucker. Intrinsic sucker musculature. Although we did not 128 W. M. K.IER AND A. M. SMITH Figure I . Schematic diagram of the microanatomy ot'the sucker of Octopus in transverse section. A. acetabulurn; AR. acetahular roof; AW. acetabular wall: C, circular muscle; CC, crossed connective tissue fibers; D, dermis; E, extrinsic muscle; EC, extrinsic circular muscle; EP, epithelium; IN, infundihulum; 1C, inner connective tissue layer; M, meridional muscle; OC. outer connective tissue layer; R, radial muscle; SI. primary sphincter muscle; S2, secondary sphincter muscle. make a systematic study of a wide range of sucker sizes and sucker locations on the arms, the general arrange- ment of the muscle and connective tissue of the suckers is the same for the different species and sucker sizes we examined. Several minor differences between genera were observed and are noted below. The acetabular and infundibular portions of the sucker consist primarily of a tightly packed, three-dimensional array of muscle fibers. The muscle fibers can be categorized by orientation into three major groups: radial muscle fibers that extend across the wall of the sucker more or less perpendicular to the inner surface; circular muscle fibers that are ori- ented circumferentially around the sucker and parallel to the frontal plane; and meridional muscle fibers that are oriented perpendicular to the radial and circular muscle fibers (Fig. 1 ). The acetabular portion consists of a wall region and a domed roof. The acetabular wall includes radial, circu- lar, and meridional muscle fibers. The acetabular roof includes radial and meridional muscle fibers but lacks circular muscle fibers. Radial muscle fibers extend be- tween their origins and insertions on an inner fibrous connective tissue layer lining the acetabulum and an outer fibrous connective tissue layer encapsulating the sucker (Figs. 1-3). As the radial fibers project toward the outer surface, they interdigitate with bundles of meridio- nal muscle fibers. In the acetabular wall, the radial mus- cle fibers also interdigitate with circular muscle bundles (Fig. 2). The circular muscle bundles extend around the perimeter of the acetabular wall. The location of the circular and meridional muscle bundles in the acetabular wall of the suckers of Eledone cirrosa is different from that of the Octopus species exam- ined in this study. In E. cirrosa, the meridional muscle bundles are located peripheral to the circular muscle bundles. In the Octopus species, however, the arrange- ment is reversed; a distinct series of circular muscle bun- dles are located peripheral to the meridional muscle bun- dles (Compare Figs. 1 and 2). In addition to the circular muscle bundles of the ace- tabular wall, a mass of circular muscle forms a sphincter located adjacent to the inner surface at the level of the narrow orifice that connects the infundibulum to the ac- etabulum (Figs. 1, 2). A secondary sphincter is also evi- dent near the junction between the outer surfaces of the walls of the acetabulum and infundibulum and has a cross-sectional area that is approximately 1 0% of the area of the primary sphincter. The meridional muscle fibers project from a point near the apex of the acetabular roof toward the sphincter muscles as an array of flat bundles that lie between the radial muscle fibers. When the outer surface of the ace- tabular roof is viewed in a grazing frontal section, the meridional muscle fiber bundles appear to be arranged in a stellate pattern (Fig. 4). Many of the meridional muscle fibers insert on the outer connective tissue layer at the level of the sphincter muscles. Some meridional muscle fibers extend into the wall of the infundibulum. The arrangement of muscle fibers in the wall of the infundibulum is similar to that of the acetabular wall de- scribed above. Radial muscle fibers extend across the wall from their origins and insertions on the inner and outer connective tissue layers of the infundibulum. The radial muscles pass between a series of flat bundles of circular muscle fibers located adjacent to the inner sur- face of the infundibular wall (Fig. 5). Meridional muscle fibers are also present in the infundibular wall (Fig. 1, Fig. 5). Many originate on the outer connective tissue layer at the level of the sphincter muscles and extend to- ward their insertion at the margin of the infundibulum while others appear to be extensions of the meridional fibers of the acetabular wall. The bundles of meridional fibers are flat and are interwoven between the radial mus- cle fibers. Sucker connective tissue. The two major components of the connective tissues of the sucker are an array of crossed connective tissue fibers embedded in the muscu- lature of the acetabular roof, and the inner and outer connective tissue capsules. Thin layers of connective tis- sue also surround the circular and meridional muscle bundles of the sucker. It is likely that the connective tis- sue fibers observed in the sucker are collagenous because SUCKER FUNCTIONAL MORPHOLOGY 129 they appear birefringent when viewed with polarized light microscopy and show staining characteristics typi- cal of collagen. The inner and outer connective tissue capsules are compact layers of fibers that enclose the sucker muscula- ture. The layers appear to be arranged as a crossed-fiber array when viewed in transverse sections that graze the inner or outer surface of the wall (Fig. 6). At the level of the sphincter muscles, fibers of the outer connective tis- sue layer penetrate into the musculature of the sucker wall (Fig. 5). These fibers branch repeatedly and extend to the primary sphincter muscle, dividing it into fasci- cles. The extension of the outer connective tissue capsule that encloses the infundibulum is thinner than that of the acetabulum. The inner connective tissue capsule extends from the acetabulum to the infundibulum without any appreciable change in thickness. In addition to the connective tissue layers encasing the sucker, crossed connective tissue fibers are present in the musculature of the roof of the acetabulum (Figs. 1, 3). These fibers extend between the inner and outer connec- tive tissue capsule at oblique angles to the radial muscle fibers. They are reminiscent of the "intermuscular" con- nective tissue fibers described by Gosline and Shadwick (1983a, b) and Bone et cil. (1981) in the mantle of squid and cuttlefish and those described by Kier (1989) and Kieret al. (1989) in the fins of squid and cuttlefish. How- ever, the angle they make with the radial fibers is not con- stant (Fig. 3). These connective tissue fibers do not occur in the acetabular wall. The boundary between the acetab- ular roof and the acetabular walls includes a particularly robust band of intermuscular connective tissue fibers, and the wall is thinner at this point (Fig. 1 ). Sucker epithelium. Several distinct zones of epithe- lium are present on the sucker (see also Girod, 1884; Guerin, 1908; Nixon and Dilly, 1977; Packard, 1988). The epithelium lining the infundibulum consists of tall columnar cells resting on a basal lamina and the inner connective tissue capsule. These cells secrete a tough, chitinous cuticle (Hunt and Nixon, 1981). The surface of the cuticle bears numerous tiny denticles or pegs, each secreted by a single columnar epithelial cell (see Nixon and Dilly, 1977). The epithelial cells lining the radial grooves of the infundibulum are cuboidal, and the cuti- cle lining the grooves lacks denticles. The cells of the epi- thelium lining the acetabulum are cuboidal. In addition, the denticles are rudimentary or absent from the cuticle lining the acetabulum. The transition between the epi- thelial surfaces of the infundibulum and acetabulum oc- curs at the level of the primary sphincter muscle (Figs. 1 , 2, 5). Another transition is observed in the groove that separates the rim and the infundibulum. The epithelial cells in the groove are cuboidal and the cuticle is thin and lacks denticles. The epithelium covering the pillows and folds of the rim is columnar and the underlying dermis is loose and folded. An additional differentiation of the epithelium was observed in a zone surrounding the sucker rim. Cells in this zone showed intense staining by Mowry's colloidal iron stain (Humason, 1979) for acid mucopolysaccharides(Fig. 7). Girod ( 1 884) described the infundibulum of the suck- ers of Octopus vulgaris as being covered by numerous small "hillocks" of tall columnar epithelial cells and cuti- cle with denticles. He describes the epithelium between the hillocks as being flattened. Although small hillocks are visible on the surface of the infundibulum or on shed sucker linings of the species we examined, no differenti- ation of the epithelium was observed between the hill- ocks. A flattened epithelium was only observed in the radial grooves. Extrinsic sucker musculature. The suckers are at- tached to the arms by a series of extrinsic muscle bundles (see also Guerin, 1908). A group of major extrinsic mus- cle bundles is associated with each sucker and originates on the connective tissue sheath surrounding the arm musculature (Kier, 1 988) and extends orally to converge on the sucker (Fig. 8). These bundles insert on the outer connective tissue capsule of the sucker at the level of the sphincter muscle (Figs. 1, 2). The extrinsic muscle bun- dles are, in turn, surrounded along much of their length by a sheet of circumferential muscle fibers (Fig. 8). In addition to the major extrinsic muscle bundles illus- trated in Figure 8, a medial group of smaller diameter extrinsic muscle bundles was observed in the region en- closed by the major extrinsic bundles. Although many are oriented parallel to the major bundles, some follow oblique courses, crossing from one side to the other. Kinematics Octopus suckers are capable of a wide range of move- ments. The animals explore their environment with their arms, holding their suckers extended and splayed out. The muscular base that attaches the sucker to the arm can elongate to twice its resting length, extending the suckers away from the arm. Sometimes individual suck- ers were observed to probe through small openings such as a screen, then extend fully and tilt up and down or side to side. If the sucker is stimulated lightly, it either extends to attach to the stimulus or withdraws, always orienting so that the infundibulum faces the object. When the octopus is active, the infundibuli of the suckers are flattened. Sucker "footprints" in wax show that the entire infundibulum is pressed firmly against the substra- tum during attachment. When the animal is at rest, the infundibuli are cone-shaped. An octopus can grip nearly any size object with its suckers. They seem to prefer large flat surfaces but can easily grip irregular objects and objects smaller than their suckers. When manipulating threads or thin sheets, the 130 W. M. K.IER AND A. M. SMITH Figure 2. Photomicrograph of a transverse section of a sucker from Eledone cirrosa in the region of the primary and secondary sphincter muscles (SI, S2) and the acetabular wall. The radial muscles (R) extend from the inner connective tissue capsule (1C) to the outer connective tissue capsule (OC). Inter- woven between the radial muscle fibers are meridional muscles (M) and circular muscles (C). An extrinsic muscle (E) inserts on the outer connective tissue capsule adjacent to the secondary sphincter muscle. The photomicrograph was made using bnghtfield microscopy of a 15 ^m-thick paraffin section stained with Milligan tnchrome. The scale bar equals 0.5 mm. Figure 3. Photomicrograph of a transverse section of a sucker of Octopus bimaculoides/bimaculatus in the region of the acetabular roof. The crossed connective tissue fibers (CC) extend across the roof from the inner (1C) to the outer (OC) connective tissue capsule at oblique angles to the radial muscle fibers. Meridional muscle fibers (M) are also visible adjacent to the outer surface of the acetabular roof. The intersection of the meridional bundles at the axis of radial symmetry is apparent in the top of the micro- SUCKER FUNCTIONAL MORPHOLOGY 131 suckers sometimes fold so that the two halves of the in- fundibulum grasp the object like a mittened hand (see also Packard, 1988). A sucker can grip a strand of fishing line and pull on it with surprising force. When it does this, the crease of the fold is usually parallel to the long axis of the arm. Suckers are often observed to fold over the corner of an object without noticeably weakening the force of attachment. A striking example of this occurs when a sucker is attached to the end of a cylinder with a smaller diameter than that of the sucker. Here the perim- eter of the infundibulum folds around the side of the cyl- inder while the remainder of the infundibulum presses flat against the end. The movies allowed us to distinguish and quantify changes in the sucker's dimensions during suction, par- ticularly the diameter of the large sphincter. We consid- ered the diameter of the orifice leading to the acetabulum to be the same as the diameter of the inner surface of the sphincter. We measured the diameter of the rim and the diameter of the orifice when the sucker was attached (x) and when it was relaxed (x0). When gripping, the rim diameter increased from its resting state (x = 1 .26xo° 94; r = 0.82) as does the orifice diameter (x = 1.48x0087; r = 0.80). The movies also showed that the roof of the ace- tabulum does not press against the substratum during at- tachment, contrary to the mechanism reported by Pack- ard (1988). Discussion Principles of forming a suction attachment Suckers attach to the substratum by forming a seal at the rim and reducing the pressure in the acetabular cav- ity. This decrease in pressure has been measured and can account for all of the attachment force of octopus suckers (A. M. Smith, in prep). The acetabular cavity is filled with water, and the ability of water to withstand this de- crease in pressure is critical to sucker function. The dis- tinction between water-filled and air-filled suckers has not been emphasized in previous studies of sucker func- tion (see Denny, 1988). A sucker filled with air has different mechanical re- quirements from one that is filled with water. An air- filled sucker must significantly increase its enclosed vol- ume to decrease the pressure in the cavity. Starting from 0. 1 MPa ambient pressure ( 1 atm), doubling the volume would halve the pressure to 0.05 MPa, increasing the vol- ume ten times would only reduce the pressure to 0.01 MPa. To create a vacuum, the cavity must be reduced to a negligible volume before attachment. The lowest possi- ble pressure inside such a sucker would be a vacuum (0 MPa). At normal ambient pressure (0. 1 MPa), the force holding this sucker and the substratum together would be 0.1 MPa multiplied by the area exposed to the vacuum. Octopus suckers operate in water rather than air, which leads to two important functional consequences: first, the sucker can decrease pressure without detectably expanding, and second, the pressures generated will not necessarily be limited to a vacuum. Water is essentially inexpansible at physiological stresses because of its cohe- sive strength. Therefore, water resists the activity of the muscles that expand the enclosed volume. Thus, if more water does not leak into the sucker, the muscles involved in generating suction contract isometrically, reducing the water's pressure. As long as the water adheres to all surfaces, the sub-ambient pressure in the water pulls the substratum tightly to the sucker. Also, as long as the wa- ter adheres to all surfaces, the sub-ambient pressure is only limited by the strength of the water-water bonds. Water columns have sustained pressures as low as -27.0 MPa in the laboratory without breaking (cavitating) (Briggs, 1950). Pressures of this magnitude are extremely difficult to achieve in practice because water does not ad- here perfectly to all solid/liquid interfaces. Nevertheless, unlike the situation in air, suckers filled entirely with wa- ter have the potential to generate pressures well below 0 MPa. In fact, pressures below 0 MPa have been mea- sured inside octopus suckers (A. M. Smith, in prep). The difference between air and water has been over- looked in previous work in which octopus suckers were assumed to operate by creating a vacuum (Girod, 1884; Guerin, 1908), or where the pressure was assumed to be limited to a vacuum (Nixon and Dilly, 1977). Parker (1921) measured the suction force from one sucker, but apparently performed this experiment in air, which would explain why he did not measure pressures lower than 0.028 MPa. The failure to make a distinction between air and wa- ter may have led to errors in the literature dealing with such diverse groups as limpets and torrential stream- dwelling vertebrates. Hora (1930) claimed that certain graph (arrows). The photomicrograph was made using brighlfield microscopy of a 10 jim-thick paraffin section stained with Milligan trichrome. The scale bar equals 0.25 mm. Figure 4. Photomicrograph of a grazing frontal section of the acetabular roof of a sucker from O. bimaculoides/bimaculatus. The stellate arrangement of the meridional muscles (M) is visible. The radial muscle fibers (R) and extrinsic muscles (E) appear in cross section in this micrograph. The photomicro- graph was made using brightfield microscopy of a 10 /im-thick paraffin section stained with Mallory's triple stain. The scale bar equals 0.25 mm. 132 W. M. ICIER AND A. M. SMITH Figure 5. Photomicrograph of a transverse section of a sucker from Eledone cirrosa in the region of the infundihulum. The layers of circular muscle bundles (C) are interwoven between the radial muscle bundles ( R ). The inner connective capsule (1C) underlies the tall columnar epithelium (EP) of the infundib- ulum. The primary sphincter muscle (SI ) is also visible. The photomicrograph was made using brightfield microscopy of a 1 5 fim-thick paraffin section stained with Milligan tnchrome. The scale bar equals 0.25 pm. Figure 6. Photomicrograph of a grazing transverse section of a sucker from Octopus bimaculnides/ himaciilutiix in the region of the acetabular wall. The connective tissue fibers of the outer connective tissue capsule (OC) are oriented in a crossed-fiber array. Radial (R) and circular (C) muscle fibers of the acetabular wall are also visible. The photomicrograph was made using polarized light microscopy of a 10 ^m-thick paraffin section stained with Picro-Ponceau and iron hematoxylin. The scale bar equals lOO^m. SUCKER FUNCTIONAL MORPHOLOGY 133 8 Figure 8. Computer-assisted three-dimensional reconstruction of a portion of an arm of Eledane cirrosa showing the extrinsic musculature associated with two suckers. In (a), the outer surface of the arm and suckers is visible. In (b), the epithelium and dermis have been removed to reveal the extrinsic muscles (E) that are surrounded by the extrinsic circular muscles (EC). In (c), the extrinsic circular muscles have been removed to reveal the course of the regularly arrayed extrinsic muscles (E). In (d) the extrinsic muscles have been removed to reveal the arm muscle (AM) and the musculature of the two suckers. The axial nerve cord (N) is also visible. fish are not using suction because the center of the adhe- sive disc is not elevated during attachment, as it must be to create a partial vacuum in air. But these suckers are water-filled and therefore there would be no detectable expansion of the cavity while generating sub-ambient pressure. The attachment of limpets has also been attrib- uted to mechanisms other than suction because their te- nacity exceeds that which can be explained if one as- sumes that water cavitates at 0 MPa (Branch and Marsh, 1978; Grenon and Walker, 198 1 ). Seawater can, in fact, endure pressures lower than 0 MPa without cavitating (A. M. Smith, in prep). Thus, suction attachment mecha- nisms may be used in both groups. Another important factor that may have been over- looked is the depth-dependence of the attachment force. The depth at which an octopus lives will have an effect on the relative pressure difference that can be created in the sucker. Just below the surface, the maximum pres- sure differential is determined by the ambient pressure (approximately 0. 1 MPa) outside the sucker and the ab- solute pressure at which seawater cavitates inside the sucker. With an increase in depth, the ambient pressure Figure 7. Photomicrograph of a transverse section of a sucker from O. joubini. The epithelium sur- rounding the sucker rim (arrow) shows intense staining of arid mucopolysaccharides. The remaining tissues of the sucker are unstained in this micrograph. The photomicrograph was made using brighttield microscopy of a 10 /jm-thick paraffin section stained with Mowry's colloidal iron stain. The scale bar equals 0.25 mm. 134 W. M. KIER AND A. M. SMITH outside of the sucker increases while the absolute pres- sure at which seawater cavitates is unaffected. The poten- tial relative pressure difference increases by 0. 1 MPa with every 10 m of depth, rather than doubling as stated by Denny (1988) in his discussion of gas-filled suckers. Since the attachment force of a sucker is the pressure differential multiplied by the area of attachment, the greater pressure differential at depth might allow deep- sea octopuses to create the same attachment force with smaller suckers, assuming that sufficient force can be produced by the sucker musculature. It is of interest in this regard that Voight (1990) has observed an inverse correlation between sucker diameter and depth of occur- rence for a variety of octopus species. Proposed fund ion of octopus suckers In the following discussion, we propose hypotheses of sucker function, based on the principles of suction at- tachment outlined above and a biomechanical analysis of the musculature and connective tissue. Further exper- imental work is required to test these proposals. Initial contact: forming ami maintaining a seal. The first step in generating suction is forming a tight seal to prevent water from leaking in and equalizing the pres- sure. This implies that the infundibulum must be flexible and dexterous enough to mold itself to a wide range of surface shapes and textures. The infundibulum is com- posed of a tightly packed three-dimensional array of muscles that allow precise bending. Kier and Smith ( 1985) outlined the basic principles of this type of system, termed a muscular-hydrostat, and described the wide va- riety of movements of which it is capable. Since the mus- cular system itself has a constant volume, contraction in one dimension must be compensated by expansion in at least one other. Contraction of the radial muscles of the infundibulum will thin the infundibulum and thereby extend the rim, increasing the circumference and the sur- face area facing the substratum. More importantly, the radial muscles can hold the rim extended by resisting the increase in thickness that must accompany retraction of the rim. If the distance from the rim to the primary sphincter cannot decrease when the meridional muscles of the infundibulum contract, the infundibulum will bend toward the arm, flattening the face of the sucker. The circumferential muscles of the infundibulum may function as antagonists to the meridional muscles by constricting the infundibulum to a conical shape. One major advantage the sucker gains by using a muscular- hydrostatic mechanism rather than hard skeletal ele- ments is the local control of movement that is possible. The effect of muscle contraction in the infundibulum is localized such that it can bend at any point. This allows it to match exactly the contours of the substratum. Once matched to the substratum, the mucus and loose epithe- lium of the rim may provide the seal. The denticles on the chitinous lining of the infundibu- lum probably play an important role in maintaining a seal at the rim margin rather than close to the orifice. If the ends of the denticles are resting on the substratum, then an interconnected, water-filled network of spaces will be formed between them. This network may provide a means of transmitting the subambient pressure of the acetabular cavity underneath the entire infundibulum, thereby pulling it tightly against the substratum. The wax impressions of attached suckers demonstrate that the en- tire infundibulum is forcefully applied to the substratum during attachment. Without such a provision for trans- mitting pressure, the seal would probably be formed at the orifice and no force would be available to hold the infundibulum against the substratum. This would dra- matically decrease the shear resistance of the sucker. To form an effective attachment, suckers must be able to resist not only forces that lift the sucker away from the substratum but also shearing forces that slide the sucker along the substratum (see Denny, 1988). This is particu- larly important since the animals appear to prefer hold- ing objects so that the arms are aligned parallel to the force and most of the suckers are thus being sheared rather than being pulled normal to the surface. The fric- tion between the rim and the substratum resists shearing forces and also prevents the rim from sliding towards the center as the pressure in the cavity drops. The denticles on the infundibulum may enhance the friction between the rim and the substratum. As long as the sub-ambient pressure presses the infundibulum against the substra- tum, the denticles provide a substantial frictional force. In shear, this force determines the tenacity of the attach- ment. The constant wear from friction may require the sucker linings to be shed periodically. Some form of denticles or roughened pads often occur on the suckers of other animals. Green and Barber (1988) reported numerous discrete papillae on the mar- ginal region of the sucker of the clingfish. These are cov- ered with a keratin-like cuticle. The authors suggest that these may provide frictional resistance to shear. It is also possible that they allow transmission of the sub-ambient pressure to the rim, as we suggest above for octopus suck- ers. Denticles or projections are also observed on the sur- faces of suckers of other aquatic vertebrates (Hora, 1 930; Nachtigall, 1974), lumpsuckers (Arita, 1967), and tad- poles (Gradwell, 1973;Inger, 1966). Nixon and Dilly (1977) proposed an adhesive function for the denticles on the infundibulum, but they are un- clear whether the proposed force comes from capillarity or suction. There is no evidence that the denticles alone are adhesive. Our analysis suggests that only the muscu- lature and connective tissue of the acetabulum are needed to generate the attachment force (see below). Also important for initial contact are the extrinsic muscles that move and orient the entire sucker. Our me- SUCKER FUNCTIONAL MORPHOLOGY 135 chanical analyses predict the following functions for these muscle groups. The major extrinsic muscles link the sucker to the arm, transmitting the force of attach- ment. They also retract the sucker. The band of extrinsic circular muscles surrounding these dorsoventral muscles will extend the entire sucker away from the arm by thin- ning the base that connects the sucker to the arm. Simul- taneous contraction of both sets of muscles will tilt the sucker, depending on the location of the active dorsoven- tral extrinsic muscles relative to the axis of rotation. If the circumferential muscles did not provide resistance, the ma- jor extrinsic muscles would only retract the sucker. The primary sphincter muscle probably serves an im- portant function in maintaining suction. As previously suggested, the extrinsic muscles transmit the force of at- tachment to the arm. These muscles converge from their origin on the arm to insert adjacent to the sphincter. Thus, in transmitting force to the arm, the extrinsic mus- cles also tend to increase the diameter of the sphincter. During attachment, the diameter of the orifice was ob- served to increase. Contraction of the sphincter restricts the extent of this increase. If the sphincter could not resist the increase, then the sucker would deform and probably lose its grip. Interestingly, Guerin (1908) stated that pe- lagic octopuses in the family Alloposidae lack a primary sphincter. His figure illustrating a histological section of an allopsid sucker does not show any large extrinsic mus- cles, only diffuse connective tissue. Another pelagic octo- pus, Japatella diaphana, appears also to lack both pri- mary sphincter muscles and large extrinsic muscles (Nixon and Dilly, 1977). The coincident lack of a pri- mary sphincter and large extrinsic muscles would be pre- dicted if the sphincter serves to resist deformation from the stress of muscles that connect the sucker to the arm. Sub-ambient pressure generation. Although the infun- dibulum is critical for making the initial contact, it is the muscles of the acetabulum that probably create the sub- ambient pressure required for attachment. The radial muscles are arranged such that their contraction would increase the enclosed volume, were it not for the resis- tance of the water. Contraction of the radial muscles of the acetabulum generates a force that tends to thin the wall. Because the wall has a constant volume, a decrease in thickness must increase the internal surface area, or overall size, of the hemisphere and cause the cavity to expand. The cavity cannot expand, however, because of the resistance of the enclosed water. In resisting this ex- pansion, the water is put in tension. The muscular-hy- drostat mechanism of the sucker allows suction attach- ment to occur even if no force is being transmitted from the arm to the sucker. Indeed, the suckers of amputated arms can still attach strongly (Rowell, 1963) as can iso- lated suckers ( Parker, 1921). The circumferential muscles and meridional muscles of the acetabulum probably function as antagonists to the radial muscles. Contraction of the circumferential muscles alone would decrease the circumference and in- crease the height of the acetabulum. Contraction of the meridional muscles alone would decrease the height of the acetabulum. When the sucker is not attached, their simultaneous contraction evenly decreases the hemi- sphere volume and thereby thickens the cavity wall. The arrangement of radial, meridional, and circumferential muscles in the wall of the acetabulum appears typical of most of the suckers from a variety of phyla as described byNiemiec(1885). An important aspect of sucker morphology that has been overlooked previously is the array of crossed con- nective tissue fibers in the musculature of the acetabular roof. Gosline and Shadwick ( 1983a, b) described an ar- ray of crossed connective tissue fibers in the mantle of squid and showed that it could serve as an elastic energy storage mechanism during locomotion and mantle ven- tilation. Perhaps the connective tissue fibers in octopus suckers also store energy. This elastic energy could main- tain sub-ambient pressure in the sucker over extended periods of time, which might account for the observation that octopuses often hold onto objects for several hours. Prior to attachment, the connective tissue fibers of the acetabular roof could be prestrained by the thickening of the acetabular muscle mass that is created by the activity of the meridional and circumferential muscles. Then, upon attachment, the stored strain energy might exert a force analogous to that created by the radial muscles. Thus, rather than expending energy by contracting the radial muscles to maintain suction, suction could be maintained by virtue of the elastic properties of the con- nective tissue fibers. Nevertheless, several aspects of the arrangement of the connective tissue fibers are perplex- ing in the context of this mechanism. For example, it is unclear why the acetabular wall lacks these fibers and why the fiber angle is not more regular. Further work is needed to clarify the function of the crossed connective tissue fibers. Acknowledgments We thank S. F. Huggins for help with histology and O. Moe for help with the kinematics. We are grateful to K. K. Smith, J. R. Voight, S. A. Wainwright, and anony- mous reviewers for comments on the manuscript. H. Crenshaw and the Duke University Department of Zool- ogy biomechanics lab group provided valuable discus- sion. The Marine Biomedical Institute of the University of Texas Medical Branch at Galveston provided assis- tance with animal maintenance and supply. We thank A. K. Harris for the use of a 16mm cine film analyzer and S. A. Wainwright for the use of a 1 6mm cine camera. This material is based upon work supported under a Na- tional Science Foundation Presidential Young Investiga- 136 W. M. ICIER AND A. M. SMITH tor Award (DCB-8658069) to W.M.K. and a National Science Foundation Predoctoral Fellowship to A. M.S. A National Science Foundation Research Experiences for Undergraduates Supplement to W.M.K.'s Presidential Young Investigator Award provided support for the par- ticipation of S. F. Huggins in the preliminary histological study. Literature Cited Arita, G. S. 1967. A comparative study of the structure and function of the adhesive apparatus of the Cyclopteridae and Gobiesocidae. M. Sc. thesis. University of British Columbia, Vancouver. Bone, Q., A. Pulsford, and A. D. Chubb. 1981 . 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Saugnapf-.epi- und hypofasciale Armmuskulaturder Kephalopoden — ein Beitrag zur funktionellen Anatomic freibe- weglicher Skelettmuskelkorper. Gegenbaurs Morphol. Jahrb 106: 90-115. Voight, J. R. 1990. Population biology of Octopus digueti and the morphology of American tropical octopods. Ph.D. Dissertation. University of Arizona, Tucson. Wells, M. J. 1978. Octopus: Physiology and Behaviour oj an Ad- vanced Invertebrate. Chapman and Hall. London. 417 pp. Young, S. L., E. K. Fram, and B. L. Craig. 1985. Three-dimensional reconstruction and quantitative analysis of rat lung type II cells: a computer-based study. Am. J Anal. 174: 1-14. Reference: Biot. Bull 178: 137-143. (April, 1990) The Limulus Blood Cell Secretes a2-Macroglobulin When Activated PETER B. ARMSTRONG1, JAMES P. QUIGLEY2, AND FREDERICK R. RICKLES3 Marine Biological Laboratory, H 'oods Hole, Massachusetts 02543 Abstract. Alpha:-macroglobulin, a protease-binding protein that is reactive with almost all endopeptidases, is present in high concentrations in the plasma of the horse- shoe crab, Limulus. Alphai-macroglobulin was demon- strated by its ability to protect the active site of trypsin from inactivation by the macromolecular active site in- hibitor, soybean trypsin inhibitor, and by reaction with an antiserum prepared against purified Limulus «:-mac- roglobulin. The blood cells also contain «2-macroglobu- lin in a form that is released when washed cells are stimu- lated to undergo exocytosis by treatment with the iono- phore, A23 1 87. Alpha2-macroglobulin is detected in the materials released from the cells during degranulation both by activity in the soybean trypsin inhibitor-protec- tion assay and by immunochemical staining of Western blots. The subunit molecular weight of the cell-associ- ated form of «2-macroglobulin. 185 kDa, is identical to that of the plasma form. The amount of a2-macroglobu- lin contained within the cells of a given volume of blood is 0.5-2% of the quantity in solution in that volume of plasma. The distilled water lysates of N-ethylmaleimide- stabilized amebocytes used to detect endotoxin (e.g., Limulus amebocyte lysate or LAL) contain relatively large quantities of active «:-macroglobulin. These prepa- rations are essentially free of the principal plasma pro- tein, hemocyanin, indicating that the cells had been well washed prior to lysis. Received 20 September 1989; accepted 18 January 1990. 1 Department of Zoology. University of California. Davis. California 95616. - Department of Pathology. Health Sciences Center, State U niversity of New York, Stony Brook, New York I 1 794-869 1 . 3 Medical Research Service, Veterans Administration Medical Cen- ter, Newington, Connecticut 061 1 1 and the Division of Hematology- Oncology, Department of Medicine, School of Medicine, University of Connecticut Health Center. Farmington, Connecticut 06032. Introduction Higher animals deploy a variety of defense systems to cope with invading pathogens that are based on compo- nents in solution in the blood or associated with the blood cells. These systems operate to restrict the growth and invasion of pathogens and to disable their toxic products. The protein, rt:-macroglobulin, is an element in the system of humoral defenses that binds proteases of all of the major classes and from diverse sources, in- cluding proteases of microbes (Barrett, 1981; Feinman, 1983;Sottrup-Jensen, 1987, 1989). This breadth of reac- tivity contrasts with the relatively narrow range of prote- ases recognized by individual active-site protease inhibi- tors (Laskowski and Kato, 1980; Travis and Salvesen, 1983). Proteases bound to «:-macroglobulin are ren- dered incapable of hydrolyzing protein substrates and, at least in mammals, are removed from the circulation when the a2-rnacroglobulin-protease complex is inter- nalized into secondary lysosomes following receptor-me- diated endocytosis (Van Leuven, 1984). Alphaj-macro- globulin is apparently of considerable evolutionary an- tiquity, because it has been demonstrated in vertebrates (Starkey and Barrett, 1982), arthropods (Armstrong and Quigley, in prep.; Quigley and Armstrong, 1983, 1985; Armstrong el ai. 1985; Hergenhahn and Soderhall. 1985;Spycherrta/.. 1987; Hergenhahn el al, 1988), and molluscs (Armstrong, unpub. data) — forms whose evo- lutionary lineages diverged approximately 0.5-0.6 bil- lion years ago. In the arthropod, Limulus (the American horseshoe crab), «2-macroglobuIin is present in the plasma at concentrations of approximately 0.3-3 pM (unpub. data), which is similar to the levels reported for humans (3.5 nM: Coan and Roberts, 1989; Harpel, 1987). The blood of Limulus contains a single type of cellular element, the granular amebocyte (Armstrong, 1985a), 137 138 P. B. ARMSTRONG ET AL. which functions as a thromhocyte. Blood clotting in- volves formation of a cellular plug of adherent amebocytes at sites of injury (Loeb, 1920; Bursey, 1977) and the release of the extracellular clotting system from exocytotic vesicles contained within the amebocyte (Bang, 1979; Miirer el ai. 1975; Armstrong and Rickles, 1982). This latter system consists of the structural pro- tein of the clot and a system of proteases that act on the apo form of the clottable protein to convert it into the form that polymerizes into the fibrils of the extracellular clot (Levin, 1985). The present report documents the presence of a:-macroglobulin in the granular amebocyte in a form that is released from the cell during exocytosis. Materials and Methods Limulus blood cells One hundred ml of blood obtained under sterile, en- dotoxin-free conditions from pre-chilled animals by car- diac puncture (Armstrong. 1985b) was collected into sterile chilled 50 ml plastic centrifuge tubes (Falcon Plas- tics, Lincoln Park, New Jersey) and centrifuged at 150 X g. The plasma was discarded and the cells were resus- pended in 20 ml of ice-cold anti-coagulant buffer [0.5 A/ sterile, endotoxin-free NaCl (Travenol Laboratories, Deerfield, Illinois), 0.01 A/ethylenediaminetetraacetate, 0. 1 A/ glucose, 0.056 M citrate buffer, final pH 4.6 (Soder- hall and Smith, 1983)]. The amount of cells in a preparation is presented as the volume of the cell pellet present at this stage. The cells were then washed 3 times with 20 ml/wash ice-cold endotoxin-free 0.5 M NaCl and resuspended in 15 ml endotoxin-free 0.5 M NaCl, 0.01 M CaCl:. Exocytosis was initiated by adding the iono- phore, A23187 (Sigma Chemicals, St. Louis, Missouri), to a final concentration of 10 n M The preparation was incubated at 22°C for 3 h to allow the blood cells to de- granulate, aggregate, and for the cell aggregate to con- tract. Approximately 13-14 ml of cell-free fluid was col- lected. This fluid contains the contents of the exocytotic granules, apparently uncontaminated by cytoplasmic constituents, because the cytoplasmic marker enzyme (lactate dehydrogenase) is present in the cells and can be released by detergent extraction of the cell pellet, but is absent from the materials released by the A23 1 87-treated cells (Armstrong and Quigley, 1 985). SDS-poh 'cicr\ -lun i iilc gel elect n tplioresis Standard techniques were used for SDS-polyacryl- amidegelelectrophoresis(Laemmli. 1970). Soluble sam- ples were dissolved in reducing sample buffer and were not boiled, to prevent heat fragmentation of the «;-mac- roglobulin (Armstrong and Quigley, 1987). and electro- phoresed at constant current on 7.5% polyacrylamide gels. We have found the coagulin clot of Limulus to dis- solve only sparingly in reducing sample buffer (Rickles, unpub. data); not surprisingly, live blood cells and the cell clot produced following A23187-stimulated degran- ulation of blood cells tailed to dissolve completely. Coag- ulin is the most abundant protein of both preparations. However, soluble proteins from the cells, including a2- macroglobulin, are dissolved under these conditions. Anti-a^-tnacroglobulin antisenim A purified preparation of the plasma form of Limulus os-macroglobulin (Quigley and Armstrong, 1985) was subjected to SDS-polyacrylamide gel electrophoresis (Laemmli, 1970; reducing conditions, 12% polyacryl- amide gel) and transferred to nitrocellulose paper by electrophoretic blotting (Towbin el ai. 1979). The posi- tion of the protein was determined by Ponceau S stain- ing. The band at 185 kDa was cut out, and the paper minced with scissors, suspended in water, and then frag- mented by ultrasonication. Approximately 200 ng of ni- trocellulose-bound «:-macroglobulin was injected with Freund's complete adjuvant subcutaneously in multiple sites into female New Zealand white rabbits. A booster dose of 200 ^g was given in incomplete adjuvant after 4 weeks, and antiserum was collected at 2-week intervals thereafter (Daino el ai, 1987). Assav tor ctz-macroglobulin activity The functional assay for «:-macroglobulin is essen- tially that of Armstrong el ai ( 1 985). Briefly, bovine pan- creatic trypsin (Sigma) was prepared as a stock solution at 1 mg/ml in 1 m.U HC1 and stored frozen until used. Protease activity was measured by the hydrolysis of the low molecular mass amide substrate, N«-benzoyl-DL- arginine p-nitroanilide (BAPNA) (Sigma). The assay for a:-macroglobulin activity depends on the ability of «2- macroglobulin to bind trypsin without inactivating the active site of the enzyme. Alpha2-macroglobulin-bound trypsin can hydrolyze BAPNA with equal efficiency to that of free trypsin (Quigley and Armstrong, 1983, Fig. 1 ). The ability of «2-macroglobulin to suppress the pro- teolytic activity of proteases apparently depends on its ability to form a molecular cage around the protease molecule that establishes a steric barrier that prevents contact between protease and target proteins. This mo- lecular cage also prevents the inactivation of the active site of the protease by high molecular mass active site protease inhibitors. Specifically, trypsin bound to Limu- lus rti-macroglobulin is protected from inactivation by the active site inhibitor, soybean trypsin inhibitor (Mr = 2 1 .000). This property is unique to the «:-macroglob- ulin family of protease inhibitors and is the basis for an assay for «:-macroglobulin activity (Ganrot, 1966) that LIMULUS BLOOD CELL SECRETES «2-MACROGLOBULIN 139 has been used to detect rt2-macroglobulin in the plasma of molluscs (Armstrong, unpub. data), Limulus, and crustaceans (Armstrong el al, 1985). The sample sus- pected of containing «2-macroglobulin is incubated with trypsin, and then saturating amounts of soybean trypsin inhibitor are added to inactivate all unbound trypsin. The determination of the rate of hydrolysis of BAPNA allows quantitation of the fraction of trypsin that is pro- tected from inactivation by virtue of its binding to the «2-macroglobulin in the sample. As far as we know, the assay is specific for the «2-macroglobulin family of prote- ase inhibitors. Preparation of Limulus amebocyte lysate (LAL) Lysates of Limulus amebocytes were prepared by hy- potonic disruption of washed amebocytes at room tem- perature with sterile, pyrogen-free distilled water, essen- tially as described by Levin and Bang (1968). All glass- ware was siliconized, sterilized, and then rendered endotoxin-free by heating at 1 80°C in a dry oven. Adult female horseshoe crabs were bled directly into an equal volume of warm (40°C) 0.5 A/NaCl, 0.005 mA/N-ethyl maleimide (NEM, Sigma). Following sedimentation, the blood cells were resuspended in warm, NEM-containing saline, and then washed twice in warm saline without NEM. Hypotonic lysis of the cell button was accom- plished by incubation in pyrogen-free distilled water (3/ 1 v/v of cells) at room temperature. The cell suspension was vortexed daily for 2 days and the resultant lysate ( pri- mary extract) was collected following centrifugation. The pellet was re-extracted by further incubation with distilled water (secondary extract). Lysate was stored at 4°C. Reactivity was determined by incubation with a standard preparation of endotoxin (E. coli. 026:B6, Difco Laboratories, Detroit, Michigan). In general, Lim- ulus amebocyte lysate prepared in this manner has a pro- tein concentration of 1.5-3.0 mg/ml and forms a gel in the presence of 10-100 pg/ml of endotoxin (Rickles et al., 1979). Results Anti-armacroglobulin antiserum The antiserum recognized specifically the 185 kDa band of a2-macroglobulin on immunoblots of purified a2-macroglobulin (Fig. 1 , lanes A. 2 and B. 1 ), whole Lim- ulus plasma (Fig. 1, lanes A. 3 and B.2), and Limulus plasma depleted of hemocyanin by ultracentrifugation (not shown). The antiserum did not cross react with «2- macroglobulin from Homanis (the American lobster) or with human «2-macroglobulin (not shown). PANEL A 1 2 3 PANEL B 1 2 205- 116- 97- 66- ^^^ 8 45- 29- Figure 1. Characterization of the anti-Limulus a^-macroglobulin antiserum by immunoblotting. The antiserum reacts specifically with a band at an apparent molecular mass of 185 kDa with purified «2- macroglobulm (lanes A. 2 and B. 1 1 and with whole plasma (lanes A. 3 and B.2). The arc-like density at about 40-60 kDa on panel B is due to a scratch on the nitrocellulose, and does not represent specific deposits of HRP reaction product. Lane A. 2 contained 2.2 ^g of protein; lane B. 1 contained 0.55 ^g of protein; and lanes A. 3 and B.2 contained 0.25 n\ of plasma. In Figures 1, 2, and 4, panel A is a SDS-polyacrylamide gel (7.5% polyacrylamide) run under reducing conditions and stained with Coomassie blue, and panel B is a Western blot of a parallel gel stained with the itnti-Lirmilus n:-macroglobulin antiserum. Immunological demonstration qfa^-macroglobulin The materials released from washed Limulus blood cells that had been stimulated to undergo exocytosis by exposure to A23187 were subjected to SDS-polyacryl- amide gel electrophoresis under reducing conditions and then electrophoretically transferred to nitrocellulose pa- per. Alpha2-macroglobulin was demonstrated by prob- ing the transfers with the anti-«2-macroglobulin antise- rum. A single band at an apparent molecular weight of 185 kDa was recognized by the antibody (Fig. 2, lanes A. 5 and B.6). The penultimate cell wash buffer con- tained no immunoreactive material (Fig. 2, lanes A.4 and B.5), indicating that the presence of a2-macroglobu- lin in the material released during exocytosis of washed blood cells was not a result of contamination by plasma. Alpha2-macroglobulin could likewise be demonstrated in blots of protein from whole, washed blood cells (Fig. 2, lanes A. 3, B.3 and B.4). Attempts to demonstrate a2- macroglobulin by immunoblotting of the proteins ex- tracted from the residual pellet of cells that had under- gone exocytosis were unsuccessful (Fig. 2, lanes A.6 and 140 P. B. ARMSTRONG ET AL. PANEL A PANEL B 1234 — - - 5 6 45- 29- Figure 2. Immunologic demonstration ofurmacroglobulin in Limw/tis blood cells and in the materials released dunng degranulation of the cells. Alpha:-macroglobulin is evident in the plasma (lanes A. 2 and B.2) and whole, undegranulated cells (lanes A. 3, B.3 and B.4) hut is not demonstrable in the penultimate wash of the cells (lanes A. 4 and B.5). Alpha;-macroglobulin is also demonstrable in the materials released from the cells during degranulation (lanes A. 5 and B.6) but not in the degranulated cells (lanes A. 6 and B.7). Lane B. 1 was loaded with 1 . 1 jig of purified I.imtilitu «i-macroglobulin. Lanes A. 2 and B.2 contained 0.25 M! of plasma; lane A. 3 contained 2 p\ of pelleted live, whole blood cells; lane B.3 contained 1 ^1 of pelleted whole, live blood cells; lanes A. 4 and B.5 contained 10 M' of buffer from the penultimate cell wash; and lanes A. 5 and B.6 contained 10 /il of releasate. B.7). indicating that most or all of the cell-associated «:- macroglobulin had been released during exocytosis. The apparent molecular weight of the subunit of cell-associ- ated rt:-macroglobulin was identical to that of the plasma form of «:-macroglobulin. The relative amounts of pro- tein in different bands of Coomassie blue-stained gels were estimated spectrophotometrically with the curve in- tegration function of a Bio Rad Model 620 scanning den- sitometer. Estimations of the relative amounts of «2- macroglobulin by the optical density of the 185 kDa band on Coomassie blue-stained gels of whole cells and plasma indicate that the cells contained in a given vol- ume of whole blood contain approximately 0.5-2% of the a:-macroglobulin contained in the same volume of plasma. Measurement ofa.2-macroglobu.lin activity The ability to protect trypsin from inactivation by soy- bean trypsin inhibitor was used to estimate the amounts of active a2-macroglobulin in samples of plasma and the materials released by blood cells stimulated to undergo exocytosis. Comparable results to the immunological studies were obtained: plasma contained large quantities of «2-macroglobulin, the cells of a given volume of blood contained about 0.5-2% of that amount of «:-macro- globulin, and the penultimate wash buffer was negative (Fig. 3). Alpharmacroglohulin in Limulus amebocyte lysate(LAL) Limulus amebocyte lysate (LAL) is the soluble materi- als recovered by distilled water lysis of N-ethyl maleim- ide-stabilized Limulus amebocytes and is used for the de- tection of lipopolysaccharides from Gram-negative bac- teria (Levin, 1979). Both immunostaining of protein blots (Fig. 4) and activity measurements (data not shown) indicated the presence of large amounts of a2- macroglobulin in preparations of LAL. Primary extracts (Fig. 4, lanes A. 2 and B.2) contained significantly more «2-macroglobulin than secondary extracts (Fig. 4, lanes A.I and B.I), on a per mg of total protein basis. The amounts of «:-macroglobulin in primary extracts is sig- nificantly larger than would be expected from the amounts present in washed, live cells. Preparation of LAL involves exposing whole blood to a warm solution of n-ethyl maleimide, followed by extensive washing of UML'LUS BLOOD CELL SECRETES «rMACROGLOBULIN 141 E c O) o (O 0.4i 0.3- 0.2- 0.1 - 0.0 20 40 Minutes 60 80 1 00 Figure 3. Demonstration of a:-macroglobulin in materials released by saline-washed Limuliis blood cells stimulated to degranulate by ex- posure to the ionophore, A23 1 87, and in plasma from the same animal. The released materials protect trypsin from inactivation by the high molecular mass active site inhibitor, soybean trypsin inhibitor. Ten ^g samples of trypsin were preincubated with 20 M' of plasma (curve B), the materials released from 1 2 ^1 of packed cells, which was the amount of cells contained in 530 /jl of whole blood (curve C). or 160 ^1 of the penultimate saline wash (curve D) for 10 min and then 20 ^g of soybean trypsin inhibitor was added. The remaining active trypsin (e.K-. the trypsin bound to the «2-macroglobulin in the sample, and thereby pro- tected from inactivation by soybean trypsin inhibitor) was assayed by its ability to hydrolyze the low molecular mass amide substrate, BAPNA. This was followed by the increase in optical absorbance at 410 nm. In the absence of added «:-macroglobulin, the hydrolysis of BAPNA is zero (not shown). Curve A is the activity of 10 /ig of trypsin in the absence of soybean trypsin inhibitor or «:-macroglobulin. In this sample, the cells from a given volume of blood (curve C) contained 0.6% as much a^-macroglobulin as the plasma from the same volume of blood (curve B). In other trials, the cells have contained as much as 2% of the total «:-macroglobulin in a given volume of blood. the cells and then their lysis in distilled water. The effi- ciency of washing is indicated by the minimal contami- nation of the LAL preparations with hemocyanin (com- pare the relative intensities of the hemocyanin band at 67 kDa and the «rmacroglobulin band at 185 kDa in plasma (Fig. 2, lanes A. 2 and B.2) with that in LAL (Fig. 4, lanes A. 2 and B.2). We speculate that «2-macroglobulin in the plasma spe- cifically becomes associated with the cells during the ex- posure of blood to n-ethyl maleimide and is released dur- ing the distilled water lysis step. The amounts of rt2-mac- roglobulin in the NEM-containing plasma phase that is the by-product of the preparation of cells for production of LAL were low or undetectable (Armstrong, unpub. data), consistent with the possibility that significant quantities of plasma a2-macroglobulin become associ- ated with the cells during treatment of blood with NEM. Discussion The blood is the principal organ involved in the de- fense against pathogens that have entered the body. In most animals, both plasma- and blood cell-mediated sys- tems participate in immunity. The «2-macroglobulin system of protease-binding proteins is a well-studied ex- ample of a humoral system of immunity, both in verte- brates (Sottrup-Jensen, 1987, 1989) and arthropods (Quigley and Armstrong, 1983, 1985; Armstrong ct a/.. 1985; Hergenhahn and Soderhall, 1985; Spycher el a/.. 1987; Hergenhahn et ai, 1988; Armstrong and Quigley, in prep.). The present report documents that the sole blood cell type of Limuliis, the granular amebocyte, also contains «2-macroglobulin in a form that is released dur- ing degranulation. The cell-associated form of «2-macro- globulin is immunologically reactive with an antiserum prepared against the plasma form of Limuliis a:-macro- globulin and has an identical subunit molecular weight. Although the plasma of a given volume of whole blood contains much more «2-macroglobulin than do the blood cells of that same volume of blood, the cell-associ- ated form may play an important role in suppression of proteases in the densely cellular clot formed by aggre- gated blood cells at sites of wound healing (Loeb, 1920; Bursey, 1977). In this situation, exocytosis into the con- fined spaces between cells would be expected to yield high local concentrations of «2-macroglobulin that might be of importance specifically because diffusion of «2-macroglobulin from the plasma into these spaces PANEL A PANEL B 123123 205- 116- 97- 66- 45- 29- L t Figure 4. Demonstration of a2-macroglobulin in preparations of Limuliis amebocyte lysate. Lanes A.I and B.I show a secondary ex- tract, lanes A. 2 and B.2 show a primary extract, and lanes A. 3 and B.3 show the materials released from live cells exposed to A23I87. Lanes A.I and B.I contain 18 n% of protein, lanes A. 2 and B.2 contain 125^g of protein, and lanes A. 3 and B.3 contain 10 n\ of released materials (45 ng of protein). 142 P. B. ARMSTRONG ET AL. might be expected to be slow. By forming a temporary barrier between the septic external milieu and the inter- nal tissues of the animal, the blood clot is a critical battle- ground between invading pathogens and the animal. The gradual release of rt:-macroglobulin as cells of the clot degranulate may play an important role in defense dur- ing the early stages of wound healing. The mammalian blood platelet — a cell with homologous function to the Limulus amebocyte — also contains «2-macroglobulin and other protease inhibitors in forms that are released by exocytosis (Nachman and Harpel, 1976; Plow and Collen, 1981). Like the Limit/us amebocyte, the concen- trations of protease inhibitors in platelets are small frac- tions of the total concentrations in whole blood (Nach- man and Harpel, 1 976; Plow and Collen, 1981). The Limiilus blood cells also release both acid-stable and acid-labile active site inhibitors of serine proteases during degranulation (Armstrong and Quigley, 1985; Nakamura el ai. 1987). The importance of «2-macro- globulin in this situation may derive from its ability to bind such a wide selection of proteases. Although the spectrum of proteases that is susceptible to the active site inhibitors of the blood cells has not been established, most active site inhibitors are reactive only to a defined subclass of proteases, in contrast to the near universal reactivity of «:-macroglobulin. Interestingly, the only protease that we have found to be unreactive to Limiilus «:-macroglobulin is the terminal protease in the blood clotting cascade (Armstrong ct ai. 1984). In Limiilus. both the clottable protein and the proteases involved in clotting are localized in the secretory granules of the blood cells and are released by exocytosis. It can be sug- gested that the unique resistance of the clotting protease to inactivation by o^-macroglobulin is a physiological adaptation to the requirement that blood clotting can proceed in a milieu containing an abundance of ai-mac- roglobulin. The active site inhibitors of the blood cells do inhibit this protease. Acknowledgments This research was supported by NIH Grants No. GM 35185 and CA22202 and the Medical Research Service of the Veterans Administration (RDIS 7446). Literature Cited Armstrong, P. B. 1985a. Adhesion and motilily of the blood cells of Limulus. Pp. 77-128 in Blood Cells of Marine Invertebrates. W. D. Cohen, ed. Alan R. Liss. New York. Armstrong, P. B. 1985b. Amebocytes of the American "horseshoe crab." Limiilus. Pp. 253-258 in Blood Cells of Marine Inverte- brates. W. D. Cohen, ed. Alan R. Liss. New York. Armstrong, P. B., and J. P. Quigley. 1985. Proteinase inhibitory ac- tivity released from the horseshoe crab blood cell during exocytosis. Bioclum. Biophys. Ac/a 827: 453-459. Armstrong, P. B., and J. P. Quigley. 1987. Limulus «:-macroglobu- lin. First evidence in an invertebrate for a protein containing an internal thiol ester bond. Biochem. J 248: 703-707. Armstrong, P. B., and F. R. Rickles. 1982. Endotoxin-induced de- granulation of the Liinii/ux amebocyte. Exp. Cell Rex 140: 15-24. Armstrong, P. B., J. Levin, and J. P. Quigley. 1984. Role of endoge- nous protemase inhibitors in the regulation of the blood clotting system of the horseshoe crab, Limulus polyphemus. Thrombos. Haemostasis (Stuttgart) 52: 1 17-120. Armstrong, P. B., M. T. Ressner, and J. P. Quigley. 1985. An «,- macroglobulmlike activity in the blood of chelicerate and mandibu- lale arthropods. J. Exp. Zoo/. 236: 1-9. Bang, F. B. 1979. Ontogeny and phylogeny of response to Gram-neg- ative endotoxins among the marine invertebrates. Prog. Clin Biol Rex 29: 109-123. Barrett, A. J. 1981 . «:-Maeroglobulin. Meth. Enzymol 80: 737-754. Bursey, C. R. 1977. Histological response to injury in the horseshoe crab, Limulus polyphemus. Can J Zoo/. 55: 1 158-1 165. Coan, M. IL, and R. C. Roberts. 1989. A redetermination of the con- centration of rt:-macroglohulm in human plasma. Biol. Client. lloppe-Seyler 370: 673-676. Daino, M., A. Le Bivic, and M. Him. 1987. A method for the produc- tion of highly specific polyclonal antibodies. Anal Biochem 166: 224-229. Feinman, R. I), (ed.). 1983. Chemistry and Biology of ay-Macroglobu- lin Annals of the New Y'ork Academy of Sciences, vol. 421. New York Academy of Sciences, New York, NY. Ganrot, P. O. 1966. Determination of «:-macroglobulin as trypsin- protein esterase. Clin. Clum. Ada 14: 493-501. Harpel, P. C'. 1987. Blood proteolytic enzyme inhibitors: their role in modulating blood coagulation and tibrinolytie enzyme pathways. Pp. 219-234 in llemostusis and Thrombosis. Basic Principles and Clinical Practice. R. W. Colman, J. Hible, V. J. Marder. and E. W. Salz, eds. J. B., Lippincott Co.. Philadelphia, PA. Hergenhahn, II. -G., and K. Soderhall. 1985. <>/ Penaeus setiferus juveniles Enzyme (substrate) Activity (lU/g wet weight) Activity UU/mg protein) Gut Carcass Ratio Gut Carcass Ratio Trypsin(BAPNA) 0.207 (±0.094) 0.023 (±0.038) 9.0 (1.0527 (±0.0235) 0.00 10 (±0.00 17) 52.3 Trypsin(TAME) 29.7 (±12.2) 1.17 (±1.43) 25.4 7.53 (±2.31) 0.05 (±0.07) 1 50.6 Carboxypeptidase A 3.08 (±0.67) 0.13 (±0.055) 23.7 0.787 (±0.258) 0.006 (±0.003) 131.2 Carboxypeptidase B 5.90 (±1.76) 0.27 (±0.25) 21.8 1.504 (±0.498) 0.0 1 3 ( ±0.0 1 1 ) 115.7 Chymotryptic-like esterase 0.27 (±0.10) 3.95 (±4.15) 0.1 0.067 (±0.022) 0.184 (±0.216) 0.4 Arylamidase 0.204 (±0.205) O.I 36 (±0.204) 1.5 0.05 17 (±0.0488) 0.0064 (±0.0093) 8.0 Esterase ( /i-naphthol acetate) 0.188 (±0.052) 0.0 18 (±0.0 16) 10.4 0.0478 (±0.0072) 0.0007 (±0.0006) 68.3 Amylase (starch) 21.5 (±3.7) 0.5 (±0.6) 43.0 5.47 (±1.01) 0.02 (±0.03) 273.5 Ratio of activity in gut tissues to activity in remaining carcass indicated. Activity expressed both as International Units of activity per gram wet weight and International Units of activity per mg protein. Mean activity (±95% confidence limit) is indicated (n = 3). Discussion The ontogenetic decrease in specific activities of diges- tive enzymes at metamorphosis coincides with degenera- tion of the gut (from M,-PL4 ) in Penaetis setijents. The subsequent increase in enzyme activities during postlar- val development coincides with differentiation of the gut into the adult form. This increase represents both an in- crease in enzyme activities in hepatopancreatic tissues, and an allometric increase in the relative size of the hepa- topancreas. Observed changes in enzyme activities dur- ing postlarval development are not the result of change in diet because diet was held constant during this period. Thus, the ontogenetic change in activities represents some other change associated with development. Table III Specific activities for digestive enzyme* for 24-h Artemia nauplii. obtained from whole-animal homogenates Enzyme (Substrate) Specific activity (lU/mg protein) Trypsin(BAPNA) Trypsin (TAME) Carboxypeptidase A Carboxypeptidase B Non-specific esterase ( /3-naphthol acetate) Non-specific esterase ( pJ-naphthol laurate) Non-specific esterase ( fJ-naphthol stearate) Amylase (starch) Amylase (glycogen) 0.0055 ±0.0012 0.27 ± 0.08 0.034 ±0.0 13 0.20 ± 0.02 0.023 ± 0.005 0.0016 ±0.0002 0.000 14 ±0.00002 0.1 00 ±0.004 0.038 + 0.018 Ontogenetic change in digestive enzyme activity The ontogenetic patterns of enzyme activity found in Penaeus sctifcrus are similar to those described for other decapod species (Van Wormhoudt, 1973; Laubier-Boni- chon et a/.. 1977; Van Wormhoudt and Sellos, 1980; Galgani and Benyamin, 1985; Biesiot, 1986). In P. setij- ents. P. japoniciis, Palaemon .terrains, and Homarusam- ericanits. specific activities of both amylase and protease are low in those developmental stages preceding the first 80 h- UJ 60 S 40 O tr o. LJ _) CD 20 m 15 6 12 14 > Activities expressed as International Units of activity per mg protein. Mean activity ± 95% confidence limit is indicated for three replicates. 51-31-31 4 7 10 14 17 21 24 "35 140 N Z M PL DEVELOPMENTAL STAGE Figure 2. Soluble protein content in developmental stages of Pe- naeus selijerus, obtained from whole-animal homogenates. Solid ver- sus broken lines indicate separate spawnings. Error bars indicate 95% confidence interval about mean value for each developmental stage. Sample size for each mean is indicated by numbers above or below bars. N. nauplis stage 5; Z. protozoeal stages 1-3: M. mysis stages 1-3; PL, age of postlarvae in days. ONTOGENY OF ENZYMES IN SHRIMP 149 0.008 0.006 0004 .71 0002 oooo E > > o < O 0.8 0.6 04 0.2 00 TRYPSIN (BAPNA) 004 003 0.02 001 000 CARBOXYPEPTIDASE A 12 51-31-31 4 7 10 14 17 21 24 35 " 140 N Z M PL TRYPSIN (TAME) 030 0.20 010 000 51-31-31 4 7 10 14 17 21 24 "35 " 140 N Z M PL CARBOXYPEPTIDASE B I--*" 51-31-31 4 7 10 14 17 21 24 35 140 N Z M PL 51-31-31 4 7 10 14 17 21 24 35 140 N Z M PL DEVELOPMENTAL STAGE Figure 3. Specific activity of trypsin (with substrate indicated), carboxypeptidase A, and carboxypepti- dase B for developmental stages o'iPenaeu* setijerus, obtained from whole-animal homogenates. Activity expressed as International Units of activity per mg protein in entire animal. Solid versus broken lines indicate separate spawnings. Error bars indicate 95% confidence interval about mean activity for each developmental stage. Sample size for each mean is indicated by numbers above or below bars. N. nauplius stage 5; Z. protozoeal stages 1-3; M, mysis stages t-3: PL. age of postlarvae in days. feeding stage. Enzyme activities increase during early lar- val development in all four species. In P. setijerus and P. japonicus, amylase activity decreases to a low level by metamorphosis, but activity remains relatively constant in P. serratus and H. americanus. In all species, amylase activity increases during postlarval development. Prote- ase activity in all four species decreases at metamorpho- sis. During early postlarval development, protease activ- ity remains low in Penaeus spp., but increases in P. serra- tus and H. americanus. There is a peak in A/P ratio for P. japonicus at Z3, but in P. setijerus the peak occurs at Mi; in both species the ratio declines to a low level at metamorphosis. Ontogeny of gut. Increase in digestive enzyme activity has been correlated with differentiation of the gut in lar- vae of both teleosts (Buddington and Doroshov, 1986) and echinoderms ( Vacquier et a/., 197 1 ). In P. setijerus, the decrease in most enzyme activities immediately after metamorphosis coincides with degeneration of the ante- rior midgut caeca into the vestigial anterior midgut di- verticulum (Lovett and Felder, 1989). Laubier-Boni- chon et al. (1977) examined whole-animal concentra- 150 D. L LOVETT AND D. L. FELDER 80 60 40 20 00 NON-SPECIFIC ESTERASE (ACETATE) 51-31-31 4 7 10 14 17 21 24 35 140 N Z M PL f 0.6 NON-SPECIFIC ESTERASE (LAURATE) 0 ° 04 X i v H > \ p 02 0 6 \6 6, * li \I ? , _12__^4 CJ fiY IT^^j^^J — * — ~"j3~ t 00 9 o L U 5 8> N 1-31 -31 4 7 10 14 17 21 24 _ 35 " 140 Z M PL 0010 0008 0006 0004 0002 0000 NON-SPECIFIC ESTERASE (STEARATE) 35 140 51-31-31 4 7 10 14 17 21 24 N Z M PL DEVELOPMENTAL STAGE Figure 4. Specific activity of non-specific esterase (with fatty acid chain of fi-naphthol substrate indicated) for developmental stages of /Ymu'in \ctilcni.\. obtained from whole-animal homogenates. Activity expressed as International Units of activity per mg protein in entire animal. Solid versus broken lines indicate separate spawnings. Error bars indicate 95% confidence interval about mean activity for each de- tions of RNA and DNA in P. japonicus and concluded that the rates of cell multiplication, cell hypertrophy, and cellular metabolic rate were at peak levels during larval development, but then dropped to low levels during the first week of postlarval life. Thus, reduced metabolic ac- tivity during the critical period may coincide with low digestive enzyme activity observed, and may reflect some accommodation to limited nutrient uptake during this transformational period of morphogenesis in the gut. The increase in enzyme activities following the critical period in P. setifcnts coincides with the ramification of lobes of the hepatopancreas into small-diameter tubules (Lovett and Felder, 1989). In Palaemon serratus, the in- crease in enzyme activity also coincides with an increase in the number of caeca in the hepatopancreas (Van Wormhoudt, 1973; Richard. 1974, thesis cited in Van Wormhoudt and Sellos, 1980), but unlike the situation in P. setijerus. the increase in number of caeca in P. ser- ratus occurs during larval development. In P. set i ferns, substantial increases in enzyme activities (particularly for trypsin, carboxypeptidase A, and amylase) occur dur- ing the fourth and fifth week of postlarval development and coincide with completion of differentiation by the hepatopancreas. Moreover, by this stage in develop- ment, the foregut has nearly attained the adult morphol- ogy and function, the posterior diverticulum has differ- entiated, and retention time of food in the gut has in- creased dramatically over that of early postlarval stages (Lovett and Felder, 1989, 1990). Thus, protraction in de- velopment of gut morphology of P. set (ferns is reflected in the protraction of ontogenetic change in digestive en- zyme activity. Ontogeny offeeding habits. Ontogenetic change in en- zyme activity can also be correlated with diet and feeding habit. In stage Z, , larvae off. sctifenis begin to feed on algae; esterase activity is maximal. We provided Anemia nauplii beginning at stage M , (although larvae of P. setif- enis will feed on Anemia beginning at stage Z, ); activi- ties of trypsin and carboxypeptidase A and B in larvae are maximal at Z,-M , . Larvae shift from being primarily filter feeders to being primarily raptorial feeders at M,, and filtering efficiency continues to decline during PL, (Emmerson, 1980, 1984); enzyme activities decline dur- ing M, and become very low at PL, . The diet in wild populations is reported to change from predominately algae in early postlarval stages to include a more substan- tial portion of animal matter in later (PL^s-PL-u) post- larval stages (Flint, 1956; Fujinaga, 1969; Sastrakusu- velopmental stage. Sample size for each mean is indicated by numbers above or below bars. N. nauplius stage 5; Z. protozoeal stages 1-3; M, mysis stages 1-3; PL, age of postlarvae in days. ONTOGENY OF ENZYMES IN SHRIMP 151 O CE Q. o> E 05 04 0.3 02 0 I 0.0 AMYLASE (STARCH) 51-31-31 4 7 N Z M 14 17 21 24 PL o < o ± 025 O UJ Q. W 020 015 010 005 000 AMYLASE (GLYCOGEN) / 7 10 14 17 21 24 35 N Z M PL DEVELOPMENTAL STAGE Figure 5. Specific activity ofamylase (with substrate indicated) for developmental stages of Pcnaeus seliferus. obtained from whole-ani- mal homogenates. Activity expressed as International Units of activity per mg protein in entire animal. Solid versus broken lines indicate sepa- rate spawnings. Error bars indicate 95% confidence interval about mean activity for each developmental stage. Sample size for each mean is indicated by numbers above or below bars. N, nauplius stage 5; Z, protozoeal stages 1-3: M, mysis stages 1-3: PL. age of postlarvae in days. mah, 1970; Jones, 1973; Chong and Sasekumar, 1981; Nelson, 1981; Gleason and Zimmerman, 1984; but see also Kitting el al, 1984; Gleason, 1986). In PL:s-PL35, enzyme activity increases substantially. Despite the correlation of ontogenetic change in en- zyme activity with change in feeding habits, ontogenetic change in activity may be developmentally cued and may reflect temporal genetic regulation of enzyme syn- thesis, rather than a change in diet. For example, ontoge- netic change of digestive enzyme activity in the first feed- ing stages of Homarus larvae occurs even in the absence of access to exogenous food substrates (Biesiot, 1986). In Anemia, ontogenetic change in enzyme synthesis is likely under genetic control, which then is modulated by diet and nutritional requirements (Samain et al., 1980). Moreover, no consistent correlation of A/P ratio with composition of diet has been found within a single crus- tacean species (Hoyle, 1973; Boucher et al.. 1976; Sa- main el al.. 1980;Mauglertfl/.. 1982b; Bamstedt, 1984; Harris et al.. 1986). Because both amylase activity and the A/P ratio in P. set (ferns increase during postlarval development, it might be inferred that postlarval shrimp become more herbivorous. However, beginning with PL5, the diet consisted entirely of Anemia. Thus, change in enzyme activity in P. set i ferns occurs without a change in diet. Dietary implications Even though we did not vary diet in the present study, we can infer that diet is not the only factor influencing enzyme activity. Attempts have been made to correlate digestive enzyme activity with diet and to use ontoge- netic change in enzyme activity as an index of trophic state to estimate the phase in development where diet formulations for cultured shrimp need to be changed 1.6 (J < O O < rr 0.8 04 00 51-31-31 4 7 10 14 17 21 24 " 35 140 N Z M PL DEVELOPMENTAL STAGE Figure 6. Ratio of amylase activity (starch used as substrate) to trypsin activity (BAPNA used as substrate), for developmental stages of Penaeits set (tents. Solid versus broken lines indicate separate spawn- ings. Error bars indicate 95% confidence interval about mean activity for each developmental stage. Sample size for each mean is indicated by numbers above or below bars. N. nauplius stage 5: Z, protozoeal stages 1-3; M. mysis stages 1-3: PL, age of postlarvae in days. 152 D. L. LOVETT AND D. L. FELDER Q 020 o 3 O (E O UJ I 015 0 10 005 51-31- 31 N Z M 10 14 17 21 24 140 PL DEVELOPMENTAL STAGE Figure 7. Ratio of volume of hepatopancreas (in early stages, vol- ume of anterior midgut caeca plus lateral midgut caeca) to total body volume for developmental stages of /V/n/fi/.v sctifcrus. Areas were mea- sured on 8 nm serial sections of formalin-fixed, paraffin-embedded specimens. Volumes were calculated by summing frusta. Error bars in- dicate 95% confidence interval about mean ratio of volumes are indi- cated for each developmental stage (n = 3). N, nauplius stage 5; Z, protozoeal stages 1-3; M, mysis stages 1-3; PL, age of postlarvae in days. carbohydrate in the postlarval diet. A similar response to elimination of starch from the diet was observed by Hernandorena (1982) in Artemia. However, such a re- sponse is contrary to an assumption that is widely held among aquaculturists: i.e.. that enzyme activity is high for those substrates most common in the diet. An additional problem associated with the use of en- zyme activity to evaluate diet is that the stage of molt cycle, nutritional status of shrimp, season, sexual condi- tion, and ontogenetic stage have been shown to affect size, histological condition, water content, and protein content of the hepatopancreas, (Cuzon el ai. 1980; Rosemark el ai. 1980; Van Wormhoudt et ul.. 1980; Van Wormhoudt and Sellos, 1980; Storch et ai. 1982; Barclay et ai. 1983; Pascual et ai. 1983; Storch and An- ger, 1983; Lee, 1984; Lee et ai. 1984; Storch et ai. 1984; Vogt et ai. 1985). Thus, the units selected to express en- zyme activity (either activity per mg protein in hepato- pancreas, activity per g wet weight of hepatopancreas, ac- tivity per mg protein in whole shrimp, or activity per g wet weight of shrimp) can affect whether significant change in digestive enzyme activity is reported (Cuzon et ai. 1980; Van Wormhoudt et ai. 1980; Barclay et ai. 1983; Lee. 1984; Lee et ai. 1984; present study). Little is known about the implications of using any one of these units to describe enzyme activity in shrimp. (Hoyle, 1973; Van Wormhoudt. 1973; Laubier-Boni- chon et ai. 1977; Cuzon et ai. 1980; Lee et ai. 1980. 1984; Van Wormhoudt et ai. 1980; Maugle et ai. 1982b;Galgani, 1983;Galgani el al . 1984; Lee and Law- rence, 1985). Despite postlarval diet being held constant in the present study, a significant ontogenetic change oc- curred in digestive enzyme activity and in the A/P ratio. Therefore, we question the validity of using the A/P ratio to predict the degree to which an organism is carnivorous or herbivorous. In those larval stages of Penaens with maximal amylase activity and A/P ratio, the diet is in- deed composed of phytoplankton. However, the signifi- cance of high amylase activity here is not clear; very few groups of marine phytoplankton use starch as a storage product, and those storage products used most widely by marine phytoplankton are not hydrolyzed by amylase. In contrast to the usual explanation for diet effects on enzyme activity, Harris et ai (1986) and Hofer (1982) propose that secretion of large amounts of enzyme may maximize the use of a scarce component in the diet. Such elevated enzyme activity could maximize hydrolysis and the resulting extraction of a dietary substrate that was present in small amounts. Thus, the substantial increase in amylase activity observed in P. setiferits during post- larval development may be a response to low levels of Enzymes present in t;ut Lack of specificity in assay substrates precludes con- clusive identification of enzymes responsible for hydro- lysis of substrates. For example, the substrates TAME and BAPNA are specific for trypsin only in the sense that they are not hydrolyzed by chymotrypsin (Hummel, 1959; Rick, 1974b). They can be hydrolyzed by both non-specific esterases and crustacean collagenase (Hess and Pearse. 1958;Pearse, 1972; Grant and Eisen, 1980; Grant et ai. 1983). The chymotrypsin-specific substrate BTEE also is subject to hydrolysis by both non-specific esterase and Type I crustacean collagenase, whereas GPANA is relatively resistant to hydrolysis by either of these enzymes ( Eisen et ai. 1973; DeVillez, 1975). Chy- moptryptic-like activity is not considered further in the present study because: ( 1 ) no activity was measured with GPANA. (2) most activity measured with BTEE oc- curred in non-gut tissues, and (3) conclusive evidence that crustaceans secrete chymotrypsin in quantities sig- nificant for digestion is lacking (DeVillez. 1975; Vonk and Western, 1984; Appendix 1 ). Both arylamidase and aminopeptidase are not considered further because: (1) no activity was found with either of the aminopeptidase substrates L-leucinamide or L-leucyl-/}-naphthylamide as substrates, (2) activity measured with the substrate 1.0 0.8 06 04 O 02 en 0.0 TRYPSIN (BAPNA) ONTOGENY OF ENZYMES IN SHRIMP 5.0 40 3.0 2.0 iV''* 1.0 0.0 153 51-31-31 4 7 10 14 17 21 24 " 35 140 N Z M PL NON-SPECIFIC ESTERASE (ACETATE) -\v-" 51-31-31 4 7 10 14 17 21 24 35 140 N Z M PL 0 50 < C O LJ- 4.0 O LL) Q. cn 30 2.0 6 if 10 A 6 f [U 7 . Y 1 \ oo • vf CARBOXYPEPTIDASE A 51-31-31 4 7 10 14 17 21 24 " 35 " 140 N Z M PL 50 40 30 20 10 AMYLASE (STARCH) 51-31-31 4 7 10 14 17 21 24 35 N Z M PL DEVELOPMENTAL STAGE Figure 8. Specific activity of trypsin (BAPNA used as substrate), carboxypeptidase A, non-specific esterase (/i-naphthyl acetate used as substrate) and amylase (starch used as substrate) corrected to Interna- tional Units of activity per mg protein in hepatopancreas. lor developmental stages ofPenaeus setilmi.i. Concentration of soluble protein was determined for total body; correction to concentration of soluble protein in hepatopancreas was estimated from ratio of hepatopancreas volume to total body volume. Solid IW.VH.V broken lines indicate separate spawnings. Error bars indicate 95% confidence interval about mean activity for each developmental stage. Sample size for each mean is indicated by numbers above or below bars. N. nauplius stage 5;Z. protozoeal stages 1-3; M, mysis stages 1-3; PL, age of postlarvae in days. LPNA was not membrane-associated (as is aminopepti- dase activity in other systems), and (3) substantial activ- ity measured with LPNA came from non-gut tissue. Be- cause there is considerable overlap in the substrates that each esterolytic enzyme can hydrolyze (Nachlas and Se- ligman, 1949), and because activity of homogenate in the present study decreased as chain length of fatty acid in the substrate increased, activity assayed with each of the three ,tJ-naphthol substrates appears to represent a single type of non-specific esterase; lipase activity appears to be absent. Enzyme activity detected in tissue homogenates may not necessarily represent activity of enzymes that will be secreted into the digestive lumen. In crustaceans, activity for proteases, amylase, chitinase, chitobiase, and non- specific esterase has been found in tissues outside of the gut (Osuna ct at.. 1977; Trellu and Ceccaldi, 1977; Mykles and Skinner, 1986; Mattson and Mykles, 1987; O'Brien and Skinner, 1987, 1988). Even though such en- zymes would contribute to activity assayed in whole-ani- mal homogenates, most enzyme activity detected in Pe- nacus xetij'enis was restricted to gut tissues. Some diges- 154 D. L. LOVETT AND D. L. FELDER live enzymes, particularly those associated with lysosomes, are involved in intracellular processes only (deDuve and Wattiaux, 1966). Furthermore, because there is no evidence to suggest that digestive enzymes in crustaceans are produced in a zymogen form (Gates and Travis, 1969; Zwilling et al.. 1969; Brockerhoff el ai. 1970; Eisen el ai. 1973; Zwilling and Neurath, 1981; Vonk and Western, 1984), enzymes that have been syn- thesized, but have not yet been secreted, also contribute to the enzyme activity measured in tissue homogenates. Nonetheless, intracellular concentration of digestive en- zymes in Palaemon directly reflected luminal concentra- tion of enzymes (Rodriguez et al., 1976). Thus, activities assayed in the present study are probable indicators of relative enzyme activities in the lumen. Acknowledgments Thanks are extended to A. L. Lawrence and his staff. Texas A&M Shrimp Mariculture Project, for generous provision ofPcniicus sctifcnix nauplii and algal cultures. S. C. Hand, University of Colorado, and E. J. DeVillez, Miami University of Ohio, provided valuable advice on enzyme assays. J. H. Spring. R. 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ONTOGENY OF ENZYMES IN SHRIMP 157 Appendix 1 eninnes for which presence or alienee (+/-) of activity has been reported in Penaeus spp. (Activity of general protease is not included) Enzyme Species Substrate" Activity Reference Trypsin Carboxypeptidase A Carhoxypeptidase B P. selilenn P. a:tecn.\ I' in/milieus P. kerathurus P monodon P mergiiiensix P occidentalis P. pen/cil/ulin P stylirostris P vannumei P setiferus P azlecus P japonicus P. kerathurus P. merguicnsis P monodon P. occidentnlis P penicillatus P stylirostris P. setijcrus P. aitecus P japonicus P kcralhurwi P merxmensis P monodon P. occidental!! P. penieillatu.'i BAEE BAPNA TAME BAPNA BAPNA TAME BAPNA TAME BAPNA TAME BAPNA TAME BAPNA BAPNA BAPNA TAME BAPNA TAME BZGPA BZGPA FALPP HPLA HPLA HPLA FALPP HPLA BZGPA FALPP BZGPA HPLA BZGPA HPLA BZGA BZGA BZGA BZGA BZGA BZGA BZGA BZGA + Gates and Travis, 1969 + Lee, 1984; Lee and Lawrence. 1982. 1985; Lovett and Felder. present paper + Lovett and Felder, present paper + Lee. 1984; Lee and Lawrence. 1982 + Cuzon et al.. 1980: Galgani, 1983: Galgani etal.. 1984, 1985; Tsai etal.. 1986; Laubier-Bonichon el al., 1977; Trellu, 1978 + Galgani. 1983; Galgani and Benyamin. 1985: Galgani et al.. 1984, 1985; Trellu, 1978 + Ceccaldi-nitroanilide; HPLA, hippuryl- L-phenyllactate; LA, L-leucinamide hydrochlonde; LNA, L-leucyl-/i-naphthylamide hydrochloride; LPNA, L-leucine-/>-nitroanilide; SAAPPNA, succinyl-L-alanine-L-alanine-L-phenylalanine-L-phenylalanine; SPNA, succinyl-L-phenylalanine nitroanilide; TAME, a-/Moluenesulphonyl-L- arginine methyl ester hydrochloride. Reference: Biol. Bull. 178: 160-174. (April, 1990) Ontogenetic Changes in Enzyme Distribution and Midgut Function in Developmental Stages of Penaeus setifems (Crustacea, Decapoda, Penaeidae) DONALD L. LOVETT* AND DARRYL L. FELDER Department of Biology and Center for Crustacean Research. University of Southwestern Louisiana, Lajavetle, Louisiana 70504 Abstract. Ultrastructure and histochemical distribu- tion of enzymes were examined in the midgut oflarval and postlarval stages of Penaeus setijerus. Acid phospha- tase and esterase activities were present in all gut tissues at all stages. Protease activity was present in the anterior and lateral midgut caeca, as well as in the anterior por- tion of the midgut trunk ( MGT) of larvae and early post- larvae (PLi-PLj ). Amylase activity could not be detected histochemically in larvae or early postlarvae. even though it was detected in assays of whole-animal homog- enates. In later postlarvae, both protease and amylase ac- tivities were present in the hepatopancreas and anterior MGT. but were absent from the anterior midgut diver- ticulum. In larvae, alkaline phosphatase activity is present throughout the midgut. suggesting that absorption is widespread. In juveniles, activity is restricted to the hepa- topancreas and regions of the MGT within the cephalo- thorax. The abdominal MGT (or "intestine") is no longer absorptive by the time the hepatopancreas has at- tained its adult form. Although epithelial cells of the MGT synthesize protein and produce electron-dense se- cretory vesicles, they are substantially different in ultra- structure from those cells in the hepatopancreas respon- sible for digestive enzyme synthesis and secretion. Epithelial cells of the larval anterior and lateral midgut caeca are structurally and functionally similar to cells of the postlarval hepatopancreas. However, the lateral mid- gut caeca retain these features as they transform into the hepatopancreas, while the anterior midgut caeca lose Received 4 October 1989; accepted 22 January 1990. * Present address: Department of Biology, Lake Forest College. Lake Forest. Illinois 60045. these functions as they degenerate into the anterior di- verticulum and change in ultrastructure during early postlarval development. The anterior and posterior mid- gut diverticula of postlarvae are similar ultrastructurally even though they differ in ontogenetic history. Introduction In crustaceans, the foregut and hindgut are chitin- lined, while the intervening midgut is uncuticularized. The midgut is thus the region in which cells are in con- tact with the lumen of the alimentary canal. It comprises a tubular portion, which we call the "midgut trunk" (MGT)1, and the various outpocketings (diverticula and caeca) of this MGT (Lovett and Felder, 1989). In adult penaeids, such as Penaeus setiferus( Linnaeus, 1767), outpocketings of the MGT include the complex hepatopancreas (= digestive caeca or midgut gland, by some authors), a single anterior midgut diverticulum at the junction of the foregut with the MGT, and the poste- rior midgut diverticulum at the junction of the MGT with the hindgut (Dall, 1967a). These adult structures arise, during ontogeny, by the progressive transforma- tion of several larval structures: a pair of anterior caeca located at the foregut-MGT junction, and a pair of lateral caeca that arise slightly posteriad to the foregut-MGT junction. During late larval and early postlarval develop- ment, the two anterior caeca decrease in relative and ac- tual size, fuse medially, and begin to form the single ante- 1 The term "intestine" has usually been used to refer to this tubular region of the midgut. But we and others consider this to be a misappli- cation of vertebrate terminology and a practice to be discouraged (Dall and Moriartv. 1983: Lovett and Felder, 1989). 160 ONTOGENY OF M1DGUT FUNCTION 161 rior midgut diverticulum of the adult. At about the same time, the adult hepatopancreas is derived by ramification of the larval lateral midgut caeca. Finally, and in con- trast, the adult posterior midgut diverticulum first makes its appearance during the third week of postlarval devel- opment. The ontogeny of the penaeid gut is described in detail elsewhere (Lovett and Felder, 1989, 1990a). The ultrastructure and function of the crustacean mid- gut is only partly known, and most work has been done with adult specimens only. The adult hepatopancreas has been well-studied in Penaeus (Al-Mohanna et at.. 1985a, b; Vogt, 1985; Vogt et a/., 1985, 1986; Al-Mo- hanna and Nott, 1986; Caceci et a/.. 1988) and in other decapod crustaceans (see Gibson and Barker, 1979; Dall and Moriarty, 1983), but relatively little attention has been given to other regions of the midgut. Functions previously attributed to the crustacean MGT include: ( 1 ) absorption of nutrients from digested food (Yonge, 1924; Reddy, 1937; Speck and Urich, 1970; Talbot ct al.. 1972; Ahearn, 1974; Quaglia ct a!.. 1976; Ahearn and Maginniss, 1977; Barker and Gibson, 1977, 1978; Brick and Ahearn, 1978; Gemmel, 1979); (2) absorption of ions and control of net water flux be- tween the midgut lumen and the hemolymph (Yonge, 1924;Croghan, 1958; Green et al., 1959; Gifford, 1962; Dall, 1965, 1967b;Geddes, 1975; Malley, 1977; Ahearn ct al., 1978; Mykles and Ahearn, 1978; Mykles, 1979, 1980. 1981; Ahearn, 1980, 1982, 1984; Wyban et al., 1980); (3) excretion of ions (Green et al.. 1959; Gifford, 1962; Dall, 1967b. 1970); and (4) secretion of the per- itrophic membrane (Georgi, 1969; Mykles, 1979; John- son, 1980; Dall and Moriarty, 1983). Some of these func- tions, in particular the absorption of nutrients, have been assigned on the basis of an assumed analogy between the MGT and the vertebrate intestine. The functions of the anterior and posterior midgut caeca and diverticula remain obscure; yet some propos- als have been made: ( 1 ) both may increase surface area of the midgut for absorption of either water at ecdysis (Holliday et al., 1980) or nutrients during digestion (Yonge, 1924; Reddy, 1937); (2) both may secrete diges- tive enzymes (Holliday et al.. 1980); (3) both may func- tion in excretion (Dall, 1967b); (4) both may function in ion and water balance (Young, 1959; Heeg and Can- none, 1966; Dall, 1967b; Mykles, 1977, 1979); (5) both may secrete the peritrophic membrane (Pugh, 1962; Dall, 1967a; Georgi, 1969; Mykles, 1979); (6) both serve as sources of replacement cells (sites of cell regeneration) for the hepatopancreas and MGT (Davis and Burnett, 1964; Johnson, 1980); (7) the anterior diverticulum may accommodate volume change during contraction of the foregut (Powell, 1974); and (8) the anterior diverticulum may contribute essential components of digestive fluid that function in activation of proteolytic enzymes or pH change (Dall and Moriarty, 1983). In most decapod crustaceans, the adult form of the gut appears immediately following metamorphosis (Felder et a!., 1985). However, in Penaeus setiferus, transforma- tion of the gut to the adult form is protracted, taking place over several weeks after metamorphosis (Lovett and Felder, 1989, 1990a). Thus, this shrimp has enabled us to correlate the development of digestive function with that of structure. Toward that end, we have investi- gated the ontogenetic changes in the distribution of di- gestive enzymes in Penaeus setiferus and have compared those changes with simultaneous ontogenetic transfor- mations in midgut ultrastructure, gross morphology, and movement. Materials and Methods Specimens examined Larvae of Penaeus setiferus were reared in the labora- tory with natural seawater (for details, see Lovett and Felder, 1990b) on a diet of algae (Isochrysis sp., Chaeto- cems gracilis, and Tetraselmis c/iuii) and 24-h Artemia nauplii, by the method of McVey and Fox ( 1 983). Begin- ning with PL5 (the fifth day of postmetamorphic life), the diet consisted entirely of Anemia nauplii. Larval stages (protozoea and mysis) were identified in accord with de- scriptions by McVey and Fox (1983). Postlarval (PLn) stages are identified by postmetamorphic age (where n = days beyond metamorphosis), as is the practice in cul- ture of penaeid shrimp. "Juveniles" examined were at postlarval stage PLU(I. Histochemical localization of enzymes Sample preparation. Because diel rhythmicity in en- zyme activity has been reported for adults of Penaeus (Van Wormhoudt et al., 1972; Cuzon et al.. 1982), all specimens were collected in mid-morning when peak en- zyme activities reportedly occur. Food was available continually and the guts of all specimens were filled with food. Specimens were embedded in Tissue-Tek O.C.T. Compound® (Miles Scientific, Naperville, Illinois), quench frozen in liquid nitrogen, and stored until use at -70°C in an ultracold freezer. Serial sections were cut at 8 Mm thickness with a Miles Cryostat II. All slides used in this study were coated with a chrome alum-gelatin subbing solution (Pappas, 1971). Sections were placed on cold (-25°C) slides (but see amylase and protease tests below) and then melted by placing a thumb on the un- derside of the slide. Slides were allowed to dry at room temperature before incubation. Control sections for al- kaline phosphatase, acid phosphatase, and esterase tests were immersed in 90°C water for 5 min before incuba- 162 D. L. LOVETT AND D. L. FELDER tion. After incubation, sections were counterstained with Mayer's haemalum; color was developed by dipping sec- tions in Scott's solution (2% MgSO4 with 0.2% NaHCO, ). Sections were mounted in glycerol gel. Reconstruction of serial sections. Distribution of sites of enzyme activity within the midgut was determined by reconstruction of serial sections. To determine the ab- dominal segment within which sites of activity occurred, the product of total number of sections multiplied by 8 ^m was compared with average total length and length of each abdominal segment for the respective develop- mental stage. Non-specific estcrase. Sections were incubated for 30 min at room temperature in a 0.01% solution of Naph- thol AS-LC acetate by a method adapted from Burstone ( 1962): substrate solution was made by dissolving 5.0 mg Naphthol AS-LC acetate in 1.0 ml N,N-dimethylform- amide. After the substrate had dissolved, 10 ml of ethyl- ene glycol monomethyl ether was added, followed by 10 ml of 0.2 M Tris(hydroxy methyl) ami nomethane hydro- chloride buffer at pH 7.1. Immediately before incuba- tion, 40 mg of Fast Garnet GBC dissolved in 29 ml of distilled water were filtered into the substrate solution. Cholinesterase activity was inhibited by adding 10~3A/ eserine to the final substrate solution. Sites of esterase activity were indicated by deep violet precipitate. While esterase activity was detected successfully with Naphthol AS-LC acetate as the substrate, neither esterase nor lipase activity was detected when several other sub- strates were used. When other substrates were used to incubate frozen sections and paraffin sections of fresh, formalin-fixed, acetone-fixed, and freeze dried speci- mens, the following results were obtained: The 5-bromo- indoxyl acetate substrate by the method of either Barr- nett and Seligman (1951) or Holt and Withers (1952) yielded a highly diffuse, faint blue precipitate that was unsuitable for study. Incubation of sections with Tween 20, 40, 60, or 80 substrates by the method of Gomori (1945, 1949) and with Tween 85 by the method of Bok- dawala and George (1964). followed by demonstration of calcium soaps with either yellow ammonium sulfide or Alizarin Red S, yielded a diffuse precipitate that could not be differentiated from that obtained in control sec- tions heated at 90°C for 5 min prior to incubation. Alkaline phosphatase. Sections were incubated for 25 min at room temperature in a 0.02% solution of Naph- thol AS-MX phosphate free acid with 0.06% Fast Red Violet LB salt at pH 8.5 by the method of Burstone ( 1962). Sites of alkaline phosphatase activity were indi- cated by magenta precipitate. Acid phosphatase. After sections had dried on the slide, they were fixed in 4°C 10% neutral formalin for 30 s and washed for 3 min to localize the reaction. Sections were incubated for 30 min at 37°C in a 0.02%- solution of Naphthol AS-BI phosphate with 0.06% Fast Red Vio- let LB salt at pH 5.2 using Burstone's (1958, 1962) method. Sites of acid phosphatase activity were indicated by a magenta precipitate. Amyluse. By a technique modified from that of Trem- blay and Charest (1968), slides coated with chrome alum-gelatin subbing were dipped in a solution of 4% pu- rified potato starch in 20 mM phosphate buffer pH 6.9 with 10 mAI NaCl and dried at room temperature. The solution had been boiled, filtered, and degassed under vacuum prior to use. Frozen sections were placed on the starch substrate film of slides that were prechilled to — 25°C. Sections were melted and air-dried at room tem- perature. Slides were then incubated at 37°C for 1-2 h in covered petri dishes lined with water-soaked filter paper. Slides were thereafter air-dried, immersed in a solution of 5:1:5 methanol:acetic acid:distilled water for 15 min, treated with Periodic Acid-Schiffs (PAS) by the method ofMcManus( 1948), and air-dried again. Sites of amylase activity were indicated by clear areas where the starch film (now stained magenta) had been digested away. Protease. Sections were incubated on the gelatin emul- sion of Kodachrome-25™ color transparency film that had been previously exposed to daylight and developed commercially. By a technique adapted from Fratello ( 1968), sections were placed on emulsion prechilled to -25°C. Sections were then melted, air-dried at room temperature, incubated at 37°C for 1-2 h in petri dishes lined with water-soaked filter paper, and then dried again at room temperature. No buffer was added to control pH. Sites of protease activity were indicated by light blue- green or white areas where the darkly colored emulsion had been digested away. Activity of specific proteolytic enzymes were not de- tected successfully. Sections were incubated with N-(«- benzoyl-DL-arginine-/3-naphthylamide) hydrochloride in the method of Glenner and Cohen (1960) to detect trypsin-like activity and with both L-leucyl-^-naphthyl- amide by the methods of Burstone and Folk ( 1956) and Loizzi and Peterson (1971) and L-leucyl-4-methoxy- naphthylamide hydrochloride by the method of Nachlas el ul. (1960) to detect arylamidase (aminopeptidase) ac- tivity. Regardless of the method used for fixation and embedment, all preparations yielded results not different from controls. Transmission electron microscopy Specimens were fixed in cold (4°C) 4% glutaraldehyde solution buffered to pH 7.2 with 0.2 M phosphate buffer and postfixed with 2%' buffered osmium tetroxide. Speci- mens were dehydrated in acetone, infiltrated by centrifu- gation at 2500 rpm (after Millonig, 1976), and embedded in Spurr's low viscosity resin (obtained from Polysci- Regions IAC/AD' of Gut rar Esterase Protozoea I - Juvenile Amylase Postlarva 35" Juvenile Protease Protozoea I - Postlarva 4 Postlarva 7 - Postlarva 21 Postlarva 35~ Juvenile ONTOGENY OF MIDGUT FUNCTION Acid Phosphatase 163 12345 6/pB -MGT; \LC/TTpl Protozoea I - Juvenile Alkaline Phosphatase Protozoea I- Mysis 2 Mysis 3 Postlarva I - Postlarva 4 Postlarva 7 Postlarva 21 - Postlarva 35 Juvenile Figure 1 . Diagrammatic representation of a lateral view of the gut in Penaeus seiifems illustrating distribution of enzymes during development. AC, anterior midgut caeca; AD, anterior midgut diverticu- lum; FG, foregut; HG, hindgut; HP, hepatopancreas; LC, lateral midgut caeca; MGT, midgut trunk (= "intestine"); PD, posterior midgut diverticulum. Abdominal segments 1-6 are numbered. Label to left indicates developmental stages included for each diagram. Solid black areas indicate regions of gut where presence of enzyme was detected in all specimens. Stippled areas indicate regions where enzyme was de- tected in some, but not all specimens. ences. Inc., Warrington, Pennsylvania). Material was sectioned both with glass and diamond knives on a Sor- val MT-5000. Ultrathin sections of 80-90 nm were stained with methanolic uranyl acetate and lead citrate and examined at 75 kV with an Hitachi H-600 transmis- sion electron microscope. Results Ontogenetic change in enzyme distribution Non-specific esterase. Esterase activity was found in all regions of the midgut in all stages examined (Figs. 1, 2b). Amylase. No amylase activity was detected histochem- ically in developmental stages before PL,5 . In late post- larval stages activity was found in the hepatopancreas, anterior portion of the MGT, and the lumen of the fore- gut (Fig. 1 ). Because of the nature of the test it could not be determined with certainty whether activity in the MGT was restricted to either the extraperitrophic or en- doperitrophic lumen. No activity was found in the ante- rior diverticulum or in any areas of the gut posteriad to abdominal segment 2. Protease. In all larval and postlarval stages, protease activity was found in the hepatopancreas and in the ante- rior region of the MGT (Fig. 1). Resolution was inade- quate to differentiate intracellular activity from luminal activity. Furthermore, it could not be determined whether activity was restricted to either the extraperi- trophic or the endoperitrophic lumen of the MGT. Ac- tivity was never found posteriad to abdominal segment 2. In larval and early postlarval stages (PL,-PL4), prote- ase activity was found in the anterior midgut caeca, but not in the foregut. When the anterior caeca had degener- ated into the anterior diverticulum, activity no longer was found in this caecal extension of the midgut, but ac- tivity then was found in the lumen of the foregut. Acid phosphatase. Acid phosphatase activity was found in all regions of the midgut in all developmental stages (Figs. 1, 2d). Alkaline phosphatase. Alkaline phosphatase activity was detected in the hepatopancreas and the anterior re- gion of the MGT in all developmental stages (Figs. 1 , 3d). However, distribution of alkaline phosphatase for the re- maining regions of the midgut becomes limited during 164 D. L. LOVETT AND D. L. FELDER Figure 2. Histochemical localization of enzymes in fresh frozen transverse sections of posterior midgut diverticulum and hindgut in Penaeus selij'erus juveniles (PL,4(I). A. control section. B, section incubated with Naphthol AS-LC acetate as substrate to indicate esterase activity. C, section incubated with Naphthol AS-BI as substrate to indicate alkaline phosphatase activity (no precipitate present). D, section incubated with Naphthol AS-MX phosphate as substrate to indicate acid phosphatase activity [in diverticulum, pre- cipitate present (arrows) along apical surfaces of cells], (hg, hindgut; pd, posterior midgut diverticulum). Scale bar indicates 1 50 ^m for all figures. ONTOGENY OF MIDGUT FUNCTION 165 development. For all larval and early postlarval stages, activity was found in the anterior midgut caeca (Fig. 3b). In PL, and PL4, alkaline phosphatase activity was always more intense in the anterior caeca than in any other re- gion of the midgut. However, no activity was detected in the anterior diverticulum of subsequent postlarval stages. Activity was found along the entire length of the MGT in larval stages Protozoea 1 through Mysis 2 (Fig. 3d, f ). However, all specimens of Mysis 3 larvae had a short region in the MGT between the middle of abdomi- nal segment 2 and the end of abdominal segment 4, in which no activity was found (Fig. 1 ); the exact location of this region varied from specimen to specimen. In PL, and PL4, no activity could be demonstrated posteriad of abdominal segment 2 in some specimens, while in all specimens of these stages no activity was detected poste- riad of abdominal segment 4. During development, the posterior limit of alkaline phosphatase activity pro- gressed anteriad until, in the juvenile (PL140), no activity was found in any portion of the abdominal MGT (Fig. 3h). Alkaline phosphatase activity was never demon- strated in the posterior midgut diverticulum (Fig. 2c). Ontogenetic change in itllrastnicture In larval and early postlarval stages, the ultrastructure of cells of the anterior midgut caeca resembled that of cells of the lateral midgut caeca (Fig. 4a, b). However, the degeneration of the anterior caeca into the anterior diverticulum was accompanied by considerable change in the ultrastructure of the epithelial cells. The cells be- came elongate and no longer contained large vacuoles. In some cells, particularly in those ventral to the lumen of the diverticulum. the cytoplasm and all recognizable organelles became electron dense (Figs. 4c, 5a). Because adjacent cells varied in electron density (Fig. 5a, b), this density was not attributable to thick sections or over- staining. Cells of the anterior diverticulum bore apical micro- villi with a glycocalyx. Golgi bodies had swollen cisternae and produced secretory granules similar to those pro- duced by the MGT. The lateral cell membranes were dis- tinctly undulatory in nature. Where the epithelium of the anterior diverticulum tapered into the MGT, a mo- saic of cell types was present (Fig. 5b). Epithelial cells of the posterior midgut diverticula (Fig. 5c) were similar in ultrastructure to those of the anterior diverticulum. In all developmental stages of Penaem set (ferns, epi- thelial cells of the MGT had apical microvilli with a dis- tinct glycocalyx (Fig. 6). Active Golgi produced electron- dense secretory vesicles, which accumulated in the apical cytoplasm. Cisternae of the smooth endoplasmic reticu- lum were often distended. Rough endoplasmic reticu- lum usually was dense and its membranes were arranged in parallel rows. Intracellular lipid droplets were found occasionally in cells of the MGT, within both the cepha- lothorax and first abdominal segment. Discussion In the early postlarval stages ofPenaeus setifenis. both the anterior and lateral midgut caeca secrete digestive en- zymes, and the entire midgut is absorptive. As the two anterior caeca degenerate into the single anterior diver- ticulum, there is tremendous change in both function and ultrastructure: the capacity for both secretion of di- gestive enzymes and absorption is lost; the epithelium changes from being ultrastructurally similar to that of the adult hepatopancreas to being ultrastructurally similar to that of the posterior midgut diverticulum, even though the latter has an independent ontogenetic origin. As the hepatopancreas differentiates and increases allo- metrically in size, the MGT loses its absorptive capacity. Contrary to some reports, the abdominal MGT (or "in- testine") does not absorb digested food substrates once the gut has attained the adult form. Enzyme distribution With a few exceptions (notably Holliday et al., 1980), the histochemical distribution of enzymes observed in Penaeus setifenis is consistent with that reported for other species of decapod crustaceans (Travis, 1955, 1957; Miyawaki et al., 1961; Davis and Burnett, 1964; Loizzi, 1966; Van Herp, 1970; Loizzi and Peterson, 1971; Momin and Rangneker, 1974, 1975; Barker and Gibson, 1977, 1978). Although arylamidase activity was reported in hepatopancreatic cells of Scylla (Barker and Gibson, 1978), neither arylamidase nor aminopeptidase activity has been demonstrated unequivocally in histo- chemical studies of any other species of decapod. In tis- sue homogenates of P. setifenis, we measured significant amylase activity for all developmental stages, but activity remained low until late in postlarval development (Lov- ett and Felder, 1990b). Lack of histochemical evidence for amylase activity in larval and early postlarval stages of P. setifenis suggests that concentrations were below the limits of detection for the technique used. Lateral midgut caeca and hepatopancreas Both acid phosphatase and esterase activities within the hepatopancreas of decapods have been associated with the synthesis and secretion of digestive enzymes by this tissue, whereas alkaline phosphatase activity in the hepatopancreas has been associated with transmem- brane transport of metabolites (Momin and Rangneker, 1974; Barker and Gibson. 1977, 1978; Lane, 1984). Al- though the exact function of alkaline phosphatase in ab- 166 D. L. LOVETT AND D. L. FELDER ' -* mgt , ^_. G • T' W *;*•> ^v :Jr' *>£*>'•' Figure 3. Histochemical localization of alkaline phosphatase activity in fresh frozen sections of Penaeus setifenis. A, C, E, G, control sections. B, D, F, H. sections incubated with Naphthol AS-BI phos- phate as substrate. A, B, transverse section through foregut and anterior midgut caeca of larval stage Mysis 2. C, D. transverse section through lateral midgut caeca and midgut trunk of Mysis 2. E. F, transverse section through abdominal segment 2 of Mysis 2. G, H, transverse section through abdominal segment 2 of juvenile (PL]40). (ac, anterior midgut caecum; fg. foregut; d, debris in lumen; Ic. lateral midgut caecum; mgt, midgut trunk; pa. posterior artery). Scale bar indicates 100 ^m for A-F and 125 ^m for G and H. ONTOGENY OF M1DGUT FUNCTION 167 V lu :••••- , Figure 4. Epithelia of larval midgut caeca and postlarval anterior midgut diverticulum in fcimciix Si'lijerus. A, lateral midgut caecum. B, anterior midgut caecum. C, anterior midgut diverticulum. (bl, basal lamina; g, Golgi bodies; gc, glycocalyx; lu. lumen; m, mitochondrion; mv, microvilli; n. nucleus; rer. rough endoplasmic reticulum; sv, secretory vesicle; tj, tight junction; v. vacuole; asterisk indicates electron dense epithelium ventral to lumen of anterior diverticulum; arrow indicates undulatory lateral membranes). A. B, larval stage Protozoea 3. C, postlarval stage PL,5. Scale bar indicates 6.8 /im for A and B and 4 ^m forC. sorption has yet to be demonstrated, tissues in which al- kaline phosphatase activity is present are generally thought to function in absorption by active transport (see review by McCombet al., 1979). Localization ofamylase and protease activity within the hepatopancreas in the present study is consistent with previous detection ofam- ylase and tryptic activity within B-cells of the hepatopan- creas of other decapod species (Malcoste et al., 1983; De- Villez and Fyler, 1986). Localization of alkaline phos- phatase in the hepatopancreas of P. set (ferns in the present study is consistent with the absorption usually attributed to this tissue (Gibson and Barker, 1979; Dall and Moriarty, 1983). Ultrastructure also has been used to infer that the hepatopancreas functions in protein syn- thesis, secretion, and absorption in Penaeus (Al-Mo- hanna et al.. 1985b; Vogt, 1985; Al-Mohanna and Nott, 168 D. L. LOVETT AND D. L. FELDER he Figure 5. Epilhelia of midgut diverticula in Pcnai'iis M-titcm*. postlarval stage PL35 . A, anterior midgut diverticulum ventral to lumen (see Fig. 5c); these cells attach to hypodermis of dorsal pyloric valve of foregut: note "normal" cell (asterisk) surrounded by electron-dense cells. B, mosaic of cells where epithe- lium of anterior diverticulum tapers into midgut trunk. C. posterior diverticulum dorsal to lumen, (hi. basal lamina; g.Golgi bodies; gc, glycocalyx; he, hemocoel; lu, lumen; m, mitochondrion; me, cell undergo- ing mitosis: mv. microvilli; n. nucleus; rer, rough endoplasmic reticulum; sv, secretory vesicle; tj, tight junction; arrows indicate undulatory lateral membranes). Scale bar indicates 4 ^m for A and C and 5.8 urn for B. 1986; Caceci ct ai, 1988). and in other decapod crusta- ceans (Gibson and Barker, 1979). In P. setiferus. substantial morphological change oc- curs when the lateral midgut caeca of larvae differentiate into the hepatopancreas during early postlarval develop- ment (see Lovett and Felder, 1989). However, except for a decrease in the number and size of lipid droplets within cells during the mysis stages of development, the epithe- lial cells of the larval lateral caeca and of the mature he- patopancreas are identical in ultrastructure. The amy- lase, protease, and alkaline phosphatase activities that are evident in the lateral caeca during early development ONTOGENY OF MIDGUT FUNCTION 169 V ^teW it^/V 'V1 >> ly " " .-^^ • >^ Figure 6. Typical epithelium of midgut trunk in Pcnacux sclilimx. (bl. basal lamina: g, Golgi bodies; gc, glycocaly.v. lu, lumen: m, mitochondrion: mv, microvilli: n, nucleus: rer. rough cndoplasmic reticulum: ser. smooth endoplasmic reticulum; sv. secretory vesicle; tj. tight junction). Scale bar indicates 3 170 D. L. LOVETT AND D. L. FELDER are also evident in the mature hepatopancreas. Thus, this region of the gut retains the functions of digestive en- zyme synthesis, secretion, and absorption throughout development. Anterior midgut caeca In larvae of P. setiferus. chyme does not appear to flow from the lateral midgut caeca into the anterior midgut caeca (Lovett and Felder, 1 990a). Therefore, protease ac- tivity in the anterior midgut caeca most likely represents enzyme that has been secreted by the anterior caeca. Ul- trastructural similarity of the anterior midgut caeca with both the larval lateral midgut caeca and the mature hepa- topancreas also suggests that the anterior caeca secrete digestive enzymes. Although chyme does not flow into the anterior caeca from the lateral midgut caeca, it does flow into the ante- rior caeca from the foregut and the anterior-most portion of the MGT. Absorption in the anterior caeca, as inferred from both alkaline phosphatase activity and ultrastruc- ture, is consistent with the observed movement of chyme into the caeca and secretion of digestive enzymes by the caeca. Anterior and posterior midgitl divert icula The absence of alkaline phosphatase activity from the anterior diverticulum of postlarvae of P. seliferus sug- gests that this diverticulum is not absorptive, while the absence of amylase or protease activity suggests that it does not secrete digestive enzymes. The apparent post- metamorphic loss of both absorption and the capacity to secrete digestive enzymes in this portion of the midgut is reflected in ( 1 ) the complete absence of chyme from the lumen of the anterior diverticulum, and (2) the extensive change in ultrastructure that occurs when the anterior midgut caeca degenerate into the anterior diverticulum. Vacuolated F-cells and B-cells, usually associated with synthesis and secretion of digestive enzymes (Gibson and Barker, 1979), are not present in this portion of the mid- gut after metamorphosis. Unlike the anterior midgut diverticulum, which devel- ops from the larval anterior midgut caeca, the posterior midgut diverticulum first differentiates as a distinct structure about three weeks after metamorphosis ( Lovett and Felder, 1989, 1990a). Absence of alkaline phospha- tase and digestive enzyme activity and absence of chyme from the lumen of the posterior diverticulum suggest that this diverticulum, like the anterior midgut diverticu- lum, does not function in absorption or digestion. Also, while the anterior and posterior diverticula differ in on- togenetic histories, their epithelia are similar in ultra- structure. Even though many functions have been proposed for the anterior and posterior diverticula (see Introduction), the precise function of the diverticula remains obscure. As mentioned, neither of these structures appear to func- tion in either the secretion of digestive enzymes or ab- sorption through active transport. However, because the epithelial cells of the diverticula in P. setiferus and the mucus-secreting cells in the intestine of mammals both have electron-dense cytoplasm (Ito, 1965), cells of the diverticula may function in secretion of a mucus-like substance. Such a mucous secretion could contribute to the formation of the peritrophic membrane, as proposed by other authors (Pugh, 1962; Dall, 1967a;Georgi, 1969; Mykles, 1979); Holliday el ul. (1980) dispute this inter- pretation. Midgut trunk Because both absorption in postlarval stages (as in- ferred from alkaline phosphatase activity) and the pres- ence of digestive enzymes in all developmental stages are restricted to the anterior portion of the MGT in P. setif- erus. we initially predicted that epithelial cells in the an- terior region of the MGT might be differentiated ultra- structurally from those in the posterior MGT. Further- more, because there is significant ontogenetic change in the distribution of alkaline phosphatase in the MGT, we also predicted that there may be ontogenetic change in ultrastructure of the abdominal MGT during larval and early postlarval development. However, essentially no difference in ultrastructure was found along the length of the MGT and no ontogenetic change in ultrastructure occurred that could be correlated with presence or ab- sence of alkaline phosphatase activity. We also could not distinguish the two types of MGT epithelial cells (light and dark) identified by Talbot et al. (1972). The distribution of acid phosphatase in P. setiferus suggests that the entire MGT is involved in active protein synthesis and secretion, and this is consistent with the observed ultrastructure. However, ultrastructural evi- dence does not necessarily indicate that digestive en- zymes present in the anterior MGT are being synthesized and secreted by the MGT. The F-cells and B-cells associ- ated with secretion of digestive enzymes in the hepato- pancreas are absent from the MGT. From our in vivo observations of flow of chyme within the gut of P. setif- erus, the observed activity of amylase and protease in the anterior lumen of the MGT likely represents enzymes discharged into the MGT from either the larval midgut caeca or the hepatopancreas. We also observed in all postlarval stages that chyme within the MGT as far pos- teriad as abdominal segment 2, is regularly "regurgi- tated" anteriad into the hepatopancreas (Lovett and Felder. 1990a). Moreover, digestive enzymes also occur in the MGT as far posteriad as abdominal segment 2, ONTOGENY OF MIDGUT FUNCTION 171 where they mix with chyme. These enzymes and the di- gesting chyme are periodically carried anteriad into the midgut caeca or the hepatopancreas where final diges- tion and absorption take place. The presence of apical microvilli, a glycocalyx, tight junctions, and a well-developed basal lamina in epithe- lial cells along the length of the MGT in adult specimens ofPenaeus and other decapod crustaceans has led some investigators to conclude that these cells are absorptive (Talbot ct a/.. 1972; Hootman and Conte, 1974; Kurata and Shigueno, 1976; Mykles, 1979). From the distribu- tion of alkaline phosphatase in P. setifen/s. it appears that the entire MGT is absorptive in the early larval stages. However, this apparent absorption is lost from the abdominal MGT during postlarval development. Other investigators also have concluded that the MGT in adults ofPenaeus and other decapod crustaceans probably play a minimal role in absorption of organic nutrients, be- cause only low rates of transport for amino acids, sugars, and vitamins could be measured across the MGT epithe- lium from the lumen (Ahearn, 1982, and citations therein; Chu, 1986). Even though small amounts of these solutes are transported from the lumen of the MGT into the cytoplasm of epithelial cells, intracellular metabo- lism of the solutes results in no net transepithelial flux into the hemolymph. From studies of carrier-mediated transmembrane transport systems for amino acids and glucose, it is also evident that relative rate of transepithe- lial solute transport in the adult decapod MGT by these carrier systems is almost two orders of magnitude lower than in the decapod hepatopancreas (Ahearn el ell., 1983, 1985, 1986; Ahearn and Clay, 1987a, b, 1988). These observations independently support the conclusion that the adult hepatopancreas is the primary area of absorp- tion and that the MGT does not function significantly in this role. From histological and ultrastructural evidence, together with demonstration of in vivo uptake of radiola- beled solutes, it is usually inferred that the hepatopan- creas is the primary site of nutrient absorption in adult crustaceans (Yonge. 1924; van Weel, 1955, 1970; Vonk, I960; Speck and Urich, 1970;Dall, 1981). The MGT in Penaeus is reported to function in ion transport and regulation of water flux from the midgut lumen to the hemolymph (Dall, 1967b; Talbot et at., 1972; Ahearn et ai, 1978; Ahearn, 1982). Evidence for such a function is not surprising given the degree to which anal drinking and antiperistaltic water move- ments occur in some decapods (Fox, 1952; Pillai, 1960; Dall. 1965; Lovett and Felder, 1990a), and osmoregula- tory function may, in part, account for observed ultra- structure of the MGT. Absence of alkaline phosphatase activity from the MGT is also consistent with an osmo- regulatory function as alkaline phosphatase activity was not detected in either the gills or branchiostegites (pri- mary osmoregulatory tissues) of P. set (ferns. Ontogeny of midgut junction Because the developing midgut tissue in embryos and nauplii ofPenaeus functions in digestion and absorption of yolk, it is not unexpected that the entire midgut might retain similar functions during larval development. Even so, these functions are gradually lost from the abdominal MGT and from the anterior midgut caecum during lar- val and postlarval development. By the juvenile stage, only the hepatopancreas and that portion of the MGT within the cephalothorax are absorptive, while only the hepatopancreas functions in digestion. A similar ontoge- netic change (from an undifferentiated and unspecial- ized larval gut to an adult gut in which functions are seg- regated) is also seen in teleost fish (Prakash, 1961; Blax- ter, 1969) and may represent a general developmental phenomenon. Dendrobranchiate shrimp such as P. setiferus may be unique among decapod crustaceans in their retention of both digestion and absorption in the anterior midgut caeca throughout larval development. In Homarm, after yolk material has been depleted during the first larval stage, the anterior caeca rapidly decrease in size. Similar to the change in the cells of the anterior caeca of P. setif- erus after metamorphosis, there is a change in the epithe- lia of the anterior caeca in Homarus after the first larval stage: cuboidal, highly vacuolated cells are replaced by the highly columnar cells characteristic of the adult epi- thelium (Hinton and Corey, 1979). Absorption in the abdominal MGT of larvae and early postlarvae may compensate for the small surface area in the anterior and lateral midgut caeca. In addition, be- cause the gastric mill of the foregut is not functional dur- ing larval development, and because food has a relatively short retention time in the gut of larvae, retention of ab- sorptive capacity along the entire length of the MGT could maximize assimilation of ingested food. As the simple lobes of the lateral midgut caeca ramify into the many tubules of the hepatopancreas, the relative surface area of this region of the gut increases substantially (Lov- ett and Felder, 1 989). Thus, with loss of absorption in the anterior midgut caeca and the abdominal MGT, there is an increase in relative surface area (and hence absorptive capacity) of the hepatopancreas. Acknowledgments Special thanks are extended to A. L. Lawrence and his staff, Texas A&M Shrimp Mariculture Project, and to C. Howell and J. Benty, Continental Fisheries, Ltd., Pan- ama City, Florida, for their generous provision of Pe- naeus setiferus larvae and postlarvae. B. E. Felgenhauer, 172 D. L. LOVETT AND D. L. FELDER R. C. Brown, and J. F. Jackson, University of Southwest- ern Louisiana (USL), and S. C. Hand, University of Col- orado, offered useful comments on the manuscript. E. J. DeVillez, Miami University of Ohio, provided extensive assistance in development of histochemical methodol- ogy. K. R. Roberts and J. L. Staton (USL) assisted in culture of algae and shrimp. Primary support for this study was provided under research grant NA85AA-D- SG 1 4 1 to D. L. Felder and D. L. Lovett from the Louisi- ana Sea Grant College Program. Additional funds were provided to D. L. Felder by research grants from the Louisiana Education Quality Support Fund, projects 86- USL(l)- 126-07 and 86-LUM( D-083-13, and by a grant from the Coypu Foundation. 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(April. 1990) Host-Zooxanthella Interactions in Four Temperate Marine Invertebrate Symbioses: Assessment of Effect of Host Extracts on Symbionts D. C. SUTTON1 AND O. HOEGH-GULDBERG:* [Sir George Fisher Cent re for Tropical Marine Studies, James Cook University, Townsville. Queensland 4811, Australia and 2Sc/wol of Biological Sciences, University of Sydney, Svdnev, New South Wales 2006, Australia Abstract. Photosynthesis and translocation of photo- synthetic products from symbiotic zooxanthellae in four species of temperate-latitude invertebrates were investi- gated in vivo and in vitro. In vivo, zooxanthellae fixed I4C and translocated a substantial proportion of fixed prod- ucts to host tissues. In vitro, the effect of host tissue ex- tracts on isolated zooxanthellae varied. Extracts of the soft coral Capnella gaboensis, lysed zooxanthellae after a relatively short exposure. Those of the zoanthid Zoan- thus robustiis and the nudibranch Pteraeolidia ianthina had little effect on translocation of organic carbon from zooxanthellae. In contrast, host extract of the scleractin- ian coral Plesiaslrea versipora stimulated the release of up to 42% of the total I4C fixed, and the magnitude of release was positively correlated with the protein concen- tration of the extract. Host extracts had no effect on pho- tosynthetic rates in algal symbionts. The effect of P. versipora extract on isolated zooxan- thellae was studied. This extract caused zooxanthellae to divert photosynthetic products from lipid synthesis to the production of neutral compounds, principally glyc- erol, and these compounds were the predominant form of carbon detected extracellularly after incubating zoo- xanthellae in this extract. Only organic compounds made during the period of exposure of zooxanthellae to host extract, and not pre-formed photosynthetic prod- ucts, were translocated. The translocation-inducing ac- tivity of host extract was almost completely destroyed by heating ( 100°C), and a preliminary attempt to fraction- Received 21 October 1986; accepted 8 January 1990. * Present address: Department of Biological Sciences, University of Southern California. Los Angeles, California 90089-037 I . ate the tissue extract revealed that the active constituent did not pass through dialysis tubing of nominal pore size 10,000 D. These results are discussed in relation to host control of symbiotic partners, and to previous reports of "host-release factors" in other invertebrate symbioses. Introduction Many marine invertebrates belonging to the phyla Mollusca, Platyhelminthes. Cnidaria, and Protozoa con- tain endosymbiotic dinoflagellates, collectively known as zooxanthellae. In nudibranch molluscs (Rudman. 1981a, 1982) and the majority of cnidarians (Trench, 1979), zooxanthellae are found within vacuoles in host cells derived from the endoderm. Zooxanthellae carry out photosynthesis within the confines of the host cell, and make significant contributions to host cell metabo- lism by translocation of organic compounds to the host (for review, see Trench, 1979). Muscatine and co-work- ers estimate that up to 95% of the carbon fixed during photosynthesis is translocated to coral hosts (Muscatine et ai, 1983, 1984), and at least some of the translocated carbon is used by the host for respiration and growth (Franzisket. 1970; Johannes, 1974; Kevin and Hudson, 1979; Kempf, 1984). Moreover, the animal provides or- ganic and inorganic nutrients, some of them metabolic waste products, to the algae (Cook, 1971; Muscatine and Porter, 1977). Most of the studies noted above concern tropical sym- bioses. In temperate latitudes, many marine symbiotic associations involve zooxanthellae, and a number of studies of these associations show that zooxanthellae contribute to metabolic processes in their hosts and to 175 176 D. C. SUTTON AND O. HOEGH-GULDBERG calcification rates (Jacques and Pilson, 1980; Jacques el al., 1983; Tytler and Davies, 1986). However, the extent and significance of //; vivo translocation of photosyn- thetic products to host tissues in these interactions have rarely been studied. Little is known about the control of translocation be- tween the partners in symbiosis. Several in vitro studies have suggested that the host may contain compounds ("host factors") that cause carbon to be translocated from the alga (Muscatine, 1967; Trench, 1971c), but no chemical entity with this function has been identified, and no mechanism for host factor action has been dem- onstrated. In some associations, host factors have been reported only in extracts of symbiotic hosts and not in extracts from aposymbiotic individuals (Trench, 197 Ic). Host factors from some (Muscatine, 1967; Yu and Die- trich, 1977), but not all (Muscatine el ul.. 1972) symbi- otic invertebrates are heat-labile, and cross-reactivity ex- periments suggest that invertebrates having zooxanthel- lae have similar host factors. Thus, host extract from the symbiotic clam Tritiacna stimulates the release of or- ganic carbon from zooxanthcllae from the coral. Pod II o- pora danuconus. and the coral extract has a similar effect on clam zooxanthellae (Muscatine, 1967). Host factors may also influence the photosynthetic rate of zooxan- thellae (Trench, 197 Ic; Muscatine el al.. 1972), and their alanine uptake (Carroll and Blanquet, 1984b; Blanquet el ul.. 1988). In summary, the distribution of host factors and their effects among invertebrates having zooxanthel- lae is obscure. This study had three objectives: the first was to deter- mine whether in vivo translocation in a range of temper- ate invertebrates occurs. To this end, four relatively abundant marine invertebrates from Latitude 34°S, New South Wales, Australia, having zooxanthellae as symbi- onts and representing diverse taxa, were chosen for ex- perimentation: the soft coral Capnclla gaboensis. the stony coral Plesiastrea versipora, the zoanthid Zoanlhus robustus. and the aeolid nudibranch Pteraeolidia ian- l/u'na. The second objective was to determine whether there was evidence, in these temperate marine inverte- brate symbioses. for host factor control of zooxanthellar processes, particularly of photosynthesis, metabolism, and translocation of photosynthetic products. The final objective was to characterize the active components of host extracts affecting zooxanthellar processes, should they be found. Materials and Methods Source and maintenance <>l animals The invertebrates used in this study were collected from the Sydney region. New South Wales, Australia (Latitude 34° S), between May 1980 and May 1981. The nudibranch Pteraeolidia ianthina was collected at Ben Buckler Point at depths of 20 m. Plesiastrea versipora. Capnella gaboensis. and Zoanthns robustus were col- lected at 5-m depth at Port Jackson. Specimens were placed in seawater in plastic bags, transported to the lab- oratory, and kept in seawater aquaria illuminated by cool white fluorescent lighting (photoperiod 12 h light/ 12 h dark; 12 ^E-irT2-s~') in an air conditioned room (21 ± 2°C). Animals were used in experiments within three weeks of collection. Isolation of zooxanthellae and preparation oj host extracts Unless otherwise stated, filtered (0.45 urn, Millipore) natural seawater was used in all experiments. Suspen- sions of zooxanthellae were prepared from each species as follows: the surfaces of small colonies (approximately 100 cm2) of P. versipora were abraded with a stainless steel brush, flooded with seawater, and scraped with a nylon toothbrush into a glass dish. For C. gaboensis. sev- eral branch tips (up to 50 g wet weight) were macerated using a mortar and pestle. The resulting slurries from each animal were passed through one layer of Miracloth (Calbiochem) and made up to 10 ml total volume with seawater. Whole individuals of Z. robustus were split longitudinally with a scalpel, and the gastrodermis, con- taining the zooxanthellae, was scraped into 10 ml of sea- water. The cerata of several individuals of Pa. ianthina were excised and homogenized in 5 ml of seawater, using a ground-glass homogenizer. Microscopic observations indicated that these techniques did not disrupt zooxan- thellae. The suspensions of zooxanthellae were centrifuged at 2000 rpm (490 X g) for 60 s (M.S.E. benchtop centri- fuge). The supernatant ("host extract") was poured off. The zooxanthellae were then washed three times by re- suspension in 10 ml of seawater followed by re-centrifu- gation. The pH of the host extract (pH = 7.5-7.9) was adjusted to that of seawater (pH = 8.1) with 0. 1 /VNaOH. The extract was stored at 5°C for not more than 20 min until used. The protein concentration of each extract was determined at the end of each experiment (Lowry et ul.. 1951), from samples frozen (0°C) following extract prep- aration. Electron microscopy Isolated zooxanthellae and small pieces of intact tissue were fixed for 3-4 h in 3% glutaraldehyde in sodium cac- odylate buffer (pH 7.3) and then washed for 10 min in each of three changes of 0. 1 M sodium cacodylate buffer. Fixed sections of coral tissues were decalcified in a solu- tion of 10% sucrose containing 3% EDTA (sodium ethyl- enediaminetetraacetic acid; Borowitzka and Vesk, ZOOXANTHELLAE IN TEMPERATE INVERTEBRATES 177 1978). Specimens were post-fixed for 1 h in 1.0% os- mium tetroxide in sodium cacodylate buffer, dehydrated through an acetone series, and embedded in Spurr's (1969) resin. Sections were cut, stained with lead acetate and uranyl acetate, and examined with a Philips-300 transmission electron microscope. Measurement of release of 4C from zooxanthellae In the intact association. Whole zoanthids, or 2 cm branch tips of C. gaboensis, were incubated in seawater containing 15 MCi'4C-ml ' (as NaH'4CO,; Amersham) at 25°C under fluorescent light (78-80 jiE-m~--s~'). Af- ter I h, the zooxanthellae and host tissues were separated (see above), and the radioactivity of each fraction was measured using liquid scintillation counting. Superna- tants were made up to a known volume, while zooxan- thellae were resuspended in 20 ml of distilled water. Three 100-^1 subsamples were taken from the superna- tants and three 50-/ul subsamples from the resuspended zooxanthellae. Each subsample was acidified with 1 00 n\ of 0. 1 A/HC1 and left in a fume hood for 4 h. Scintillation fluid (10 ml) was added to each subsample and the vial shaken to ensure mixing. The scintillation cocktail con- tained 0.2 g POPOP, 3.0 g PPO, and 0.5 1 Teric-10 dis- solved in toluene ( 1 1). In vivo experiments with P. versi- pora were not undertaken because the host tissues could not be completely extracted, nor all the zooxanthellae removed from the calcareous skeleton. In vitro. Freshly isolated zooxanthellae (106-mr') were incubated in 2 ml of host extract or seawater in 20 ml glass scintillation vials on a linear shaker (Grant SS40, 100 strokes -min"1) under fluorescent light (78-80 pE- m~:-s~'). At the beginning of each experiment, 10 /iCi- ml ' NaH'4CO3 was added and the vials shaken to en- sure complete mixing. Triplicate 50-^1 samples were taken immediately following mixing for the determina- tion of specific activity. Each 50 jul was added to 10 ml of scintillation fluid (see above) made basic by the addi- tion of 0. 1 ml of 1 .0 M NaOH. The radioactivity of each sample was counted for 5 min in a Packard Tri-Carb scintillation counter. Counts were corrected for quench- ing and background radioactivity and expressed as mg C using calculated specific activities. Calculation of specific activities was based on the total inorganic carbon content of seawater ( 2.52 X 10~: mgC-ml~', Skirrow, 1975). Following incubation, triplicate 50-^1 samples were re- moved from each treatment and filtered under vacuum (0.45 /urn. Millipore). The filters were washed three times under vacuum with 0.65 ml of seawater to give a total filtrate volume of 2.0 ml. Three 100-^1 subsamples from each filtrate were acidified with 100 n\ 0. 1 M HC1 before adding 10 ml of scintillation fluid. The filters supporting zooxanthellae were dissolved in 1 .0 ml 2'-methoxyetha- nol before adding 100 n\ 0. 1 M HC1 and 10 ml of scintil- lation fluid. The samples were counted as described above. The percentage of photosynthetic products released from the zooxanthellae during incubation was calculated from the ratio of the filtrate activity (I4C released by the algae) to the total activity (filtrate plus filter, total fixed I4C). Measurement of photosynthetic rates Photosynthetic rates in zooxanthellae in each treat- ment were determined from the total 14C fixed during the experiment (i.e., the radioactivity of filter and filtrate combined), the specific activity and the total chlorophyll content (determined for each vial at the end of each ex- periment). Rates were calculated as '4C-carbon fixed per mg total chlorophyll per hour. Total chlorophyll was measured using the methods of Jeffrey and Humphrey (1975). Identification of labeled compounds Zooxanthellae were incubated in 2 ml of seawater or host extract with 25 nCi • mP ' NaH14CO3 . After 1 h, the zooxanthellae were removed from suspension by centrif- ugation, and resuspended in 3 ml of distilled water. Su- pernatants and suspensions of zooxanthellae were ex- tracted by the methanol/formic acid/chloroform proce- dure of Barnes and Crossland (1978). The resulting chloroform phase (lipid fraction) was made up to 20 ml with chloroform, while the methanol/formic acid phase was dried at 65°C, and redissolved in 20 ml of distilled water. The radioactivity of each fraction was measured by scintillation counting as described above. The methanol/formic acid extract was separated into neutral, acidic, and basic fractions by ion-exchange chro- matography (on Sephadex SP and QAE) using Redg- well's (1980) method. The eluted fractions from the ion- exchange columns were dried and resuspended in 0.5 ml ethanol (neutral compounds and organic acids) or pyri- dine (amino acids and phosphate esters). Compounds in each ion-exchange fraction were separated by thin-layer chromatography on cellulose plates (Machery-Nagel Cel 300, 10 cm X 10 cm). Plates were spotted with 60 ^1 of sample and 20 ^ig of each of a set of nonradioactive stan- dards. Basic fractions (amino acids) were chromato- graphed twice in the same direction in pyridine:dioxane: NH4OH (25%):water (2:2:1:1, v/v; G.O. Kirst pers. com.). The acidic (organic acids) and neutral fractions were chromatographed twice in the same direction in EDTA:NH4OH:water:n-propanol:isopropanol:n-buta- nol:isobutyric acid (0.25:20:190:70:15:15:500, w/v/v/v/ v/v/v; Feige et al., 1969). After drying, the compounds on each plate were visualized using a range of chemically 178 D. C. SUTTON AND O. HOEGH-GULDBERG sensitive sprays. Ninhydrin spray reagent (Gelman cata- log No. 72818) was used to detect amino acids, aniline/ xylose was used for organic acids (Smith, 1960), and sil- ver nitrate was used for monosaccharides, carbohy- drates, and phosphate esters (Smith, 1960). Plates were also exposed to X-ray film (Kodak X-Omat S) for 8 weeks; the film was then developed to determine the dis- tribution of radioactively labeled compounds. To mea- sure the amount of I4C incorporated into a given com- pound, the cellulose powder from each spot was scraped oft' and the radioactivity determined by liquid scintilla- tion counting, as described above. Effect of host extracts on zooxanthellae from other invertebrates Experiments were undertaken to assess the effects of host extracts on zooxanthellae from other animals used in this study. C. gaboensis zooxanthellae and host extract were not included. Extracts and zooxanthellae were pre- pared as described above. At the beginning of the experi- ment, zooxanthellae from each host were resuspended in ex- tracts of P. versipora. Pa. ianlliina, '/.. robiistus, orseawater. Incubation conditions with I4C, and subsequent analyses, were as described above for in vitro studies. Effect of P. versipora host extract on pre-tormed products oi photosynthesis To obtain additional information on the biochemical effects of host extracts on zooxanthellae. experiments were performed to determine if previously formed pho- tosynthetic products are subsequently released during in- cubation in host extract. Zooxanthellae were incubated for one hour in seawater containing 5 j/Ci-ml ' NaH14CO1 then washed three times by centrifugation and resuspension. The zooxanthellae were then incu- bated for 1 h in host extract, at which time release of the "pre-formed" labeled products (formed prior to expo- sure to host extract) was determined. In a parallel experi- ment, zooxanthellae were first incubated for 1 h in sea- water without I4C, washed as above, then incubated in host extract, this time with NaH14CO,. After 1 hofincu- bation, release of labeled products (formed during incu- bation in host extract) was determined. Effect of boiling and dialysis on P. versipora host extracts To determine the heat-sensitivity of P. versipora ex- tract, a freshly prepared sample was incubated in a water bath (100°C) for 10 min then cooled to room tempera- ture. The ability of heated extract to stimulate the release of carbon from zooxanthellae was compared with that of unheated host extract. To determine the effect of dialysis on host extract ac- tivity, a freshly prepared sample was first centrifuged for 3 min at 12,000 rpm (27,000 X g; 4°C; M.S.E. High Speed 18). The supernatant was filtered (0.45 j/m; Milli- pore HA), then dialysed (Selby's, type 20, nominal pore size 10.000 D) for 6 h with rapid stirring at 4°C (two changes of 1 1 buffered seawater). The ability of this treated extract to stimulate release of I4C products from zooxanthellae was compared with that of undialysed ex- tract stored at 4°C for 6 h. Dialysis tubing was boiled in deionized water for 2 h. and rinsed extensively in seawa- ter prior to use. Results Photosynthesis and the release of organic I4C from zooxanthellae In the intact association (see Table I). Zooxanthellae in C. gaboensis, Z. robust us, and Pa. ianthina photosyn- thesized at rates ranging from 0.109 to 0.221 mg C-mg chlorophyll ' -h '. Dark fixation rates varied from 2.7% to 6.4% of rates in the light. After one hour, a significant portion (up to 47%) of the fixed 14C-carbon was found associated with host tissues. The percentage of total fixed l4C-carbon detected in tissues of Z. robust us during July was significantly lower than at other times of the year. As noted previously, in vivo experiments with P. versipora were not conducted due to problems in complete extrac- tion of host tissues from the calcarious skeleton. In vitro (see Tables I la, lib and HI). Zooxanthellae isolated from all the hosts photosynthesized in seawater at rates that ranged between 0.503 and 0.886 mg C-mg chlorophyll ' • h ' (Table Ha). Dark fixation rates in sea- water were 2.0-4.0% of those in light. Photosynthetic rates for zooxanthellae in host extracts were not signifi- cantly different from those in seawater (P < 0.05), except for C. gaboensis. In this case, rates in host extract were less than 1% of those in seawater, suggesting damage to algal cells or inhibition of photosynthesis. Subsequent microscopic examination revealed that all cells were lysed after incubation for one hour in this host extract (Fig. la). In view of this observation, zooxanthellae incu- bated in extracts of the other hosts were also examined microscopically (Fig. Ib, P. versipora), but they showed no signs of damage and were indistinguishable from those fixed in the host. Zooxanthellae from all hosts retained approximately 95% of the organic I4C fixed during incubation for 1 h in seawater. In host extracts, zooxanthellae released differ- ing proportions of the organic I4C fixed during 1 h of incubation (Table lib). For P. versipora, the percentage of fixed carbon found outside zooxanthellae in host ex- tract was significantly higher than that released in seawa- ter (P < 0.01 ) and ranged from 10.9 to 42% of the total ZOOXANTHELLAE IN TEMPERATE INVERTEBRATES Table I Pholosvnthelic rates in. and percentage translocalion ii/'/ixed "C-pmducts from, zooxanthellae in vivo 179 Host Experiment number' % Fixed l4C-products translocated2 Photosynthetic rate (light)-3 Dark fixation rate2-3 Zoanthus robustus 1 35.2+ 11.9 0.221 ±0.092 0.006 ±0.0 12 2 42.2 ± 5.2 n.d.4 n.d. 3 11.8± 6.2 0.1 26 ±0.022 0.008 ± 0.009 Pteraeolidia ianthina ? 1 23.8+ 6.2 0.179 + 0.080 0.007 ± 0.004 2 47.5+ 14.4 0.1 66 ±0.026 0.006 ± 0.003 3 25.1 ± 4.0 0.1 09 ±0.02 7 0.007 ±0.0 17 Capnella gabocnsis 1 18.1 ± 3.5 0.1 85 ±0.074 n.d. 2 19.1 ± 3.1 0.135 + 0.036 n.d. ' Experiment numbers represent investigations done in March ( 1 ), May (2), and July (3) 1981. 2 Mean ± 95% confidence interval; n = 3 for all experiments. 3 mgC- nig chlorophyll '-h '. 4 n.d. — not determined. 5 Data for Pa ianthina from Hoegh-Guldberg and Hinde, 1 986. 14C fixed. The magnitude of release from P. versipora zooxanthellae was positively correlated with the protein content of the host extract (Fig. 2, P. versipora, r = 0.54, P < 0.05). For Z. robust us and Pa. ianthina, zooxanthel- lae in host extracts released only a small proportion (less than 10%) of fixed l4C-carbon. The proportion released was significantly (P < 0.05) but only slightly higher than that released in seawater, even at concentrations of host extract (as determined by protein concentration, Fig. 2) that caused in excess of 30% release in experiments with P. versipora. For C. gaboensis, between 60 and 90% of the organic 14C fixed during 1 h in host extract was subse- quently found outside the cells. However, as noted pre- viously, this host extract caused lysis of zooxanthellae and inhibited photosynthesis. The proportion of fixed l4C-carbon released from P. versipora and Pa. ianthina zooxanthellae in seawater or host extracts was monitored over 4 h of incubation (Fig. 3), and did not change significantly during that period. To investigate the possible similarities of host factors Table II Photosvnthetic rates in. and percentage translocation of fixed "C-products front, isolated :ooxanthellae in seawatcr and host extract ' a) Photosynthetic rate (mg C-mg ch 1 '-h ') Host Incubation medium Host extract Seawater. light Seawater, dark Zoanthus robusius Pteraeolidia ianthina Capnella gaboensis Plesiastrea versipora 0.438 ±0.231 (7) 0.578±0.301 (19) 0.002 (4) 0.919 + 0.612(12) 0.503 ±0.287 (7) 0.520 ±0.298 (19) 0.886 + 0.625(3) 0.877 ±0.594 (12) 0.018 ±0.009 (4) 0.023 + 0.003(6) n.d. 0.018 + 0.001 (5) b) Percentage of fixed '4C-products translocated Incubation medium Host Host extract Seawater. light Zoanthus robustus Pleraeolidia ianthina Capnella gaboensis Plesiastrea versipora 7.88 ± 1.32(7) 6.31+ 3.15(19) 70.11 ± 15.40(4) 26.76 ± 15.85(12) 2.97 ±0.88 (7) 2.42 + 0.34(19) 3.45 + 0.92 (4) 4.83±0.51 (12) 1 Mean ± 95% confidence interval; number of experiments conducted for each host is shown in brackets; U)11 zooxanthellae -ml ' incubation medium (breach treatment; incubation time — 1 h. 180 D. C. SUTTON AND O. HOEGH-GULDBERG "' - ' '' L*>». . : Figure 1. Ultrastructurc ol'/oo\anthellac: (a) Isolated I'rom C'apne//a gahoenxi.'i and incubated for one hour in host extract. Note lysis and internal cellular disruption, (b) Isolated from Plexiaslrea ri>\amhellae in host extract anil extract \ <>/ non-limi invertebrates ' Source of host extract Source of zooxanthellae Plesiastrea vcrsipora (2.60)2 Zoanthus robustus (2.75) Pteraeolidia ianlliina (2.30) Seawater P. versipora / rohnsllis I' ianlliina 27.18 + 7.11 30.80 ± 5.62 3.97 ±3.11 8.61 ± 1.00 8.52 ±0.75 8.11 ±4.02 6.49 ±1.19 11.81 ±3.52 7.28 ± 3.37 4.01 ±0.62 2.61 ±0.85 2.57 ±0.41 1 Mean ± 95'' confidence interval: n = 3. : Numbers in brackets refer to host extract protein concentration in mg(N)-ml '. ZOOXANTHELLAE IN TEMPERATE INVERTEBRATES 181 o 20- Plesiastrea versipora 0 20- toantttus robustus • I > PteraeoJidia ianthina 01234 Host extract protein concentration ( mg [N]. ml"1 ) Figure 2. Release of photosynthetically fixed l4C-organic carbon from zooxanthellae in host extract as a function of host extract protein content. Plesiaslrea versipora. r = 0.54, significant at P < 0.01, y = 6.9(X) + 12.1. Pleraeolidia ianthina, r = 0.70, significant at P < 0.05, y = 2.36(x)- 0.76. ter (Table IV). Neutral compounds, and to a lesser extent organic acids, were the dominant soluble labeled com- pounds detected outside the zooxanthellae in both sea- water and host extract treatments. About 30% of the ex- tracellular label in each treatment was in the form of glycerol (Table V), representing about 10% and 2% of the total 14C fixed by zooxanthellae in host extract and seawater, respectively. There were 3-5 unidentified com- pounds detected extracellularly in each treatment, repre- senting about 10% and 0.3% of the total 14C fixed by zoo- xanthellae in host extract and seawater, respectively. In addition to neutral compounds, labeled glycollate, pyru- vate, malate, and leucine were detected extracellularly in host extract but not seawater. Labeled fructose and aspartate were detected extracellularly in seawater but not host extract. Labeled alanine was found in both treat- ments, but was not the major labeled amino acid detailed extracellularly in either case. The major amino acid pres- ent was not identified. Effect oj P. versipora host extract on pre-formed products of photosynthesis Zooxanthellae incorporating I4C into photosynthetic products during an initial treatment of incubation for 1 h in seawater with label, retained most of those ("pre- formed") labeled products when subsequently incubated in host extract for 1 h. In contrast, zooxanthellae having an initial treatment in seawater without label but which incorporated 14C into photosynthetic products during subsequent incubation for 1 h in host extract, released a significant proportion of labeled products formed during that incubation (Table VI). Effect of boiling on host extract activity Heating of P. versipora extract at 100°C for 10 min resulted in a significant loss of release-inducing activity (Table Vila). However, zooxanthellae incubated in Figure 3. Release ot'photosynthetically fixed 14C-organic carbon by zooxanthellae with time during extended exposure (4 h) to host extract. Zooxanthellae isolated from Plesiastrea versipora (O) and Pleraeolidia ianthina ([]) and incubated in seawater; zooxanthellae isolated from P. versipora (•) and Pa. ianlhina (•) and incubated in host extract. 182 D. C. SLITTON AND O. HOEGH-GULDBERG Table IV Incorporation of'4C into looxanlhellae lipiii, amino acid, organic acid, and neutral compound clashes, and percentage of each class detected extracellularly alter incubation fur one hour in seawater or Plesiastrea versipora extract # Class of compound Zooxanthellae in seawater Zooxanthellae in host extract Intracellular Extracellular1 Total Intracellular Extracellular' Total 1 Lipid 75.4 0.2 75.6 22.5 0.5 23.0 Amino acid 8.5 0.5 9.0 11.7 1.1 12.8 Organic acid 3.9 0.1 4.0 11.7 9.6 21.3 TOTAL 96.0 4.0 100.0 67.9 32.1 100.0 2 Lipid 54.1 0.6 54.7 9.0 5.3 14.3 Amino acid 114 0.4 11.8 13.1 2.3 15.4 Organic acid 9.1 1.0 10.1 13.5 18.6 32.1 Neutral 20.5 2.9 23.4 27.0 11.2 38.2 TOTAL 95.1 4.9 100.0 62.6 37.4 100.0 3 Lipid 53.3 0.8 54.1 24.7 0.4 25.1 Amino acid 10.9 0.5 11.4 12.9 0.8 13.7 Organic acid 9.1 1.3 10.4 12.8 7.7 20.5 Neutral 20.4 3.7 24.1 24.2 16.5 40.0 TOTAL 93.7 6.3 100.0 74.6 25.4 100.0 * Experiment number. 1 "Total" extracellular for each experiment = percent I4C released from Zooxanthellae. heated host extract still released slightly more l4C-prod- ucts than did cells incubated in seawater. Effect of dialysis on host extract activity The release-inducing activity of P. versipora extract did not decrease when the extract was dialyzed against seawater tor 6 h at 4°C (Table Vllb). Discussion In this study, aspects of the interaction between four temperate marine invertebrates and their symbiotic zoo- xanthellae were examined. Extracts of the stony coral P. versipora may contain "host factors" that control trans- location of photosynthetic products from symbiont to host. In the other invertebrates examined, no evidence of a "host factor" effect on translocation of photosyn- thates was found. Therefore, these observations extend to temperate symbioses the likelihood that host factors are not a universal property of symbiotic associations in- volving Zooxanthellae. We also found that host extracts may affect Zooxanthellae in previously unreported ways, apparently through their effect on metabolic pathways. Thus, in at least some symbioses, the host may have a much greater control over zooxanthellar processes then previously thought. The transfer of fixed carbon //; vivo from symbiont to animal tissues is significant in the nutrition of the host and has been investigated for a number of Zooxanthellae/ invertebrate symbioses. In tropical invertebrates, zoo- xanthellae release between 24 and 27% of total fixed car- bon to the host in sea anemones (von Holt and von Holt, I968a), 26-55% in corals (Muscatine and Cernichiari, 1969; Muscatine et at.. 1984), and 20-42%. in zoanthids (von Holt and von Holt, 1968b; Trench, 197 la). Studies of translocation in temperate invertebrates are few, al- though significant translocation has been reported for the two temperate sea anemones, Anthopleura elegant is- sima(56%. Trench, \97la)andAnemoniasitlcata(6Q%, Taylor, 1969), respectively. One objective of the present study, therefore, was to determine whether there was evidence for translocation of photosynthetic products from Zooxanthellae to host in diverse temperate invertebrates. Results showed that Zooxanthellae /;; vivo photosynthesized at rates compa- rable to those reported for Zooxanthellae in tropical in- vertebrates (Porter, 1976; Scott and Jitts, 1977; Musca- tine el a/.. 1984), and that a variable but significant pro- portion of total fixed carbon was subsequently transferred to the host tissues. Thus, the proportion translocated ranged from 1 1.8% to 35.2% for Z. robus- tus. 23.8% to 47.5% for Pa. ianthina. and 1 8.0%. to 1 9. 1 % for C. gaboensis. Difficulties encountered in achieving complete separation of tissues from the calcareous skele- ton of P. versipora prevented any statement being made concerning photosynthesis and translocation in this as- sociation when it is intact. The possibility of seasonal variation in the translocation of fixed carbon to the host ZOOXANTHELLAE IN TEMPERATE INVERTEBRATES 183 Table V I'ercentaKe ol total extracellular fixed I4C present u.i specific compounds alter incubation ofzooxanthellae lor one hour in \eumiteror Plcsiastrea versipora extract ' Percentage of total I4C released into Percentage of total I4C released into Compounds seawater(%) host extract (%) LIPID 12.7 1.1 AMINO ACIDS Alanine 3.4 0.1 Aspartate 0.4 - Leucine - 2.3 Unidentified 4.1 1.0 ORGANIC ACIDS Lactate 2.1 8.5 Oxaloacetate 6.1 - Pyruvate - 2.9 Glycollate _ 5.4 Malate - 3.8 Unidentified 12.4 9.7 NEUTRAL Glycerol 33.5 31.5 Glucose 14.4 1.7 Fructose 6.1 - llnidentified 4.8 32.2 TOTAL 100.0 100.0 1 % recovery of 14C from incubation medium was 85% and 95% for seawater and host extract, respectively. was suggested for Z. robiistus, but was not demonstrated conclusively. Therefore, if the symbioses examined in this study are representative of temperate interactions in- volving zooxanthellae, it is apparent that they are similar to their tropical counterparts in terms of short-term movement of substantial amounts of fixed carbon from symbiont to host. The contribution of symbionts to the energy demands of the hosts in this study and in temper- ate invertebrates in general remains to be elucidated. This study, and previous ones addressing the question of ways in which the host may exert control over zooxan- thellae in symbiosis, relied on in vilro experimentation using host extracts and isolated zooxanthellae. Interpre- tation of results in studies of this nature is made difficult due to the possibility of experimental artifacts as a conse- quence of extraction or incubation procedures. Lytic effects of host extracts on isolated zooxanthellae have been reported previously (Steele and Goreau, 1 977), and, in the present study, extracts of C. gaboensis caused both lysis ofzooxanthellae and inhibition of photosynthesis. However, for P. versipora. Pa. ianthina. and Z. robnstns, the microscopic and biochemical evidence suggests that isolation from these hosts and subsequent in vitro experi- mentation in host extracts had no detrimental effects on zooxanthellae. First, the photosynthetic rates of isolated zooxanthellae were equal to or higher than rates mea- sured for zooxanthellae in the intact association, and were comparable to rates determined for isolated zoo- xanthellae from other marine invertebrates (Burris, 1977; Dunstan, 1982; Muller-Parker, 1984). Second, zooxanthellae in seawater retained more than 95% of the organic carbon fixed during four hours of incubation. Third, following incubation in host extract or seawater, zooxanthellae were microscopically indistinguishable from zooxanthellae /'// vivo. Many studies, principally of tropical invertebrates, have suggested that the host may exert control over its symbiotic partner in at least two ways, namely by affect- ing the photosynthetic rate in zooxanthellae and by stim- ulating release of photosynthetic products. A second ob- jective of this study was to seek evidence for similar host control of these processes in temperate invertebrates, and to determine whether there was evidence for host control of other zooxanthellar processes. Table VI Effect oj Plesiastrea versipora extract on release of pre- formed l4C-plwlosyntltelic products from zooxanthellae % Release of l4C-products during I h in "subsequent treatment" Initial treatment Pre-formed '4C-products present Subsequent treatment Experiment 1 Experiment 2 SW ^. sw + 14c rn . — «• SW + I4C — * HE + 14C — + SW — * + HE 5. 54 ±2.83 22.43 ±2.97 10.68 ±2. 71 9. 73 ±2. 53 5.95 ±2. 19 32.19±2.12 4.60 ± 0.95 3.92 + 0.81 1 Zooxanthellae (lOVml) incubated for 1 h in the listed treatments. SW = seawater; HE = host extract: I4C = NaH4CO,, ; Zooxanthellae removed from "initial treatment" and incubated lor 1 h in the listed "subsequent treatments." 1 Mean ± 95% confidence interval, n = 3. 184 D. C. SUTTON AND O. HOEGH-GULDBERG Table VII Efl'cct of JOO'C and dialysis on MC release-inducing activity of Plesiastrea versipora /im; extract ' a) 100°C % Release of l4C-products from zooxanthellae Experiment 1 Expenment 2 Seawater 4.67 ± 1 14 4. 70 ±0.95 Host extract 30.37 ± 1 13 47.68 ± 1.13 100°C-treated host extract2 8.53 ± 1 1 1 12.68 ± 1.15 b) Dialysis % Release of l4C-products from zooxanthellae Expenment I Seawater Fresh host extract Dialyzed (6 h. 4°C) extract' Undialyzed (6 h, 4°C) extract3 6.40 ± 1.94 28.50 ± 3.78 37.40 + 3.21 36.50 ± 1.59 1 Mean ± 95% confidence interval; n = 3. : Fresh host extract treated at lOOVfor 10 mm. 1 Extract dialyzed at 4°C for 6 h against 2 changes of phosphate butter, or held at 4°C for 6 h (undialyzed extract). No evidence was found that any of the host inverte- brates in this study stimulated enhanced photosynthetic rates in their symbionts. Zooxanthellae in the intact asso- ciation had rates the same as. or lower than, those freshly isolated, and there was no difference in rates in vitro for isolated zooxanthellae in host extract or seawater. This result contrasts with two previous ones, the first for zoo- xanthellae from A. elegantissinui. which were reported to fix I4C in host extract at rates an order of magnitude higher than in seawater (Trench, 1 97 1 c), and the second by Muscatine el al. (1972) who observed an opposite trend in experiments with the hydrozoan Millcpora alci- cornis. One explanation for the different results in these three studies may be found in the experimental proce- dures involved. In the present study, the pH of host ex- tracts was measured and adjusted to that of seawater at the beginning of each experiment. This may be signifi- cant, particularly considering the recent demonstration of a correlation between increased photosynthetic rate in zooxanthellae and high incubation pH (Hoegh-Guld- berg, unpub. data). In the other studies noted above, the pH of the host extract was not reported, but it may have been sufficiently different from seawater controls to ac- count for the observed differences in photosynthetic rates. A second explanation is that hosts' ability to influ- ence photosynthetic rates in their symbiotic partners var- ies, and that the results of this and previous studies reflect that variability. The second way in which hosts may exert control over zooxanthellae is through stimulation of release of photo- synthetic products. Evidence that host tissues contain factors controlling release in invertebrates having zoo- xanthellae has been found in one temperate anemone [.-1. elegantissima (Trench, 1971c)] and in several tropical corals (Muscatine, 1967; Muscatine el al, 1972). Only for P. versipora was strong evidence found for the pres- ence of host release factors in this study. Relative to sea- water controls, host extract of P. versipora stimulated re- lease of a large proportion (up to 42%) of the carbon fixed during photosynthesis in that extract. The rate of release remained constant over four hours of incubation, and the magnitude of release was correlated with the total protein concentration of the extract. It is likely that the constant rate of release reflects the stability of the host extract factor during the incubation period. Unpublished data suggests that removal of zooxanthellae from host extract and subsequent incubation in seawater with 14C causes an immediate reversion to levels of release charac- teristic of those in seawater. Therefore, host factor ap- pears to cause only a temporary (during exposure) effect on release patterns. Preliminary attempts to chemically characterize an ac- tive component from the host extract of P. versipora demonstrated that the release-inducing activity did not pass through dialysis tubing (nominal pore size 10,000 D) and was almost completely destroyed (approximately 80% reduction) by heating. The correlation of the magni- tude of release with protein concentration, constant rate of release of fixed products, sensitivity to heat, and non- dialyzability are consistent with the host extract contain- ing an active chemical constituent, possibly protein- aceous in nature and stimulating release of photosyn- thetic products from zooxanthellae. It is possible that "host factor" activity in this and previous studies is an artifact of the experimental procedure. However, the identification of a chemical having release-inducing ac- tivity in vitro will be a first major step in assessing the role of such chemicals in intact associations. Results of experiments with P. versipora suggested the possibility of a third, and previously unreported, level of control by the host over zooxanthellar processes. Ex- tracts of this host had a marked effect on the metabolism of zooxanthellae, resulting principally in the incorpora- tion of photosynthetically fixed carbon into glycerol and other neutral compounds (predominantly monosaccha- rides). This contrasted with the incorporation of most of the photosynthetically fixed carbon into lipids in cells in- cubated in seawater. This is particularly interesting in view of the observation that only carbon fixed in the presence of host extract was released, while photosyn- ZOOXANTHEl.LAE IN TEMPERATE INVERTEBRATES 185 thetic products formed prior to exposure to extract were retained during incubation in extract. This latter result suggests that pre-formed products have entered path- ways or pools where they are not affected by host ex- tracts. Therefore, it may be that these results demon- strate a degree of host control over metabolic processes in the symbiont, whereby organic carbon, which is nor- mally incorporated into lipids in free-living dinoflagel- lates, is diverted in symbiosis to pathways that result in the formation of products that may be more readily translocated to the host. Glycerol constituted a large proportion of the algal photosynthetic products detected outside zooxanthellae after incubation in P. versipora extracts. It was not possi- ble, using available methods, to demonstrate conclu- sively that the glycerol, or other labeled compounds de- tected extracellularly, was translocated, and not formed in the incubation medium as a result of heterotrophic fixation or the activity of host enzymes on unidentified compounds released from zooxanthellae. However, non- zooxanthellae (heterotrophic) fixation in host extracts of other marine invertebrates is negligible (Trench, 1971b), and glycerol is thought to be the major product translo- cated from zooxanthellae in marine invertebrates (Trench, 1979; Battey and Patton, 1984); up to 90% of the organic carbon translocated in short-term incuba- tions has been reported to be in this form. Other com- pounds translocated include lipids (Patton cl ai. 1977a; Kellog and Patton, 1983; Battey and Patton, 1984) and amino acids (Trench, 197 Ib, c). Glycerol is a lipid-solu- ble substance that moves easily through biological mem- branes (Davson and Danielli, 1952; Dainty and Ginz- burg, 1964; Wright and Diamond, 1969). If the translo- cation of glycerol is not restricted by a membrane barrier, it seems unlikely that host factors act by affecting mem- brane permeability to glycerol at the interface between host and symbiont, as has been suggested (Trench, 1979) in previous studies. An alternative possibility is that host factors operate by influencing glycerol catabolism and anabolism, thereby determining the concentration gradi- ent of glycerol between host and symbiont. Experiments with the other invertebrates used in our study provided no strong evidence for the presence of host factors influencing translocation of photosynthetic products from zooxanthellae. This raises the question of whether temperate invertebrates in general possess host factor activity. C. gaboensis extract inhibited photosyn- thesis and lysed freshly isolated zooxanthellae. This re- sult clearly demonstrates the possibility of artifacts in ex- periments of this nature, and points to the need for as- sessment of algal condition and physiology to detect such artifacts. Extracts prepared from Z. robustus so/A Pa. ian- t/iinu stimulated little release of carbon from their zoo- xanthellae. These latter results are in contrast with the results obtained for P. versipora and with the substantial release detected for Z. robustus and Pa. ianthina in the intact association. One explanation is that the active con- stituent or a required cofactor is destroyed during the iso- lation procedure for Z. robustus and Pa. ianthina. Indi- rect evidence that this may be the case for Z. robustus comes from the observation that zooxanthellae from this host released a significant proportion of photosynthetic products when exposed to P. versipora extract. This ob- servation would also suggest a similarity in host factors in these two invertebrates, a phenomenon previously re- ported for two tropical invertebrates (Muscatine, 1967). A second explanation is that host factors are absent in Z. robustus and Pa. ianthina. 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Determination ot alkaline- phosphatase expression in endodermal cell lineages ot an ascidian embryo .... 222 Byrne, Roger A., Erich Gnaiger, Robert F. McMa- hon, and Thomas H. Dietz Behavioral and metabolic responses to emersion and subsequent reimmersion in die freshwater bivalve, Corbu iiln flwninea 25 1 Deaton, Lewis E. Potentiation ol hvpoosmotic ( ellular volume regula- tion in the cjiiahog, Mi'in'tiinni iin'ru'iniriii, by 5-hy- droxytryptamine, FMRFamide, and phorbol esters 260 Edwards, Samuel C., Anne W. Andrews, George H. Renninger, Eric M. Wiebe, and Barbara-Anne Bat- telle File-lent innervation to Lm/ulm eyes in vivo phos- phoi ylates a 1 22 kl) protein 267 Price, D. A., K. E. Doble, T. D. Lee, S. M. Galli, B. M. Dunn, B. Parten, and D. H. Evans The- sequencing, synthesis, and biological actions of an ANP-like peptide isolated from the brain of the killihsh l-'ii>i,liilii\ heteroditus 279 Sanders, N. K., andj. J. Childress Adaptations to the deep-sea oxygen minimum layer: ox\gen binding by the hemocyanin of the bathype- lagii mysid, Gnathophausia m^i'ii\ Dohrn 286 GENERAL BIOLOGY Bidwell, Joseph P., Alan Kuzirian, Glenn Jones, Lloyd Nadeau, and Lisa Garland The effect of strontium on embryonic calcification ol Aplysia californica Boyd, Heather C., Irving L. Weissman, and Yasu- nori Saito Morphologic and genetic verification that Montn c\ Bt>tr\llit.\ and Woods Hole Bi>lr\llit.\ are the same spe- c ic-s . 231 SHORT REPORTS Clark. Wallis H., Jr., Ashley I. Yudin, John W. Lynn, Fred J. Griffin, and Muralidharan C. Pillai |ell\ la\et formation in pcnaeoidean shrimp eggs .. Margulis, Lynn, Michael Enzien, and Heather I. McKhann Revival ol Dobell's "Chromidia" hypothesis: chro- m.itin bodies in ihe amoebomastigote Paratetramitus /l/i;mH\ Index to Volume 1 78 . 300 305 Reference: Biol. Bull 178: 187-194. (Juno. 1990) Electromyographic Record of Classical Conditioning of Eye Withdrawal in the Crab RICHARD D. FEINMAN, RAFAEL H. LLINAS. CHARLES I. ABRAMSON, AND ROBIN R. FORMAN' Department of Biochemistry, State University of New York Health Science Center at Brooklyn, Brooklyn, New York 11203. ' Department of 'Neurology, Medical College of Virginia, Richmond, I 'irginia 23298, and Marine Biological Laboratory, Woods Hole, Massachusetts 02543 Abstract. Classical (Pavlovian) conditioning of the eye withdrawal reflex of the green crab, Carcinus maenas. was studied by recording electromyograms ( EMGs) from the main abductor muscle of the eye (19a). The EMG record was a reliable indicator of the response, and it was always correlated with physical movement of the eye, whether evoked by the unconditioned stimulus (a puff of air to the eye), or by the conditioned stimulus (a mild vibration of the carapace). The EMG was used to study the acquisition of conditioned responses in animals with an immobilized eye. Six of eight experimental animals developed responses to the conditioned stimulus in a manner similar to that for animals with freely moving eyes; unpaired controls showed few responses. The re- sults indicate that eye movement is not required for learning. Behavioral tests after conditioning and after the eyes had been freed supported this conclusion. The re- sults exclude theories of classical conditioning of eye withdrawal that invoke a role for stimuli due to eye movement (such as a change in visual field). Introduction The eye withdrawal reflex of the crab is one of the sim- ple invertebrate behaviors in which learning can be demonstrated (Abramson and Feinman, 1987; Abram- son et al., 1988; Abramson and Feinman, 1988; Apple- ton and Wilkens, 1990). Classical (Pavlovian) condition- Received 18 January 1990; accepted 22 March 1990. Abbreviations: Electromyogram (EMG); conditioned stimulus (CS); unconditioned stimulus (US); conditioned response (CR); and uncon- ditioned response (UR). ing of the response is brought about by pairing a pre- viously neutral stimulus (vibration of the carapace) with an aversive stimulus (an air-puff" to one of the eyes). The air-puff" [unconditioned stimulus (US)] invariably causes eye retraction. After several pairings of the US with the vibration [conditioned stimulus (CS)], eye retraction be- gins to appear during CS presentations. The responses can be recorded in several ways. In addition to direct ob- servation, movement can be recorded by optical or ca- pacitive methods (Sandeman, 1968; Forman and Brum- bley, 1980; Miall and Hereward, 1988), or by the force generated during retraction (Erber and Sandeman, 1989; Appleton and Wilkens, 1990). Electromyograms (EMGs) are also easily recorded (Burrows and Horridge, 1968) and, in this report, we describe the use of EMGs recorded from the main abductor muscle of the eye (muscle 19a) as an indicator of the response. The method allows us to record responses in the restrained eye, and we use it to show that physical movement of the eye is not required for learning. One of the virtues of this system is that some of the physiology has already been characterized (Burrows, 1967; Sandeman, 1967, 1969b) and, therefore, the neu- ronal substrate of conditioning may be accessible. Sev- eral features of eye withdrawal make it desirable for such an analysis. Retraction is mediated by only two motor neurons, one of which is identified and has a giant axon (Burrows, 1967; Sandeman, 1967, 1969a; Burrows and Horridge, 1968); the activity of this unit is the signal of greatest amplitude in the EMG recorded from muscle 19a. Studies of eye withdrawal have shown that there is no requirement for proprioceptive feedback; whether this is true under conditions where learning occurs is un- known. Although less well characterized, the sensory 187 188 R. D. FEINMAN ET AL. afferents are also known and are believed to make largely monosynaptic contacts with the motor neuron (Sande- man, 1969a, 1969b). The role of eye movement also bears on long-standing problems in the psychology of learning. The eye with- drawal reflex can be trained in a signalled avoidance pro- cedure in which US presentation can be avoided, if the eye is retracted during the CS, which acts as a "warning signal" (Abramson el ai, 1988). Acquisition of condi- tioned eye withdrawal in avoidance followed a time course similar to that for classical conditioning, suggest- ing that animals might not benefit from being able to control the contingencies of reinforcement. In other words, the animal might effectively have been in a classi- cal conditioning experiment in which some USs were omitted. The paradox is that controls that were subjected to the same sequence of USs and omissions did poorly, whereas, if the contingency between eye withdrawal and absence of US were not important, they should have done as well as the experi mentals. A similar result has been observed for vertebrates in some learning proce- dures (Moore and Gormezano, 1961;Gormezano, 1965; Woodward and Bitterman, 1973). One theory that has been proposed to explain these results is that animals are receiving a compound CS composed of the vibration plus the change in sensory input (such as visual field) that occurs as a consequence of the eye movement. The re- sults reported here suggest that the consequences of the eye movement do not play a necessary role in classical conditioning, and therefore, that the theory cannot ex- plain the similarity of classical conditioning and avoid- ance, at least in the crab eye withdrawal reflex. Materials and Methods The general experimental setup for classical condi- tioning has been described (Abramson and Feinman, 1988). The CS was a low amplitude 200 Hz vibration administered to the carapace via a needle attached to a loudspeaker. The US was a low intensity puffof air deliv- ered to the eye to be conditioned. In the experiments de- scribed here, a 1-s presentation of the CS was followed immediately by a 0.1-s presentation of the US. In gen- eral, the eye was re-elevated after the retraction; in cases where this did not occur, the animal was gently tipped or one of the legs was moved to cause the eye to come back up. For recording myograms, a single hole was made, with the tip of a hypodermic needle, in the cuticle sur- rounding the eye, and two 50-^ wires were inserted into muscle 19a and attached to the cuticle with cyanoacry- late glue. Placement of electrodes was confirmed by dis- section of formaldehyde-fixed samples. The insertion of the EMG electrodes had a sensitizing effect, and animals would respond to a level of vibration that was normally without effect. Thirty minutes after implanting the elec- trodes, this sensitivity was sufficiently reduced so that there was no response to three or four successive stimuli. Scoring of conditioned responses in myographic records of animals with restrained eyes was done blind; a naive observer was instructed to score EMG patterns during the CS that resembled those seen during the US. Results Electromyographic measurement ofacqitisitiim The first experiment demonstrated the feasibility of using the EMG record to follow conditioning. Four ex- perimental animals and four controls had EMG elec- trodes implanted in muscle 19a of one eye; the eye moved freely after this manipulation. The experimentals were subjected to 50 paired presentations of stimuli as described in Materials and Methods; controls were given 50 presentations of unpaired stimuli. Panel A of Figure 1 shows EMG records of several trials for one of the ex- perimental animals. The characteristic spiking pattern due to the activity of the fast retractor motor neuron of the optic nerve is reliably seen in response to presenta- tion of the US. Slow tonic activity is also seen in some traces in panel A. These are due to the activity of a smaller neuron of the oculomotor nerve; the tonic firing of this unit correlates with the eye being held down (San- deman, 1964; Burrows, 1967; Burrows and Horridge, 1968). Muscle 19a is more sparsely innervated by this neuron than by the larger retractor neuron, and the tonic activity is not seen in every preparation. After several tri- als, a pattern of spiking activity similar to that caused by the US is now evoked during the CS. This pattern in the CS or US was always correlated with observed retraction of the eye. Two features of the EMG record were not obvious from simple observation of the gross behavior. First, as is evident in Figure 1, the conditioned responses (CRs), when they appear, are frequently more robust than the unconditioned response (UR). In addition, although not a feature of all sessions, the UR frequently showed habit- uation even as the CR developed (data not shown). This phenomenon has been studied more thoroughly by Ap- pleton and Wilkens (1990). The pattern of acquisition seen in the present work is qualitatively similar to the acquisition of CRs as previously described (Abramson el ai, 1988; Abramson and Feinman, 1988). There were few, if any, spontaneous eye retractions (or bursts of pha- sic activity in the EMG record) during the intervals be- tween stimuli presentation. To assess the effect of the insertion of electrodes, the behavior was compared to that of a second group of four experimental and four unpaired control animals that had never had EMG wires implanted. Responses of the experimentals and the controls were tallied and the aver- CLASSICAL CONDITIONING IN THE CRAB 189 cs us TRIAL 1 TRIAL 2 TRIAL 14 TRIAL 17 «1* TRIAL 26 CS US B cs us TRIAL 1 TRIAL 7 TRIAL 9 TRIAL 13 TRIAL 25 CS US Figure 1. Electromyographic record of classical conditioning. EMGs were recorded from muscle 19a of the eye to be conditioned. A. Results for a typical animal with a freely moving eye. B. Results while the eye is physically restrained. Large amplitude spikes are due to activity of the fast phasic motor neuron of the optic nerve. Slow tonic activity evident in traces in panel A are due to a neuron of the oculomotor nerve which more sparsely innervates 19a and whose activity correlates with maintenance of the retracted state. The CS duration is 1 s. The vertical bar corresponds to 200 ,uV except in TRIAL 1 of panel B where it represents 100 ^V. Animals were trained with paired presentation ofCSand US (top and bottom traces). Animals in panel B had the eye temporarily immobilized with a rubber band. age responses for each five-trial block were plotted (first panel of Fig. 2). The behavior of the two sets of animals, with and without EMG wires, is manifestly similar: the paired animals of each group showed an increase in the probability to respond reaching a plateau probability of 50-60%, whereas the corresponding unpaired groups showed a much lower tendency to respond (see below for statistical comparison). Thus, learning is fundamentally the same in animals with and without EMG electrodes; for qualitative comparisons to animals with restrained eyes, these two groups were pooled and considered as a population of eight animals trained with freely moving eyes. However, there were some differences. First, Figure 2 shows that the EMG animals were sensitized, as indi- cated by their higher probability to respond at the outset of training (first 5-trial block). The mean probability of response for EMG animals in this period was 0.35 (SD 0.25) compared to 0.05 (SD 0.10) for unoperated ani- mals. A second difference is the somewhat greater vari- ability in the EMG animals. To see this difference we plotted individual animal data as a cumulative record, or running total, in Figure 3 (panels A and B). Usually 190 R D. FEINMAN ET AL. 10 12 14 16 18 20 22 24 26 28 30 32 NUMBER OF 5-TRIAL BLOCKS Figure 2. Effect of implanting EMG electrodes during acquisition on behavioral performance. Group data for behavior in ACQUISI- TION, RETENTION, and RE-ACQUISITION of classical condition- ing. Data points are averages of four animals each. Filled symbols: ani- mals receiving paired stimuli during ACQUISITION. Open symbols: animals receiving specifically unpaired stimuli during ACQUISITION. Two populations were used. Triangles: normal unoperated animals; Circles: animals with EMG wires implanted. In RE-ACQUISITION, dotted line is first day performance of the average of the (8) experimen- tal animals and is included for comparison. Probability of response is calculated as the total number of responses per animal per five-trial block. applied to operant conditioning experiments, a cumula- tive record is a good method for looking at trial-by-trial data. It is evident that, again, the groups are very similar, but inserting the EMG wires introduces variability in the pattern of response. In summary, the EMG record is a reliable method for following conditioning — the large differences between paired and unpaired groups are maintained — but the process of inserting electrodes may have a somewhat sensitizing effect on the CS responses. Pattern of behavior after conditioning As a second method of assessing the effect of training, we recorded a profile of behavioral responses after condi- tioning. For animals with EMG leads, wires were cut. All animals were returned to the home tank and then all (paired and unpaired controls) were tested for responses in three behavioral procedures. First, after 4 h, animals were given 50 CS-only presentations (second panel of Fig. 2 ). Then, after an additional 20 h, they were re-tested for responses to 10 CS-presentations (third panel of Fig. 2). Immediately after these 10 CS-only trials, animals were subjected to a second training session (last panel). During this second training period, the unpaired controls from the first day were given paired presentation of stim- uli to determine whether this population was. in fact, ca- pable of learning and whether there was an effect of the previous day's experience as controls. It is evident trom Figure 2 that: the paired group showed substantial reten- tion after 4 h as measured by the CS-only responses, and that extinction is fairly rapid; unpaired controls showed few CRs; and in both cases there was a considerable vari- ation among animals. There is also a rebound of the ex- perimentals' response to the conditioned stimulus after 24 h; the unpaired group, again, showed few responses. The last panel in Figure 2 indicates an enhanced re-ac- quisition of the task by the subjects that had been experi- mentals on the first day; this is consistent with earlier re- V) LU w o O. V) til cc 5 3 o 40 30 20 10 40 30 20 10 10 20 30 40 50 10 20 30 40 50 10 20 30 40 50 TRIAL NUMBER Figure 3. Cumulative record for acquisition of conditioned re- sponses. Results are shown for all experimental (paired CS. US) sub- jects. A. Normal subjects, unoperated. B. Freely moving eyes with EMG electrodes implanted. C. Animals with EMG leads and condi- tioned eye immobilized. In B and C, the dotted line represents the aver- age of the records for the four animals in A. Data were smoothed, for graphic clarity, by averaging over three trials at a time. CLASSICAL CONDITIONING IN THE CRAB 191 ports (Ahramson and Feinman, 1988). Likewise, con- trols from day-one now showed a high probability to respond, indicating that there was nothing unusual about this group and that their performance was not re- pressed by their previous experience as unpaired con- trols, again consistent with original observations. Figure 2 shows that this day-two acquisition by controls has a very similar time dependence to the day-one acquisition by experimentals (dotted line), indicating that the con- trols were also not sensitized and had not fortuitously made a CS-US association. This general pattern of re- sponses was similar for both groups: normal animals and those with EMG electrodes. Electromyographic record of conditioning of a restrained eye With the behavioral pattern of acquisition, retention, and re-acquisition as background, we next prepared 16 new animals with silver wire electrodes in the eye and now restrained one eye (to be conditioned) with rubber bands. Eight of these animals were subjected to the paired presentation of stimuli as above, while the other eight served as controls and were given specifically un- paired CS, US presentations. Conditioned responses were scored from the EMG record. Activity during CS presentations that resembled those during the US were considered conditioned responses. Figure IB shows characteristic EMG patterns typical of these animals. Six of the eight experimental animals showed development of a conditioned EMG response in a manner similar to the groups with freely moving eyes. None of the unpaired controls showed the normal acquisition, although one animal gave several responses during the first few trials, presumably due to the sensitizing effect of the manipula- tions. Panel C of Figure 3 shows the cumulative records for the eight animals in the experimental paired group. Some animals showed behavior clearly similar to that of animals whose eyes were not restrained (panels A and B), and some are actually sensitized compared to normals. Two animals made few responses, and one initially showed good acquisition but stopped responding at trial 32. Thus, six of the eight animals showed a pattern of responding similar to animals with freely moving eyes for more than 60% of the training session. Figure 5 shows that these six animals also gave more total responses that any unpaired animal in the experiment. Using these arbi- trary criteria, we would say that six of the subjects were conditioned. There is also greater variability of individ- ual animals with restrained eyes (first panel of Fig. 5). The two experimental animals that did not learn (see above; Fig. 3 A, B) did show small bursts of phasic activ- ity during the CS presentations. In animals with freely 0246 10 12 14 16 18 20 22 24 26 28 30 32 NUMBER OF 5-THIAL BLOCKS Figure 4. Effect on behavioral performance of immobilizing the eye during acquisition. Group data for animals trained with immobilized eyes compared to animals with freely moving eyes. Data points are av- erages of eight animals each. Filled symbols: experimentals; open sym- bols: unpaired controls. Broken line: average of corresponding data from Figure 2 [data from 4 normal and 4 freely moving eye with elec- trodes were pooled and averaged for each of the two groups (paired and unpaired)]. Data points in ACQUISITION are EMG responses: other data, retention and re-acquisition of classical conditioning, are re- corded behaviorally. In RE-ACQUISITION, dotted line is first day per- formance of the average of experimental animals redrawn for compan- moving eyes, these would correlate with small twitches of the eye, but are not normally scored as full responses. This suggests that even the animals that did not meet the criterion of EMG responses that resembled those to the US may have acquired some association from the train- ing. This idea was strengthened by their subsequent per- formance in the behavioral tests described below. Behavioral tests after acquisition After the acquisition trials, the eyes were freed, the EMG leads were cut, and the animals were returned to their home tanks. They were then tested, as were animals trained with freely moving eyes, for responses in the be- havioral tests: retention after 4 h and after 24 h, and re- acquisition in a second training session. The results are shown in Figure 4, where they are compared to the aver- aged data for the two groups trained with freely moving eyes. When the qualitative behavior of the animals trained with restrained eyes is compared for retention and reacquisition to that for animals with freely moving eyes (Figs. 2, 4), similar profiles are found, although, as noted above, the response to CS-only presentations var- ies substantially. During re-acquisition, behavior of the animals trained with restrained eyes is remarkably like that for animals with moving eyes: all experimentals show enhanced probability of responding, and all con- trols now subjected to paired training behaved like day- one experimentals. This behavioral performance of the 192 R. D. FEINMAN ET AL experimentals suggests that learning took place during day-one acquisition even in the case of the two animals where an EMG response was not evident. Summary of statistical analysis The major conclusions bearing on acquisition were that the groups presented with paired stimuli showed an increased probability to respond to the CS when com- pared to controls, and that the effect of EMG electrodes was somewhat sensitizing in terms of individual perfor- mance, although there were no differences over the course of the training. These conclusions are supported by an analysis of variance conducted over the 10 five- trial blocks of acquisition. For normal unoperated ani- mals, differences between paired and unpaired groups was significant, F( 1 ,60) = 265.08, P < 0.000 1 , as was the Block effect, F(9,60) = 5.67, P < 0.0001, and the Group X Block interaction. F(9,60) = 5A5,P< 0.000 1 . For ani- mals with electrodes and freely moving eyes, there was a significant Group effect F(l,60) = 125.21, P < 0.0001, no significant Block effect F(9,60) = 1 .52, P > 0.25, and no significant Group X Block interaction F(9,60) = 0.691, P > 0.25. For animals with electrodes and the eye restrained, there was a significant Group effect F(l.lOO) = 24.09, P < 0.0001, no Block effect F(9,100) = 1.42, P > 0.10 and no Group x Block interaction, F(9,100)= 1.39, P> 0.10. With regard to sensitization, the effect was limited to the initial trials. As training continued, the group differ- ences between animals with electrodes and those without was not significant. As noted above, paired animals with electrodes responded more to the CS at the outset of training than those without electrodes. A somewhat sim- ilar trend was observed for unpaired animals: unpaired animals with electrodes and the eye restrained made more responses during the first five CS presentations (mean probability 0.50, SD 0.35) than either the un- paired animals with electrodes and eye freely moving (mean 0.10, SD 0.12) or unoperated unpaired animals (mean .05, SD 0.1). Overall, however, analysis of vari- ance conducted over the 10 five-trial blocks of acquisi- tion revealed no group differences between animals with electrodes and those without: a comparison of animals with electrodes versus unoperated animals reveal no Group effect F(l,60) = 0.631, P > 0.25, a significant Block effect F(9,60) = 4.15, P < 0.005, and no Group X Block interaction F(9,60) = 1.63, P > 0.10. Also, no significant Group, Trial, or Interaction effects (P> 0.10) were obtained for animals with electrodes and freely moving eyes versus those with electrodes and the eye re- strained. An overall analysis of variance conducted over the 10 five-trial blocks for the three unpaired groups indi- cated no Group effect F(2.90) = 1 .37, P > 0.25, no Block effect F(9,90) = 1.45, P > 0.05, but a significant Group X Block interaction F( 1 8.90) = 2. 19, P < 0.0 1 ). The sig- nificant interaction reflects the fact that two of the four animals in the unpaired group with electrodes and re- strained eyes responded substantially during the first five CS presentations and that such responding decreased over the course of further unpaired training. The major conclusion about the behavior of animals that had been trained with eyes restrained is that the per- formance in reacquisition is similar to the groups with freely moving eyes. Also, the unpaired controls with re- strained eyes were capable of learning as shown in reac- quisition, were not repressed due to unpaired pre-expo- sure, and had not fortuitously made a CS-US associa- tion. The acquisition performance of all paired groups was enhanced during reacquisition. For paired animals without electrodes, analysis of variance indicated sig- nificant Group effect F(l,60) = 17.31, P < 0.0001, a Block effect F(9,60) = 6.42, P < 0.0001, and a Group X Block interaction F(9,60) = 2.73, P < 0.01. Analysis of the reacquisition performance of paired animals with electrodes and the eye free to move revealed a significant Group effect F( 1,60) = 12.64, P < 0.005. no Block effect F(9,60) = 0.676, P > 0.25, and no Group X Block inter- action F(9,60) = .676, P> .25. A significant Group effect was also obtained in paired animals with electrodes and the eye restrained F(l,60) = 96.50, P < 0.0001. There was a Block effect F(9,60) = 2.02, P < 0.05, but no sig- nificant Group X Block interaction F(9,60) = 0.546, P >0.25. As Figures 2, 4, and 5 suggest, the performance of un- paired animals was greatly enhanced when they received paired training. Analysis of variance of unpaired animals with no electrodes revealed a significant Group effect F( 1 ,60) = 79.34, P < 0.000 1 , but no Block effect F(9,60) = 1.02, P > 0.25, or Group X Block interaction F(9.60) = 1.07, P> 0.25. Analysis of unpaired animals with elec- trodes and the eye free to move indicated a significant Group effect F(l,60) = 98.97, P < 0.0001, Block effect F(9,60) = 3.75. P < 0.005, and Group X Block interac- tion F(9,60) = 2.66, P < 0.025. Unpaired subjects with electrodes and the eye restrained (the eye was free to move during the reacquisition phase) also had a signifi- cant Group effect F( 1 ,60) = 93.28, P < 0.000 1 , no Block effect F(9,60) = 0.709, P > 0.25, but a significant Group X Block interaction F(9,60) = P< 0.0001. Discussion The major goal in this work was to determine the role of eye movement in classical conditioning of the with- drawal reflex. We wanted to determine, first, if eye move- ment is necessary for classical conditioning of the eye withdrawal; that is, whether any animals are capable of CLASSICAL CONDITIONING IN THE CRAB 193 50 2 in 20 ACQUISITION ° j • 8 • n • 8 * n 9 n | « O • D n • n D • o , 0 • 9 * B • (RE-')ACOUISmON PR UNP UNP Figure 5. Total responses in acquisition and re-acquisition (50 tri- als). Data for animals with freely moving eyes are shown with open symbols and includes two subgroups: normal animals (circles) and ani- mals with EMG electrodes (squares). Each experimental population, paired group (PR) and unpaired (UNP). includes 4 normals and 4 EMGs. although some data points overlap. Data for animals with re- strained eyes during acquisition are shown as filled circles. There were eight animals in the PR groups and 8 in the UNP group, although some points overlap. Note that the PR group in ACQUISITION is given a second day of training in RE-ACQUISITION, but the group labeled UNP was a control only on the first day. In RE-ACQUISITION it was now presented with paired stimuli as described in the text. learning when the eye is restrained. Our results show clearly that some animals can, in fact, learn with immo- bilized eyes. Having established that eye movement is not necessary, we next asked whether any animals ever used signals that arise from eye movement. Two of the eight animals with restrained eyes showed no normal ac- quisition of the EMG response. Was this because of ex- perimental error, such as damage to the eye from im- planting electrodes? Or did these two particular animals normally use a strategy of acquisition that required eye movement accounting for their poor performance when the eye was restrained? Although we cannot rule out an effect of eye movement on these animals, there is evi- dence that some learning took place even for these ani- mals. When tested behaviorally, all animals performed like normals, particularly in re-acquisition. Also, small EMG bursts were evoked by CSs (but did not appear in the interval between stimuli). Thus, damage to the mus- cle might have non-specifically reduced the appearance of conditioned responses. Moreover, the eye withdrawal reflex, under normal conditions, proceeds without pro- prioceptive feedback (Burrows, 1967; Sandeman, 1967), although we do not know whether feedback can affect learning. In the optokinetic response, furthermore, mo- tor output to the eye is driven by the difference in veloc- ity of the eye and of the target (Horridge and Sandeman, 1964; Sandeman el ai. 1975; Erber and Sandeman, 1989). This motor output is normally overridden by the eye withdrawal (Burrows and Horridge, 1968), but we cannot exclude the possibility that such a signal could be used by some crabs as part of a strategy in classical conditioning. Comparison of classical conditioning and signalled avoidance Our work supports the idea that classical conditioning of the eye withdrawal is simply dependent on the integra- tion of the two sensory stimuli (vibration and air-puff). The results also bear on the study of this reflex in sig- nalled avoidance, and the apparently paradoxical behav- ior of controls in that procedure. The problem may be summarized as follows. Signalled avoidance is designed as an operant procedure. If the animal makes a response during the presentation of the (signal) CS, the US is omit- ted, that is, the animal can avoid the aversive stimulus by its own behavior. However, animals that learn in an avoidance procedure may be undergoing a predomi- nantly Pavlovian process; the procedure is identical to a classical conditioning experiment in which some USs have been omitted. Thus, the animal may learn during the CS-US pairings, but may not associate the occasional omission with its own behavior. In our study of signalled avoidance in the eye withdrawal in the crab, we found that animals learned well, but acquisition curves were es- sentially the same as those for animals in classical condi- tioning (Abramson et a/.. 1988), suggesting a Pavlovian interpretation. The apparent paradox is that "yoked" controls presented with the same pattern of CSs and USs (some of which are now omitted) as the experimental an- imals, but independent of the responses they made, did not perform as well. Since these controls receive the same number of CSs and USs as the experimentals — the only difference is the contingency between stimuli and the an- imal's behavior — they should do as well (if the mecha- nism is truly Pavlovian ). One of the following hypotheses could explain these seemingly paradoxical results. One possibility is that learning in both the avoidance procedure and "classical conditioning" procedure is ac- tually the same and substantially operant; reinforcement is provided by attenuation of the air-puff when the eye is retracted during the CR. If this is true, the yoke controls are behaving as per experimental design. The results pre- sented here exclude this mechanism because animals can learn when their eye is retracted and there is no attenua- tion of the air-puff. A second hypothesis is that avoidance learning is actu- ally Pavlovian in mechanism; the yoke controls are giv- ing erroneous data due to one of several possibilities. First, it is possible that yoke controls are actually sub- jected to different stimuli than experimentals as de- scribed by Woodward and Bitterman (1973). According 194 R. D. FEINMAN ET AL. to this theory, there are actually two CSs: CS+ and CS-. These are compound stimuli composed of the vibration and some sensory information about whether the eye is up or down (for example, visual field). For experimen- tals, CS+ is (the state of eye-up) + vibration, which is predictably followed by the US; CS- is (eye-down) + vi- bration, predictably followed by an omission; these are randomized when vibration is presented to yoke con- trols. We have excluded this theory for classical condi- tioning and. therefore, for a Pavlovian interpretation of avoidance, by showing that animals can be conditioned with the eye restrained. We favor an alternative explanation: that both classi- cal conditioning and avoidance are Pavlovian in mecha- nism, but that the process involves two conditioned states, one of which has a higher probability of response than the other and is more resistant to extinction. Such a mechanism resembles the Markov chain model for conditioning (Theios and Brelsford. 1966). Experimen- tal animals in avoidance, then, receive omissions at times when they are most resistant to extinction (high proba- bility state), whereas for yokes, omissions are random- ized. A similar explanation for experimental-yoke differences was proposed by Gormezano ( 1965) for the rabbit nictitating membrane. Thus, the current work on classical conditioning al- lows us to exclude two of the possible explanations for signalled avoidance learning in the crab eye. We cannot, however, exclude the possibility that the mechanism of learning is actually different for the two procedures. Pos- sibly the rates of acquisition for avoidance are the same as in classical conditioning because they share a common rate-determining step, probably at the output end of the behavior. For example, there may be a maximum rate of change in properties of the motor neuron. If this were so, the yoke controls would be performing as expected. At this point, we favor the Pavlovian interpretation. From the biological point of view, an all-or-none defensive re- flex, such as eye withdrawal, probably does not require the subtle information about the effects of the behavior that an operant mechanism would impart. In summary, EMGs recorded from muscle 19a of the eye can be used to study the acquisition of classical con- ditioning in animals with freely moving and immobi- lized eyes. Experiments using this method show that eye movement is not required for learning. Acknowledgments This work was supported in part by grant BNS- 8819830 from the National Science Foundation, funds from Scott, Sperry & Hanson, Inc., and funds from the Research Foundation of the State University of New York. Literature Cited Abramson, C. I., P. M. Armstrong, R. A. Feinman, and R. D. Feinman. 1988. Signalled avoidance in the eye withdrawal reflex of the green crab. J. Exp Anal. Be/niv 50: 483-492. Abramson, C. I., and R. D. Feinman. 1987. Operant punishment in the green crab, C 'arcinus maciuis Behav. Neural Biol. 48: 259-277. Abramson, C. I., and R. D. Feinman. 1988. Classical conditioning of the eye withdrawal reflex in the green crab. J. Nt'iirosci 8: 2907- 2912. Appleton, T., and .1. L. VVilkens. 1990. Habitation and sensiti/ation and the effect of serotonin on the eyestalk withdrawal reflex of ('. Biol. 138: 541-544. Moore,.). \V.,and I. Gormezano. 1961. Yoked comparisons of instru- mental and classical eyelid conditioning. J Exp. Psych. 62: 552- 559. Sandeman, D. C. 1964. Functional distinction between oculomotor and optic nerve in Carcinus. Science 201 : 302-303. Sandeman, D. C. 1967. Excitation and inhibition of the reflex with- drawal of the crab Carcinus. J. Exp. Biol 46: 475-485. Sandeman, I). C. 1968. A sensitive position measuring device for bio- logical systems. Comp. Biocht-m Physio/. 24: 635-638. Sandeman, D. C. 1969a. Integrative properties of a reflex motoneu- ron in the brain of the crab Carcimts mat-nas. Z IVnj/ Physinl 64: 290-464. Sandeman, D. C. I969b. The synaptic link between the sensory and motoneurones in the eye-withdrawal reflex of the crab. J Exp. Biol. 50: 87-98. Sandeman, D. C, J. Erber, and J. Kien. 1975. Optokinetic eye move- ments in the crab, ( 'arcimtx nuicnas. I. Eye torque. / Comp Phys- 10! 101:259-274. Theios, J., and J. \V. Brelsford Jr. 1966. A Markov model for classi- cal conditioning: applications to eye-blink conditioning in rabbits. Psych Rc\- 73: 393-405. \\oodward, \V., and M. E. Bitterman. 1973. Pavlovian analysis of avoidance conditioning in the goldfish (Caraxsius auraliis). ./ Comp Physiol. Psychol. 82: 123-129. Reference: Bio! Bull 178: 195-204. (June, 1990) Behavioral Responses of Crustacean Larvae to Rates of Temperature Change RICHARD B. FORWARD JR. Duke University Marine Laboratory. Beaufort, North Carolina 28516 and Zoology Department, Duke University, Durham, North Carolina 27706 Abstract. The ontogeny of behavioral responses of lar- vae of the crabs Rhithropanopeus harrisii and Neopa- nope sayi to rates of change in temperature were ana- lyzed using a video system. A temperature decrease evoked an ascent in both species. The threshold rates of decrease for Stages I and IV zoeae of R. harrisii, and Stage I zoeae of TV. sayi, were 0.06, 0.1, and 0.09°C min"1, respectively. Stage IV zoeae of N. sayi were unre- sponsive to any rate of decrease. Larvae descended upon a temperature increase. For Stages I and IV zoeae of R. harrisii and Stage I of N. sayi the threshold rates of tem- perature increase were 0.07, 0.24, and 0.1 8°C min ', re- spectively. Stage IV zoeae of N. sayi were again unre- sponsive. In general, there was an ontogenetic change in responsiveness as Stage IV zoeae of both species were less sensitive than Stage I zoeae. The average absolute amounts of temperature change needed to evoke a re- sponse was independent of the rate of change at rates above threshold and ranged from 0.29 to 0.49°C for both species. A consideration of larval sinking rates and ascent speeds, as well as normal environmental temperature gradients, shows that larvae of both species can respond to the rates and amounts of temperature change found in their environments. These responses constitute a neg- ative feedback system that could be used to regulate depth relative to temperature. Introduction Temperature change produces measurable alterations in the directional responses to light (phototaxis) and gravity (geotaxis), and the activity of crustacean larvae (for general reviews see Thorson, 1964; Forward, 1976; Sulkin. 1984). In phototactic studies, only narrow beams Received 27 November 1989; accepted 1 March 1990. of light have been used as a stimulus source, rather than a light field that simulates the underwater angular light distribution. For Callinectes sapidus, larval phototaxis was not affected by temperature changes of 10°C (Sulkin and Van Heukelem, 1982). The only clear effect on Rhi- thropanopeus harrisii larvae was a slight increase in nega- tive phototaxis by Stage I zoeae upon a temperature in- crease (Ott and Forward, 1976). Reductions in tempera- ture within the range encountered by larvae did not alter phototaxis in any zoeal stage of R. harrisii. Nevertheless, there was a pronounced positive geotaxis by Stage IV zo- eae of/?, harrisii at high temperatures (30 and 35°C) and a sinking response by Stage I zoeae (Ott and Forward, 1976). Similarly, C. sapidus descended by passive sink- ing upon exposure to temperatures of 27.5°C or greater (McConnaughey and Sulkin, 1984). Activity, as measured by linear swimming speed, has the pattern of an increase in speed with an increase in temperature (Sulkin et ai, 1980; Kelley et a!.. 1982) up to a certain high temperature where inactivity (sinking) occurs (Welsh, 1932; Yule, 1984). In contrast, the swim- ming speed of Stage I zoeae of C. sapidus was not modi- fied by a temperature decrease (Sulkin et a/.. 1980). Several studies suggested that responses to high tem- peratures did not result from sensitivity to a rate of tem- perature increase but rather to an absolute upper temper- ature (Ott and Forward, 1976; McConnaughey and Sul- kin, 1984). Although the upper temperature may vary with species, this generalization was substantiated by measurements of behavioral responses in sharp thermo- clines. If the upper temperature in the thermocline was above this limit, then larvae ascent stopped at the ther- mocline. Alternatively, larvae ascended through the thermocline if the upper temperature was below the ab- solute upper limit. Remarkably, 10°C thermoclines had 195 196 R. B. FORWARD JR. no inhibitory effect on an ascent, which has led to the conclusion that, for many species, temperature gradients in nature will not prevent upward movements (Kelley et ai, 1982; Sulkin et a/.. 1983; McConnaughey and Sul- kin, 1984). Considering these past studies, larval crustaceans seem relatively unresponsive to temperature changes. Never- theless, the behavioral responses that do occur upon changes in temperature can be summarized. A tempera- ture increase to temperatures at and above the absolute upper limit evokes negative phototaxis, positive geotaxis, and sinking, all of which lead to downward movement. An ascent does not occur upon a reduction in tempera- tures. Activity decreases with decreasing temperature and at extremely low temperatures larvae are totally in- active (e.g., Ott and Forward, 1976). A limitation of past studies is that larval behavior was studied at very sharp thermoclines and upon exposure to step changes in temperature. Sharp thermoclines can exist in nature, but most often larvae encounter a rate of change in temperature that depends upon the vertical gradient and rates of vertical movement. The present study was undertaken ( 1 ) to determine the lowest rates (threshold) of temperature change that evoke ascent and descent responses, (2) to measure the absolute amount of temperature change that must occur before larvae re- spond, and (3) to compare these rates and absolute amounts to those a larva could encounter in the water column. The study compares larvae of the crabs Rliithro- panopem harrisii and Neopanope stiyi (family Xanthi- dae). These were selected because both live as adults in estuaries, but the behavior of/?, harrisii larvae results in retention in upper estuarine areas (Cronin, 1982), whereas N. sayi larvae undergo development in lower es- tuarine and coastal areas (Sandifer, 1 975; Dittel and Epi- fanio, 1982; Salmon eta/., 1986). Thus the larval species are taxonomically related, but they develop in different areas where they are potentially exposed to different tem- perature regimes. Materials and Methods Ovigerous specimens of Rhithropanopeus harrisii (Gould) were collected from the Neuse River estuary (North Carolina) from July to August 1989. Crabs were placed in 20 ppt seawater, which was passed through a 5- fim filter. Ovigerous Neopanope sayi (Smith) were col- lected from the Newport River estuary (North Carolina) from August to September 1989, and females were held in 32 ppt seawater, which was the approximate salinity at the collection site. Larvae of both species were reared at the same salinity in which the crabs were maintained at a temperature of 25°C. This acclimation temperature was chosen because it approximates the average summer D Figure 1. Horizontal view of test chamber consisting of equal size cylindrical upper (1). test (2). and lower (3) sections (not drawn to scale). A — insulated input/output Tygon tubes connected to peristaltic pump; B — stirring paddle connected to a variable speed stirring motor, C — 75 n mesh plankton netting; D — O-nng; E — thermister probe con- nected to meter with digital readout; F — square water-tilled chamber surrounding the test chamber; G — magnetic stirring bar; H — magnetic stirrer; I — video camera; J — far-red illumination light; K — thermal in- sulation. The video camera and thermisters were oriented perpendicu- lar to each other in the actual chamber. temperatures where the larvae undergo development (Stefansson and Atkinson, 1967; Kirby-Smith and Bar- ber, 1979). Specimens were reared in a controlled environmental chamber (Sherer, Model CEL4-4) on a 14:10 LD cycle. Throughout development, larvae were transferred daily to clean seawater and fed newly hatched Anemia spp. nauplii. Experiments were conducted with Stages I and IV zoeae, to test for an ontogenetic change in responsive- ness because each species has four zoeal stages. All exper- iments were performed in mid photophase to avoid com- plications due to biological rhythms in behavior. Larvae were light-adapted to room fluorescent light (intensity = about 1 W irT2) prior to all experiments. In most cases a minimum of five groups of larvae, each from a separate female, were tested in each experimental situation. Experimental approach Larval responses to different rates of temperature change were measured in a chamber having three vertical cylindrical sections (section height = 2.5 cm; diameter = 2.5 cm; Fig. 1 ). For temperature increase, high temper- ature water (above 25°C) was added to the upper section LARVAL RESPONSES TO TEMPERATURE 197 and mixed by a slowly rotating paddle. Larvae were con- fined to the middle section by plankton netting (75 n mesh) at the upper and lower boundaries; their behavior was monitored and recorded with a closed circuit televi- sion system. For viewing, animals were illuminated with far-red light (maximum transmission 775 nm). to which larvae are not responsive (Forward and Cronin, 1979). The lower section was used for temperature decreases. Low temperature water (below 25°C) was added and mixed with a magnetic stirring bar. Preliminary mea- surements of larval swimming indicated that slow stir- ring in the upper and lower sections had no apparent effect on movement. Test water was initially the same water as that used for rearing larvae. This water was pumped through a coil of Tygon tubing situated in a separate water bath (Forma Scientific, Model 2095) and then into the test chamber. The section of tube from the bath to the chamber was insulated with a foam wrap. To insure that there were constant amounts of water in all chamber sections and constant flow through the center section, the waters of different temperature were delivered to the appropriate end section by a variable speed peristaltic pump ( Buchler Instruments), and water was extracted at the same rate from the other end section by the same pump. For exam- ple, to induce a temperature decrease, low temperature water was pumped into the lower section while water was removed from the upper section at the same rate. Dye studies indicated laminar flow of water through the net- ting into the center section. Also, the maintenance of constant water levels in all chambers prevented hydro- static pressure changes during experimentation. This procedure was important because larvae of both species are very sensitive to pressure changes ( Forward and Wel- lins, 1989; Forward el al, 1989). The rates of temperature change were varied through differences in temperature between the input and accli- mation temperature water and pumping rate. In most experiments the temperature difference remained con- stant and pumping rate was varied. The actual tempera- tures in the upper and lower subsections of the larval sec- tion of the test chamber were measured with two thermister probes (YSI; Model 423; Time constant 1 .45 s) connected to separate digital meters (Omega Engineer- ing, Inc.; Model 450-ATH; accuracy 0. 1°C). The digital readouts from the probes were viewed by a second video camera and inserted in the video picture with a video screen splitter (Vision Industries, Inc.; Model U2705P). A record of time was also inserted into the picture by a Field/Frame Counter (QSI Systems, Inc.). In this way larval behavior, temperature, and time were recorded si- multaneously on videotape. The actual rates of change in temperature were calculated from temperature mea- surements by the probe closest to the chamber section (upper or lower) where test seawater was added. Mea- surements by the lower probe were used for temperature decreases and upper probe for temperature increases. Specific rates of temperature change were determined di- rectly from the experimental records. In each experiment the rate of temperature change quickly increased up to the maximum for each flow rate or temperature differ- ence condition and then remained approximately uni- form through the time when responses were measured. Experimental procedures The same general procedure tested for responses to temperature increases and decreases. Larvae were held in the rearing water in finger bowls (10.3 cm diameter) situated in a separate water bath that was maintained at the acclimation temperature (25°C). The room tempera- ture was also kept at about 25°C. A group of approxi- mately 75 Stage I or 25 Stage IV zoeae was placed in the test chamber in water from the maintenance finger bowl. Thus, the initial temperature in the test chamber was very close to 25°C. The peristaltic pump and videotape recorder were started after 1 min in darkness. Tempera- ture changed at a specific rate and was first detected about 3 min after the pump was activated. The experi- ment continued until the temperature changed about 1.0°C. Larvae were then removed, the chamber rinsed with water at the acclimation temperature, and a new group of larvae placed in the chamber. The procedure was repeated. Larvae were only tested once at each rate of temperature change. If larvae were retested at a second rate on any particular day, the minimum time between testing was about 2.5 h. Larvae remained at 25°C in the water bath between tests, and there was no obvious change in behavior with multiple tests. To establish that the observed responses were not induced by water flow through the chamber, larvae were tested using the forego- ing procedure at the maximum test flow rate with accli- mation temperature water. In this way larvae experi- enced flow but no temperature change. This control also tested for changes in larval distributions over time due to random activity. Analysis All experiments were conducted with the test chamber illuminated only with far-red light. Because they were functionally in darkness in this situation, the possible be- havioral responses to changes in temperature were changes in activity or geotaxis. To analyze for behavioral responses, the test (larval) section of the test chamber was divided into three equal horizontal subsections by a template placed over the video screen. The number of larvae in each subsection was counted before (control) and after each 0. 1"C 198 R. B. FORWARD JR. A. Rhithropanopeus harr/sii B Rhithropanopeus * harnsii bottom -4- I. • toP -J---4- — i — — i — — r D Neopanope sayi " top bottom 01 03 05 07 09 Temperature Decrease (°C) 01 03 05 07 Temperature Increase (°C) 09 Figure 2. Percentage of Stage I zoeae of Rhithropanopeus harrisii(&, B)and Neopanope sayi (C, D)in the top (dashed line) and bottom (solid line) subsection of the larval section of the test chamber. Responses were measured after different absolute amounts of temperature change upon temperature decreases (A, C) and increases (B, D). These absolute temperatures are those in the bottom I A, C) and top(B. D) subsections of the test chamber. The rates of temperature change were 0.28 (A), 0.19 (B). 0.23 (C), and 0.28 (D)°C min~'. Means and standard errors are plotted and the replicate sizes are 7 (A, B), 5 (C), and 6 (D). An asterisk indicates the lowest absolute temperature change to evoke a percent response that was significantly different (/' < 0.05; Dunnett's /-test) from the control level, which is plotted at 0°C. change. Control counts were made 30 s before the first 0. PC change in temperature. The percentage of larvae in each subsection was calculated from these data. Re- sponse level was considered the percentage of larvae in the subsections after a 0.5°C change in temperature, be- cause responses are clearly evident by this absolute amount of temperature change (Fig. 2). Ascent and descent responses were expected upon a temperature decrease and increase, respectively. Thus the change in the percentage oflarvae in the bottom sub- section was monitored upon a temperature decrease, and, in the upper subsection, upon a temperature in- crease. Arcsine transformed data were used for statistical tests and to calculate means, standard deviations, and standard errors. Back transformed means and standard errors are plotted in the figures. If paired observations were made before (control) and upon stimulation (exper- imental) of each group oflarvae, a /-test for paired com- parisons was used to test for differences (P < 0.05). In cases where a control was compared to responses at different times after the beginning of stimulation, then the Dunnett's Mest for multiple comparisons with a con- trol was used to test for significant differences (P < 0.05; Dunnett, 1964). A Z statistic testing differences between two proportions (Walpole, 1974) was used to test for differences between control and experimental distribu- tions of individual trials. Results Response lime course The change in the percentage oflarvae in the lower or upper subsections of the experimental chamber, upon an increase or decrease in temperature, respectively, is the response time course. Representative patterns are shown for Stage I zoeae at rates of temperature change that I ARVAI RESPONSES TO TEMPERATURE 199 evoked strong responses (Fig. 2). Initially the larvae were approximately evenly distributed in the test chamber, as the percentage of larvae in the top and bottom subsec- tions was close to 33%. However, because the true values were not always 33%, the initial distribution was deter- mined for each group of larvae, and the mean used as the control level for comparison with percentages upon a temperature change. An ascent occurred upon a temperature decrease as indicated by a decrease in larvae in the bottom subsec- tion and increase in the top subsection (Fig. 2A, C). For both R. harrisii (Fig. 2A) and N. sayi (Fig. 2C), signifi- cant changes co-occurred in the bottom and top subsec- tions after a 0.3-0.4°C absolute temperature change. Thus, larvae leave the bottom subsection, and ascend to the top subsection, when the temperature decreases (Fig. 2A.C). With a temperature increase, there was a descent; the percentage of larvae in the top subsection decreased, while it increased in the bottom subsection (Fig. 2B, D). For both species, the percentage of larvae changed sig- nificantly in the top subsection after a 0.3°C absolute temperature change, and after a 0.4°C change for larvae in the bottom subsection. This pattern (Fig. 2B, D) indi- cates that larvae left the top subsection and aggregated in the bottom subsection. These response patterns were used to establish the ana- lytical methods for the experiments. The percentage of larvae in the bottom subsection was monitored upon a temperature decrease. Because cooled water entered the test chamber at the bottom, larvae in the bottom subsec- tion were initially exposed to the temperature decrease and responded first. Similarly, the percentage of larvae in the top subsection was monitored upon a temperature increase, because warmer water entered the test chamber from above. For both temperature decreases and in- creases, larval distributions were monitored before (con- trol) and after a 0.5°C absolute change in temperature (experimental). The results shown in Figure 2 indicate that strong responses are evident by this amount of tem- perature change, and preliminary analysis showed that if larvae had not responded by the 0.5°C change, then they did not respond at greater absolute temperature changes. Temperature decrease Responses upon a temperature decrease were not due to fluid flow through the test chamber. Larvae were sub- jected to the maximum experimental flow rate, but not to a temperature decrease. Distributions were measured at the average time after the beginning of flow for the control and experimental measurements at this flow rate. The mean percentage of larvae in the bottom section never changed significantly with flow. This result also in- dicates random larval movements did not produce the observed responses. In contrast, larvae ascended upon a temperature de- crease. The lower rates of temperature decrease (thresh- old) to induce a response by Stages I and IV zoeae of R. harrisii (Fig. 3A, C) and Stage I zoeae of N. sayi (Fig. 3B) were 0.06, 0. 1, and 0.09°C min ', respectively. A signifi- cant response was not displayed by Stage IV zoeae of N. sayi at rates up to 0.45°C min ' (Fig. 3D). Thus, there was an ontogenetic change in sensitivity by both species, in which Stage I zoeae were more sensitive than Stage IV. Tempera! lire increase Larvae descended upon an increase in temperature (Fig. 4). This response was not due to fluid flow or ran- dom movements. Using techniques for measuring re- sponses to flow as described in the previous section, the mean percentage of larvae in the upper section did not change significantly between the control and experimen- tal times (Fig. 4; plotted at rate 0° min"1). The threshold rates for Stages I and IV zoeae ofR. harrisii (Fig. 4A, C) and Stage I zoeae of A', sayi (Fig. 4B) were 0.07, 0.24, and0.18°Cmin~', respectively. Stage IV zoeae of A', \uyi were not responsive to any rate of temperature increase upto0.35°Cmin~' (Fig. 4D). These results indicate that, for both species. Stage I zoeae respond to slower rates of temperature increase than Stage IV zoeae. Absolute temperature change The absolute amounts of temperature change neces- sary to produce a significant response upon temperature decreases and increases were determined for each larval stage (Fig. 5). Determinations were made at those rates that produced a significant response (Figs. 3, 4). For each trial, the proportion of control larvae in the bottom (tem- perature decrease) or top (temperature increase) subsec- tions was compared to the proportion of larvae after each 0.1°C change, until a significant difference was evident (P < 0.05; Z statistic for testing differences between two proportions). Mean absolute temperature values were then calculated for each rate (Fig. 5). Mean values did not vary significantly with rate of temperature change (one-way ANOVA) within each species, zoeal stage, and direction of temperature change. Thus an average value was calculated for a temperature increase and decrease at each zoeal stage (Table I). Mean values varied over a narrow range from 0.28°C to 0.49°C. Discussion The general responses of both test species of larvae were an ascent upon a temperature decrease and descent upon a temperature increase. Since all experiments were 200 R. B. FORWARD JR. 50- 30- Q_ 20" 10- A Rh/thropanopeus harrisu Stage I C Rhithropanopeus harrisii Stage IV 3--J-4-- B Neopanope sayi Stage I I- -, D Neopanope soy/ Stage IV 01 0.2 03 04 1.0 20 3.0 4,0 Rate of Temperature Increase (°C/min) Figure 4. The percentage of Stages I and IV zoeae of Rhithropanopeus harrisii ( A. C) and Neopanope say/ (B, D) in the top subsection of the test section before (dashed line) and after a 0.5°C absolute tempera- ture increase (solid line) at different rates of temperature increase. The control for flow as measured at the fastest test flow rate is plotted at 0°C/min '. Means and standard errors are plotted. The average replicate size in A, B. C, and D are 6, 6, 7, and 7, respectively. Asterisks indicate the slowest rate of temperature increase at which there is a significant difference between control and test mean percentages (P < 0.05; t- test for paired comparisons). moclines during vertical movements by Eurypanopeus depressus (Sulkin el ai, 1983) and Callinectes sapulns larvae (McConnaughey and Sulkin, 1984). R. harrisii larvae are retained in upper estuarine areas (Cronin, 1982). Kirby-Smith and Barber (1979) mea- sured environmental factors in an area (South River, North Carolina) close to the collection site for ovigerous R harrisii where larvae consistently occur. Daytime temperature at the surface and bottom during the sum- mer reproductive months of July and August (1974- 1976) indicate that a temperature difference existed 80% of the measurement times. A conservative assumption is that temperature changed continuously from the surface to the bottom. Under these conditions, the average gradi- ent was 0.9°C m~'. The threshold rates of detection by larvae and speeds of vertical movement were used to calculate the minimal gradient a larva could perceive. A conservative measure of speed of downward movement is larval sinking speed because larvae can also actively swim down. IfR. harrisii sink continuously, then the minimal temperature de- crease they can detect is 0.32°C m~' for Stage I zoeae and 0.22°C irT1 for Stage IV zoeae (Table I). Using average ascent rates, the minimal increase in temperature they could detect is 0.1 9°C nrT1 for Stage I zoeae and 0.63°C m ' for Stage IV (Table I). Because these values are be- low the average gradient calculated from the measure- ments of Kirby-Smith and Barber (1979), R. harrisii lar- vae can detect changes in temperature in their environ- ment. N. sayi larvae inhabit low estuarine and coastal envi- ronments (Sandifer, 1975: Dittel and Epifanio, 1982; Salmon el al.. 1986). Pinschmidt (1963) measured sur- face and bottom temperatures in the Beaufort Inlet, which connects the Newport River estuary (where ovi- gerous N. sayi were collected) and the coastal waters. Measurements in July and August ( 1 960- 1 96 1 ) indicate temperature differences were present 50% of the time. 202 R. B. FORWARD JR. o o 6 o 0) I O) O />/'i' sayi (E, F) larvae. Means and standard errors are plotted. The average replicate size for all means was 5. Again assuming a continuous change in temperature, the average gradient was 0.3°C rrT1. Stefansson and Atkin- son (1967) extensively measured temperature in the coastal area seaward of the Beaufort Inlet at specific depth intervals. During the summer months, tempera- ture was approximately equal in the upper 10 m, but at times differences approaching those measured by Pin- schmidt ( 1 963) were evident at deeper depths. The gradients that can be perceived by N. sayi larvae were calculated using the same procedure as for R. har- risii larvae. The detectable gradient in temperature de- crease for Stage I zoeae is 0.34°C m"1 and for a tempera- ture increase is 0.94°C m~' (Table I). Thus Stage I zoeae of A', sayi should be able to detect the average environ- mental gradients in temperature decrease, but require extreme gradients in temperature increase. An additional consideration is the functional signifi- cance of responses to temperature change. Larvae of both species can respond to a temperature increase and decrease. The threshold rates are in the range of 0.1 °C min ' (2 X 10~3°C s"1), and the necessary absolute amounts of change are less than 0.5°C (Table I). Larvae do not respond to temperatures above some very high absolute upper limit as suggested by past studies (Ott and Forward, 1976; McConnaughey and Sulkin, 1984). These responses could be used to avoid extreme, adverse environmental temperatures. Since high temperatures usually occur near the surface and low temperatures oc- LARVAL RESPONSES TO TEMPERATURE Table I Calculation oj minimal delectable temperature change per in 203 Threshold rate Minimum detectable gradient Absolute amount of temperature decrease Mean sinking rate of temperature decrease temperature decrease res-') (mm s ') ("Cm-') ro Rhithropanopeus harrisii Stage 1—1. OX 10~3 3.1 0.32 0.38 Stage IV— 1.7 x KT3 7.8 0.22 0.49 Neopanope savi Stage I— 1.5 X 10~3 4.4 0.34 0.34 Minimum detectable gradient Absolute amount of Mean ascent rate of temperature increase temperature increase Temperature increase (mm s"') ('Cm-') (°C) R. harrisii State I— 1.2 X 10'3 6.3 0.19 0.28 Stage IV— 3.9 X 10'3 6.2 0.63 0.29 N. savi Stage I— 3.0 x ifr3 3.2 0.94 0.41 Threshold rates are from Figures 3 and 4. Mean sinking (Latz and Forward, 1977) and ascent speeds (Forward and Wellins, 1989) for R. harrisii are at 20 ppt (rearing salinity), while those for N. savi are at 32 ppt (Forward el al . 1989). The minimum detection rate in °C m~' is calculated as (threshold rate/sinking-ascent rate) 1000. The absolute amounts of temperature increase are mean values from Figure 5. cur at depth, the ascent response upon a temperature de- crease would move larvae upward out of cool water into warmer water. The opposite responses occur upon a tem- perature increase. Nevertheless, the high sensitivity of larvae to temperature change suggests that these re- sponses may have an additional function than avoidance of extreme conditions. Temperature could be used as a cue to regulate depth at a particular optimum tempera- ture or as a cue for depth maintenance in a particular water mass that has a characteristic temperature. With the present study it is possible to evaluate the relationships of larval responses to environmental fac- tors. For R. harrisii, responses to rate of change in light (Forward, 1985), hydrostatic pressure (Forward and Wellins, 1989), salinity (Forward, 1989), and tempera- ture (this study) have been determined. Upon descend- ing in a stratified water column, light intensity decreases, pressure increases, salinity increases, and temperature decreases. At rates of change that are within the range larvae can encounter while descending, each of the changes in these environmental factors induces negative geotaxis or an activity increase that results in an ascent. In contrast, the opposite environmental changes upon an ascent produce weak responses, at best. R. harrisii lar- vae are unresponsive to rates of increase in light intensity (Forward, 1985) and rates of decrease in salinity (For- ward, 1989) they are likely to encounter underwater. In darkness, a sinking response occurs upon a pressure de- crease, but the threshold rate is much higher than that for a pressure increase (Forward and Wellins, 1989). Similarly, this study indicates larvae can respond to both increases and decreases in temperature, but the thresh- olds were always higher for responses to a temperature increase (Table I). For N. sayi. responses to rates of changes in salinity (Forward, 1989), pressure (Forward el al.. 1989), and temperature (this study) have also been studied. Consid- ering Stage I zoeae, a pronounced ascent is also induced by changes in these factors that are likely to occur upon descending in the water column. The opposite environ- mental changes produce weaker responses. N. sayi larvae are unresponsive to decreases in salinity (Forward, 1989). They respond both to pressures increases and de- creases, but the threshold for a pressure increase was lower than that for a pressure decrease (Forward el al., 1989). Finally, this study shows that larvae respond to temperature increases and decreases, but the threshold rate is higher for a temperature increase (Table I). Thus both R. harrisii and N. sayi larvae have asymmetrical responses to changes in environmental factors. These re- sponses may keep larvae up in the water column and re- duce the likelihood that they will encounter the bottom and its associated benthic predators. Acknowledgments This material is based on research supported by the National Science Foundation under Grant No. OCE- 8603945. I thank Mr. M. Wachowiak for his technical assistance and Dr. D. Rittschof for critically reading the manuscript. 204 R. B. FORWARD JR. Literature Cited Cronin, T. \V. 1982. Estuanne retention of larvae of the crab Rhithro- panopeus liarnsn. Estiun Coast. Sei. 15: 207-220. Dittcl, A. L., and C. E. Epifanio. 1982. Seasonal abundance and verti- cal distribution of crab larvae in Delaware Bay. Estuaries 5: 197- 202. Dunnett, C. \V. 1964. New tables tor multiple comparisons with a control. Biometrics 20: 282-291. Forward, R. B., Jr. 1976. Light and diurnal vertical migration: photo- behavior and photophysiology of plankton. Pp. 157-2(19 in Photo- chemical and rhotohiological Reviews, Vol. 1 . K. C. Smith, ed. Ple- num Press, New York. Forward, R. B., Jr. 1985. Behavioral responses of larvae of the crab Rhithropanopetix hurrisii (Brachyuran; Xanthidae) during diel ver- tical migration. Mar. Biol. 90: 9-18. Forward, R. B., Jr. 1988. Diel vertical migration: zooplankton photo- biology and behavior. Occanog. Mar II ml Annii Rev 26: 361-393. Forward, R. B., Jr. 1989. Behavioral responses of crustacean larvae to rates of salinity change. Kiol. Bull 176: 229-23X. Forward, R. B., Jr., and T. W. Cronin. 1979. Spectral sensitivity of larvae from intertidal crustaceans. J. Comp. Physio/. 133: 31 1-315. Forward, R. B., Jr., and C. A. \Yellins. 1989. Behavioral responses of a larval crustacean to hydrostatic pressure: Rhithropanopeui har- r/.w(Brachyura: Xanthidae). Mar. Biol 101: 159-172. Forward, R. B., Jr., C. A. Wellins, and C. U. Buswell. 1989. Behavioral responses of larvae of the crab Neopanope \ayi to hydro- static pressure. Mar. Ecu/ Prog Ser. 57: 267-277 Kelley, P., S. D. Sulkin, and \V. F. Van Heukelem. 1982. A dispersal model for larvae of the deep sea red crab (ieryon quinquedens based upon behavioral regulation of vertical migration in the hatching stage. Mar Biol. 72: 35-43. Kirby-Smith, \V. \\ '.. and R. T. Barber. 1979. The Water Quality Ramifications in Estuaries of Converting Forest to Intensive Agri- culture. University of North Carolina — Water Resource Research Institute Report No. 148. Pp. 1-70. Latz, M. I., and R. B. Forward Jr. 1977. The effect of salinity upon phototaxis and geotaxis in a larval crustacean. Biol. Bull 153: 163- 179. McConnaughcy, R. A., and S. D. Sulkin. 1984. Measurements of the effects of thermoclines on the vertical migration of larvae of Calli- necles sapidus (Brachyura: Portunidae) in the laboratory. Mar. Biol. SI: 139-145. Ott, F. S., and R. B. Forward, Jr. 1976. The effect of temperature on phototaxis and geotaxis by larvae of the crab Rhithropanopeus harri.sii (Gould). ./. Exp. Mar. Bio/. Ecol. 23: 97-107. Pinschmidt, VV. C., Jr. 1963. Distribution of crab larvae in relation to some environmental conditions in the Newport River estuary. North Carolina. Ph.D. thesis. Duke University. Durham. North Carolina. Sandifer, P. A. 1975. The role of pelagic larvae in recruitment to pop- ulations of adult decapod crustaceans in the York River estuary and adjacent lower Chesapeake Bay. Virginia. Estuar. Coastal Mar. Sci. 3: 269-279. Salmon, M., \V. H. Seiple, and S. G. Morgan. 1986. Hatching rhythms of fiddler crabs and associated species at Beaufort, North Carolina. J Crust. Biol. 6: 24-36. Stefansson, U., and L. P. Atkinson. 1967. Physical and Chemical Properties of the Shelf and Slope Waters of North Carolina. Techni- cal Report. Duke University Marine Laboratory. Pp. 1-230. Sulkin, S. D. 1984. Behavioral basis of depth regulation in the larvae of brachyuran crabs. Mar Ecol Prog. Ser 15: 181-205. Sulkin, S. D., and \V. Van Heukeltm. 1982. Larval recruitment in the crab ( 'allinectes sapulus Rathbun: an amendment to the concept of larval retention in estuaries. Pp. 459-475 in Estuanne Compari- sons. V. Kennedy, ed. Academic Press, New York. Sulkin, S. D., \V. Van Heukelem, and \V. Kelley. 1983. Behavioral basis of depth regulation in the hatching and post-larval stage of the mud crab Eurypanopeus ilcpi cssus. Mar Ecol. Prog. Ser. 11: 157- 164. Sulkin, S. D., \V. Van Heukelem, P. Kelley, and L. Van Heukelem. 1980. The behavioral basis of larval recruitment in the crab Calli- nectes .sapiilit.s Rathbun: a laboratory investigation of ontogenetic changes in geotaxis and barokinesis. Biol. Bull. 159: 402-417. Thorson, G. 1964. Light as an ecological factor in the dispersal and settlement of larvae of marine bottom invertebrates. Ophelia 1: 167-208. Walpole, R. E. 1974. Introduction to Statistics. Macmillan. New York. Welsh, J. H. 1932. Temperature and light as factors influencing rate of swimming of larvae of mussel crab Pinnotheres macitlalu.s (Say). Biol. Bull. 63:310-326. Yule, A. B. 1984. The effect of temperature on the swimming activity of barnacle nauplii. Mar. Biol Lett. 5: 1-11. Reference: Biol Hull 178: 205-209. (June. 1940) Food Aversion Learning by the Hermit Crab Pagurus granosimanus KEITH WIGHT, LISBETH FRANCIS, AND DANA ELDRIDGE Biology Department, Bates College, Lewiston, Maine 04240 Abstract. The common intertidal hermit crab Pagurus granosimanus learns in one or two trials to reject an at- tractive, novel food (beef) when illness is induced by lith- ium chloride injected one hour after the animal accepts and eats the beef. Crabs fed a familiar food (fish) before lithium chloride injection do not learn to avoid the fish. Nor do they learn to reject beef when injected with a so- dium chloride solution, or when punctured with a hypo- dermic needle one hour after their first and second beef meals. Because many crustaceans are scavengers and generalist feeders, they must commonly encounter a wide variety of toxic foods. Quickly acquired and long- lasting aversion to a new food eaten a few hours before the onset of a serious physiological upset could cause these animals to avoid such hazardous foods in the fu- ture. Food aversion learning has never before been re- ported in a crustacean. Introduction From Baja California to Alaska, the common inter- tidal hermit crab Pagurus granosimanus lives on rocky substrates between -1.0 and +0.8 meters, relative to mean lower low water (Nyblade, 1974; Abrams, 1987). Like most hermit crabs, P. granosimanus is an omnivo- rous detritivore that feeds actively on a wide range of plant and animal foods (Orton, 1927; Roberts, 1968; Hazlett. 1981). For an opportunistic feeder living on wave-swept shores, the particular food available, its nu- tritional value, and the risk of toxicity can vary season- ally, from place to place, and even from tide to tide. This should favor the evolution of sensory capacities and learning mechanisms that allow the animal to be both selective and flexible in its choice of foods. Food aversion learning is a kind of associative learning Received 3 August 1989; accepted 16 March 1990. that is particularly appropriate for opportunistic feeders (Wilcoxon et a/., 1971; Garcia et al., 1974; Garcia and Hankins, 1977; Gustavson, 1977; Zahorik and Houpt. 1 98 1 ). It is distinguished from classical or operant condi- tioning on the basis of several distinctive characteristics (reviewed and discussed in Barker et al., 1977). ( 1 ) One or a very few conditioning trials are commonly effective. (2) Learning can occur in spite of long delays between ingestion and the resulting illness. (3) The resulting aver- sion has a long extinction time. (4) Only particular as- pects of the food are associated with the illness. (5) Novel foods are much more readily associated with the sickness than are familiar foods. As generalist feeders and scavengers that distinguish foods primarily by chemoreception ( Hazlett, 1968, 1971; Zimmer-Faust, 1987), hermit crabs may benefit from a learning mechanism similar to the taste aversion learn- ing of rats. We demonstrate here that hermit crabs (Pa- gurus gransimanus) quickly learn to reject a novel and attractive food when severe illness is induced by lithium chloride injected about an hour after they first eat that food. Materials and Methods Large animals (wet weight 0.48-1.65 grams without the shells) were collected from rockpools at Cattle Point on San Juan Island. Washington, and held in aquaria supplied with running seawater at the Friday Harbor Laboratories. After removing the apex of each shell with a belt sander, we divided the crabs haphazardly into 6 groups of 1 5 animals each. Each group was held in a plas- tic mesh ( Vexar) cage divided into separate 10 cm square compartments for each animal. The cages were raised 4 cm off the bottom of the aquaria so the animals could not browse on accumulated detritus. The foods used were fresh ground beef and fresh fish 205 206 K. WIGHT ET AL (sole) that were frozen raw. Only the amount needed was thawed each day to maintain equal freshness throughout the experiment. The crabs were hand-fed twice a day (morning and evening): we offered them tiny pieces of freshly thawed food on the end of a dissecting probe. Un- eaten food that fell through the plastic mesh was re- moved from the aquaria half an hour later. The crabs were fed fish for at least two days before treatments be- gan. On treatment days we fed them, moved them into separate finger bowls an hour later, and removed them from their shells by gently prodding the abdomens with a thin piece of plastic coated wire inserted through the hole at each shell apex. Injections were done with a microliter syringe with a fixed needle that was wiped with alcohol between injec- tions. Ten-microliter doses were injected into the thorax dorsally at the joint between the thorax and abdomen. Solutions used were 1.1 M lithium chloride (LiCl) and 1.1 M sodium chloride (NaCl) in glass distilled water. On the first day of the experiment (day 0), four of the six groups were fed heel': of these, one group was injected one hour later with lithium chloride ( LiCl), a second with sodium chloride (NaCl), the third merely pierced with the hypodermic needle but not injected, and the fourth only removed from the shell. The two remaining groups were fed fish; and one hour later one group was injected with lithium chloride, and the other with sodium chlo- ride. Only animals that accepted the test food when it was next offered (24 h after the first treatment) received a sec- ond treatment on the following day (day 1 ). The crabs' responses to food were tested twice daily for the next 1 1 days (days 2-12) without further treatment. They were offered bits offish in the morning and beef at night on the tip of a probe, and each animal's response was scored as either acceptance or rejection (described in the results below). To reduce the amount of handling during treatment, the animals were not weighed initially. Instead, on day 10 of the experiment, surviving animals were removed from their shells and weighed individually to the nearest hundredth of a gram. Results Food acceptance and rejection responses When accepting food from a probe, hermit crabs usu- ally touch the probe with the second antennae or the dac- tyls of the walking legs, grasp the food using the chelipeds and sometimes also with the walking legs, then pass it toward the mouth, usually using the minor, left cheliped. Both chelipeds may be used to tear off bits that can be ingested, or the whole mass may be manipulated and held against the inner mouthparts by the third maxilli- peds. When rejecting the food, the crabs generally flick the second antennae back and away after contacting the probe. Sometimes they push the food away vigorously with the chelipeds and back away; and sometimes they hesitantly grasp it with the minor cheliped, pass it to the mouthparts, manipulate it for a few seconds, and then eject it forward and upward using a jet of water. Dosage and effects of lithium chloride The mean wet weight overall for the animals was one gram (sd = 0.3, n = 89). To avoid excessive handling, the animals were each given the same size injection; and thus the per weight dose of LiCl varied from 250 to 970 mg/ Kg wet weight (per treatment). This dose of LiCl caused limb trembling, uncontrolled movement, and periods of immobility when the animals usually lay on their backs. All of these animals found and reaccepted their shells within two to three hours. The crabs that were injected with NaCl tended periodically to curl tightly into a ball, sometimes remaining immobile for several minutes; but they reaccepted their shells within half an hour. Those that were stabbed with the needle but not injected returned to their shells within 1 5 minutes with only occasional periods of immobility; and the crabs that were only removed from their shells usu- ally reaccepted them immediately. Induced aversion to a novel food All of the animals injected with LiCl after their first encounters with beef developed an aversion to beef (Figs. 1 A, 3 A). Two-thirds refused beef after only one LiCl in- jection. The five that were injected again, after their sec- ond beef meal, all refused beef when it was offered for the third time. Without additional injections, the num- ber refusing beef continued to be significantly higher than for the controls through day thirteen (G-test with the Williams correction, P< .05). On day 14 the number that refused beef was not significantly higher than in the control groups (G-test, P > .50). As individuals, these animals were also more con- sistent in refusing beef than were the animals in other treatment groups (Fig. 3). Extinction of the response generally required more than a week — on average, the beef-LiCl treated animals refused beef for 6.9 ± 3.0 con- secutive days within the first 11 -day period following treatment (days 2 through 12), (Fig. 3A). This was sig- nificantly longer than for any other treatment group (!- test, P 4 .001). This group rejected beef more consis- tently than it rejected fish (/-test, P « .00 1 ). Although about twice as many animals in the beef- NaCl and beef-puncture control groups received a sec- FOOD AVERSION LEARNING BY HERMIT CRABS 207 v a 0 2 4 6 8 10 Days after initial injection •a — LiCI + beef •• — NaCI + beef -• — Puncture + beef a— Shell off + beef o> a 12 Days after initial injection Figure 1. Number ofindividual hermit crabs (Pagurus granosima- nus) that rejected daily feedings of (A) beef and (B) fish. The legend applies to both graphs. Four groups of 1 5 animals each were treated on day zero, one hour after eating a novel food (beef); those that accepted beef the next day received a second treatment. One treatment group was only removed from the shell; a second was also punctured with a hypodermic needle. Two other groups received injections, one with lithium chloride, and the other with sodium chloride. ond treatment, neither group developed an aversion to beef (Fig. 1A). Significantly more of the animals in the fish-Nad treatment group rejected beef on first encoun- tering this new food, 1 2 h after their treatments on day 0 and day 1 (comparison with the beef-shell removal con- trol group; G-test, P < .0 1 and P < .025 for day 0 and day 1, respectively; Fig. 2B); however, none of these animals showed a long-term aversion to beef (Fig. 3C). Consistent acceptance of a familiar food All of the groups continued to accept fish throughout the test period (Fig. 1 B). Of the two groups that were in- jected after eating this familiar food, neither learned to reject fish (Fig. 2A). Mortality Six animals died during the experiment: one from the beef-LiCl group on day 4; three from the fish-NaCl group on days 2, 9, and 10; one on day 1 1 from the group that was simply removed from the shell; and one from the fish-LiCl group on day 10. Discussion When injected with LiCI one hour after their first beef meals, hermit crabs (Pagurus granosimanus) learned in one or two trials to avoid this novel food while continu- ing to eat a familiar food (fish). This aversion to beef commonly lasted for more than a week under laboratory conditions. Hermit crabs rely strongly on chemorecep- tion in locating food (Hazlett, 1968; Zimmer-Faust, 1987). Crustaceans can learn using chemoreception as a cue. Fine- Levy et al. ( 1988) found that the spiny lobster can learn to associate a particular smell with the presence of a predator. It is likely, then, that food is identified and avoided on the basis of chemoreception in response ei- ther to water-borne chemicals (smell) or to direct contact (taste). Further work is required to determine what spe- cific food cues are used in this learned avoidance of a specific food. In laboratory experiments with vertebrates (reviews in a E 14 - 12 - 10 - 8 - 6 4 - 2 - 0 • 2 1 - 1 - 1 - 1 - 1 - • - 1 - 024 68 Days after initial injection 10 E 3 Z 14 - 12 - 10 - 8 - 6 - 4 - 2 - 0 • 2 Days after initial injection Figure 2. Number of individual hermit crabs (Pagurus granosima- mi.v) that rejected (A) fish and ( B) beef on each day following treatment. Animals were treated on day zero, one hour after eating a familiar food (fish); those that accepted fish the next day were given a second treat- ment.The two treatment groups of 1 5 animals each were injected either with lithium chloride or with sodium chloride. 208 K. WIGHT ET AL o z o z 6 - 2- 6 - SUCI + beef NaCI + beel M Puncture + beef Q Shell off + beel 1 2 3 4 5 6 7 8 91011 Consecutive days beef rejected • LiCI + beef D NaCI t beel ^ Puncture + beef G Shell off + beel 1 2 3 4 5 6 7 8 Consecutive days fish rejected 1011 • fish, fish rejection 1 6- , D NaCI + fish, fish rejection El UCI + fish, beel rejection "D 0 NaCI + lish. beef rejection o n 1 II [ 01 234567891011 Consecutive days food rejected Figure 3. Consistency of food rejection by individual hermit crabs (PiiKiini.t xriiiuKiiiiinnis) in six treatment groups (identified in the leg- ends). Columns show the maximum number of consecutive days that each animal rejected a particular food during the eleven days following treatment: (A) rejection of a novel food (beef) by animals treated one hour after their first beef meal (or after the first and second beef meals, for those that accepted beef on the day after their first treatment). (B) fish rejection by the same animals, and (C) fish and beef rejection by different animals that were treated after eating a familiar food (fish). Barker el ill-. 1977), the effects of LiCI injection are gen- erally assumed to mimic the symptoms of illness caused by ingesting a toxic substance. The doses used in our study caused fairly severe and long-lasting general symp- toms. Moreover, the food aversion was clearly caused by the effects of the LiCI, and not by osmotic shock or the tonic effects of the injection, because the group injected with the same molar concentrations of NaCI after first eating beef did not develop an aversion to beef. Because animals that developed an aversion to beef continued to accept fish, the aversion is specific, and not merely a gen- eralized, post-trauma avoidance of food. Nor can it be explained as non-specific neophobia (general avoidance of unfamiliar food after an illness), because most of the specimens injected with lithium after a fish meal ac- cepted beef within a few days. One of the two control groups injected with NaCI solu- tion showed significantly increased rejection of beef for two days following treatment. This response is puzzling because it was inconsistent (i.e., it did not occur in both NaCI injected groups), and because none of the obvious explanations seem to fit. The animals in this group were not significantly smaller than in the other sodium-in- jected group, which rules out the possibility of unusually high osmotic or tonic stress. If the response were due to non-specific neophobia caused by the treatment, the group injected with lithium after eating fish should also have rejected the beef; but they did not. Whatever the cause of this transient reaction to a new food, it clearly is not long-lasting food aversion of the kind shown by the beef-Lid treatment group. Food aversion learning is known to occur commonly among vertebrates (Garcia e tai. 1974;Gustavson 1977), and has also been described for a mollusc (Gelperin, 1975) and two insects (Dethier, 1980; Bernays and Lee, 1988). Characteristic of this type of associative learning (Garcia and Hankin. 1977) is rapid aversion to a new food (one or two trials in this case) despite a considerable time lag between ingestion and the onset of illness (in this case, an hour). Also typically, a novel food (in this case, beef) is more readily associated with subsequent internal disorders than is a familiar food (fish). Relatively long extinction times (one or two weeks, here) are also typical. Most hermit crabs are omnivorous detritivores feeding on fine particles from the sediment as well as on larger morsels of animal matter (Orton, 1927; Roberts, 1968). Thus they are undoubtedly exposed to a wide variety of foods, and presumably also to a wide spectrum of toxins, including rotting debris that can be infested with toxic microorganisms, and macroorganisms that can manu- facture or sequester toxins. While food aversion learning has never before been described among crustaceans, many (including the hermit crabs) can learn by classical conditioning (review by Corning el a/., 1973). The ability to associate delayed illness with a particular food could be quite advantageous, and might be rather common among the Crustacea. FOOD AVERSION LEARNING BY HERMIT CRABS 209 Acknowledgments We thank the director of the University of Washing- ton's Friday Harbor Laboratories for use of the facilities, and the faculty, staff, and colleagues there for encourage- ment and useful discussion. Literature Cited Ahrams, P. A. 1987. Competitive interactions between three hermit crab species. Oecologia 72: 233-247. Barker, L. M., M. R. Best, and M. Domjan (eds.) 1977. Learning Mechanisms in Food Selection. Baylor University Press, Houston. 632 pp. Bcrnays, E. A., and J. C. Lee. 1988. Food aversion learning in the polyphagous grasshopper Schistocerca arnericana. Physiol. Ento- innl. 13: 131-138. Corning, W. C., J. A. Dyal, and A. O. D. Willows (eds.) 1973. Invertebrate Learning. Vol. 2. Plenum Press. New York. 284 pp. Dethier, V. G. 1980. Food-aversion learning in two polyphagus cater- pillars, Diacrisiu virginica and Kxtigmene c<»i.t>niu. I'hysiol. Ent. 5: 321-325. Fine-Levy, J. B., Girardot, M. N., Derby, C. D., and Daniel, P. C. 1988. Differentia] associative conditioning and olfactory discrim- ination in the spiny lobster Panulinix argux. liehav. Neural Biol 49: 315-331. Garcia, .)., W. G. Hankins, and K. \V. Rusiniak. 1974. Behavioral regulation of the milieu interne in man and rat. Science 185: 824- 831. Garcia, J., and \V. G. Hankins. 1977. On the origin of food aversion paradigms. Pp. 3-43 in Foraging Behavior. A. C. Kamil and T. D. Sargent, eds. Garland STPM Press, New York. Gelperin, A. 1975. Rapid food aversion learning by a terrestrial mol- lusk. Science 189: 567-570. Gustavson, C. R. 1977. Comparative and field aspects of learned food aversions. Pp. 23-43 in Learning Mechanisms in Food Selection. L. M. Barker, M. R. Best, and M. Domjan, eds. Baylor University Press, Houston. Hazlett, B. A. 1968. Stimuli involved in the feeding behavior of the hermit crab Clibananus villains (Decapoda, Paguridea). Cntsia- mjmil5:305-310. Hazlett, B. A. 1971. Chemical and ehemotactic stimulation of feed- ing behavior in the hermit crab Pelrochirus diogenex. Camp. Bio- chem. 39A: 665-670. Hazlett, B. A. 1981. The behavioral ecology of hermit crabs. Ann. Rev.Ecol.Syst. 12: 1-22. Nyblade, C. F. 1974. Coexistence in sympatric hermit crabs. Ph.D. Thesis, University of Washington, Seattle. Orton, J. H. 1927. On the mode of feeding of the hermit crab L'ui>u- gurus hernhardus and some other decapoda. J. Mar Biol. Assoc. V.K 14:909-921. Roberts, M. H. 1968. Functional morphology of mouth parts of the hermit crabs, Pagurus Umgiearrnis and Pagurm pollicarpis. (. 'lie.su- peake Sri. 9:9-20. Wilcoxon, H. C., W. B. Dragonin, and P. A. Krai. 1971. Illness in- duced aversions in rat and quail: relative salience of visual and gus- tatory cues. Science 111: 826-828. /.ahorik, D. M., and K. A. Houpt. 1981. Species differences in feeding strategies, food hazards, and the ability to learn food aversions. Pp. 289-3 1 1 in Foraging Behavior. A. C. Kamil and T. D. Sargent, eds. Garland STPM Press, New York. Zimmer-Faust, R. K. 1987. Crustacean chemical perception: towards a theory on optimal chemoreception. Biol. Bull. 172: 10-29. Reference: Biol. Bull 178: 210-216. (June, 1990) Effect of Calcium on the Stability of the Vitelline Envelope of Surf Clam Oocytes GEORGE DESSEV AND ROBERT GOLDMAN Department oj Cell. Molecular and Structural Biology, Northwestern University Medical School. 303 East Chicago Avenue, Chicago, Illinois 6061 1. and Marine Biological Laboratory, Woods Hole. Massachusetts 02543 Abstract. Fertilization and parthenogenic activation of oocytes of the surf clam, Spixula solidissima. require the presence of calcium in the extracellular medium. Here we report that the depletion of calcium causes a dramatic increase in the stability of the vitellinc envelopes (VE). On the basis of this effect, we have developed a method of isolating intact VE and have studied their morphology, composition, and properties. Experiments using 45Ca: + have revealed that isolated VE bind calcium in a weak, but specific way. These findings suggest that the function of calcium may be to maintain the oocyte surface in a fertilization-competent state, while the reactions subse- quent to the initial activation event, and leading to nuclear envelope breakdown (NEBD), may not require calcium. In support of this hypothesis, we have demon- strated that hypertonic conditions induce the oocytes to undergo NEBD in the absence of extracellular calcium. Introduction Development of the oocytes of most animal species is discontinued at a certain stage of meiosis, usually at the G2/M border (Masui and Clarke, 1979; Mailer and Krebs, 1 980; Mailer, 1985). The oocytes can remain in a state of arrest for long periods and then resume the mei- otic process as a response to external signals. In most ver- tebrates, these signals are hormone-like substances (Mailer and Krebs, 1980; Mailer. 1985). In S/>/.s -nla. prog- ress into M-phase is induced by fertilization or by a num- ber of physical or chemical stimuli, such as UV-light or changes in the ionic composition of the medium (Allen, Abbreviations: VE. vitelline envelopes; NEBD, nuclear envelope breakdown; MFSW, millipore-nltered seawater; CFSW. calcium-tree seawater; MPF, M-phase promoting factor. 1953). Whatever the nature of these signals, one of their early biochemical effects is believed to be the activation of a pleiotropic enzymatic system, termed M-phase pro- moting factor (MPF) (Masui and Markert, 1971; Smith and Eckert. 1971; Wu and Gerhart, 1980; Arion et al.. 1 988; Draetta <'//0/., 1 988). Activation factors, such as fertilization, UV light, and KCl require extracellular calcium (Allen, 1 953). We have shown that other agents, such as hypertonic solu- tions of glycerol and NaCl, are capable of inducing matu- ration in the absence of calcium. Since glycerol and NaCl are chemically different, they are likely to act via osmotic changes, forcing membrane depolarization to occur even when the surface is structurally altered by depletion of calcium and is not susceptible to the action of other acti- vation factors. The chemical nature of the calcium binding to the VE, which is both weak and specific, as well as the nature of the structural and functional changes in the VE induced by calcium, remain interesting subjects for further studies. Acknowledgments We wish to thank Dr. A. Telser for help in the prepara- tion of the drawings. Research support was provided by NCI. Literature Cited Allen. R. D. 1953. Fertilization and artificial activation in the egg of the surf clam. Spi.tula soliiiissiina. Biol. Bull. 105: 213-239. Arion, D., 1,. Meijer, L. Brizuella, and D. Beach. 1 988. cdc2 is a com- ponent of the M phase-specific histone H 1 kinase: evidence for identity with MPF. Cell 55: 37 1-378. Ashwell, G. 1966. New colonmetric methods for sugar analysis. Meih. Enzymol 8: 85-95. Dessev, G., and R. Goldman. 1988. Meiotic breakdown of nuclear envelope in oocytes of Spisula sulidissima involves phosphoryla- tion and release of nuclear lamin. Dcv. Biol. 130: 543-550. Dessev, G., R. Palazzo, L. Rebhun, and Goldman R. 1 989. Disassem- bly of the nuclear envelope of Spisula oocytes in a cell-free system. Dc\: Biol. 313:496-504. 216 G. DESSEV AND R. GOLDMAN Draetta, G., F. Luca, J. Westendorf, L. Bri/.uella, J. Ruderman, and D. Beach. 1989. cdc2 protein kinase is complexed with both cyclin A and B: evidence for proteolytic inactivation of MPF. Cell 56: 829-838. Dube, F., R. Golsteyn, and I,. Dufresne. 1987. Protein kinase and meiotic maturation of surf clam oocytes. Biochem. Biophys. Res. Ctmim. 142: 1072-1076. Dunphy, \V. G., L. Brizuella, D. Beach, and J. Newport. 1988. The Xeiiopiis cdc2 protein is a component of MPF, a cytoplasmic regu- lator of mitosis. CV//54: 423-431. Dunphy, \\ ., and J. \V. Newport. 1988. Mitosis-inducing factors are present in a latent form during interphase in the \enopus embryo. ./ Cell Biol. 106: 2048-2056. Eckberg, \V. R. 1983. The effect of quercetin on meiosis initiation in clam and starfish oocyles. Cell Differ. 12: 329-334. Eckberg, \V. R., E. Z., Szuts, and A. G. Carrol. 1987. Protein kinase C activity, protein phosphorylation and germinal vesicle break- down in Spisula oocytes. Dcv. Biol. 1 24: 57-64. Einkel, T., and D. Wolf. 1978. Fertilization of surf clam oocytes: the role of membrane potential and internal pH. Bu>l Bull 155: 437. Gautier, J., C. Norbury, M. Lohka, P. Nurse, and J. Mailer. 1988. Purified maturation-promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle control gene cdc-\Cell 54: 433-439. Jaffe, L. F. 1983. Source of calcium in egg activation: a review and a hypothesis. Dc>\ Biol. 99: 265-276. Jaffe, L. F. 1985. The role of calcium explosions, waves and pulses in activating eggs. Pp. 127-165 in Biology of Fertilization, Vol. 2. C. B. Metz and A. Monroy, eds. Academic Press. New York. Laemmli, U. K. 1970. Cleavage of structural proteins during the as- sembly of the head of bacteriophage T4. Nature 227: 680-685. I.ohka, M. J., M. K. Hayes, and J. L. Mailer. 1988. Purification of maturation-promoting factor, an intracellular regulator of early mi totic events. Proc. Nail. Acad Set. L 'S.\ 85: 3009-30 1 3. Longo, F. J., and E. Anderson. 1970. An ultrastructural analysis of fertilization in surf clam, Spisula xoluliitiniu I. Polar body forma- tion and development of female pronucleus. J. Ultramr. Res. 33: 495-514. Lowry, O. M., N. J. Rosebrough, A. R. Farr, and R. G. Randall. 1951. Protein measurements with the Folin phenol reagent. J Bi«l. (.'hem. 193:265-275. Mailer, J. L. 1985. Regulation of amphibian oocyte maturation. Cell Differ. 16:211-221. Mailer, J. L., and E. G. krebs. 1980. Regulation of oocyte matura- tion. Curr Tup Cell Reg. 16: 217-31 1. Masui, Y., and II. J. Clarke. 1979. Oocyte maturation. Inl Rev. Cv- lo/Sl: 185-282. Masui, Y., and C. L. Marker!. 1971. Cytoplasmic control of nuclear behaviour during meiotic maturation of frog oocytes. J. Exp. Zool. 177:349-356. Moreau, M., M. Doree, and P. Guerrier. 1976. Electrophoretic intro- duction of calcium ions into the cortex ofXenopus lucvis oocytes triggers meiosis reinitiation. J. Exp. Zool. 197: 443-449. Murray, A. V\ '., and M. \V. Kirschner. 1989. Dominoes and clocks: the union of two views of the cell cycle. Science 246: 6 14-62 1 . Rebhun. I.. I. 1962. Electron microscope studies on the vitellme membrane of the surf clam, Spisula solidissimu. J. Ultrastr. Res 6: 107-122. Rebhun, I,., and I. K. Sharpies. 1964. Isolation of spindles from the surf clam. Spi.xitliixnlnli.xMmu. J Cell Biol. 22:488-496. Sehorderet-Slatkine, S., M. Schorderel, and E. E. Baulieu. 1976. Initiation of meiotic maturation in Xenopus lae\'is oocytes by lan- thanum. \iintre 262: 289-290. Schuet/., A. W. 1975. Induction of nuclear breakdown and meiosis in Spisula solidissima oocytes by calcium ionophore. J. Exp. Zool. 191: 443_44(,. Smith, I.. I)., and R. E. Ecker. 1971. The interaction of steroids with Riina pipicns oocytes in the induction of maturation. De\\ Biol. 25: 233-247. \V u, M., and J. C. Gerhart. 1980. Partial purification and character- ization of the maturation-promoting factor from eggs ofXenopus laevix. Oev Biol. 79: 465-477. Reference: B/0/, Bull 178: 217-221. (June. Regulation of Tissue Growth in Crustacean Larvae by Feeding Regime JOHN A. FREEMAN Department oj Biological Sciences, University oj South Alabama, Mobile. Alabama 36688 Abstract. Growth of the posterior dorsal carapace, the underlying epidermal cells, and the lateral thoraco-ab- dominal muscle was examined in the second instar Pa- laemonetes pugio under different feeding regimes. Con- trol larvae (continuous feeding) and larvae fed for the first two days of the molt cycle demonstrated a mean molt increment (MI) of 10.6 and 11.1%, respectively. The muscle in these control larvae grew in width by 6.7%. Starved second instar larvae showed a MI of 3.2% and an increase in muscle width of 1.3%. Larvae fed on only one day of the molt cycle had Mis of 5.5-6.6%— values significantly different from that of the control lar- vae. Muscle growth in partially fed larvae was intermedi- ate (3.9-4.5%) between those of fed and starved larvae. The increase in the density of the epidermal cells was proportional to the MI for the control and starved larvae, and for larvae fed on day 2; larvae fed only on day 1 or day 3 grew less or more, respectively, than the MI pre- dicted from the increase in cell density. The results show that nutritional state is a strong regulator of tissue growth in shrimp larvae. Introduction Food and nutritional state have long been known to affect growth and development of crustacean larvae (Hartnoll, 1982; McConaugha, 1985, for review). Food intake regulates the rate of molting or molt cycle dura- tion ( MCD), molt increment (MI, growth at ecdysis), and rate of development in larvae (Knowlton. 1974; McCo- naugha, 1982; West and Costlow, 1988). In some species, growth, molting, and development are affected differen- tially by restricted feeding conditions. Several studies have suggested that a hierarchical partitioning of the nu- trients for growth, molting, and development may exist, although the mechanism controlling this selection pro- Received 19 December 1 989: accepted 28 March 1990. cess is not understood (Knowlton, 1974; Anger and Dawirs, 1981;LeRoux, 1982; McConaugha, 1982, 1985; Anger, 1984; West and Costlow, 1988). Thus far, little is known about the regulatory mecha- nisms that determine how the level of food consumption may control growth of the integument and tissues. As- pects of growth of the epidermis have been examined in adult shrimp (Tchernigovtzeff, 1965), juvenile crabs (Freeman et a/.. 1983), larval brine shrimp (Freeman, 1986), and Daphnia (Halcrow, 1978). In those studies, the nutritional state and feeding history of the animal were not considered. To study growth regulation in crustaceans, it will be necessary to determine how the epidermis and muscle — the two tissues with the greatest mass and interaction with the integument — grow during the molt cycle. In this study, instar II Palaemonetes pugio larvae were reared under different feeding regimes to examine growth of the epidermis and muscle with respect to feeding and to fur- ther define the relationship between the growth of the tissue and the carapace. Materials and Methods Larvae were hatched from egg-bearing females col- lected locally and maintained individually in the labora- tory. The artificial seawater, (Instant Ocean, Aquarium Systems, Ohio), was maintained at 15 ppt and at 24°C (room temperature). Under these conditions, the dura- tion of the larval molt cycle was approximately three days. Instar I (SI) larvae were fed brine shrimp nauplii, and the water was changed daily. Upon ecdysis to instar II (SII), the larvae were placed in containers with or with- out food for the appropriate test period (see Results); the water was changed daily. The data were excluded where cannibalism (indicated by partially eaten larvae) oc- curred. 217 218 J. A. FREEMAN J, I LU (J < Q. < cc < u 6O i 55 5O 45 : i 511.1 CON NO-F D-1-F D-2-F D-3-F D-1.2F 15 10. t- lil UJ tr FEEDING REGIME Figure 1. Effect of the instar II feeding regime on the carapace length (open bars; left ordinate) and molt increment (diagonal stnped bars; right ordinate) of the resulting instar III larvae. The carapace length of day 1, instar II larvae is shown for comparison (SII. 1: single bar). Each bar represents the mean and one standard deviation of 56-162 larvae. Abbreviations for this and all other figures: CON, control larvae fed throughout the molt cycle; NO-F. starved larvae: D-I-F. D-2-F. D-3-F. D-1.2F. larvae fed. respectively, on day 1 , 2, 3, or on days I and 2. Measurement of the carapace length (CL) and muscle width (MW) in living larvae was done with a calibrated ocular micrometer. Larvae were immobilized on a slide in a drop of water. The CL was determined by measuring the distance between the posterior edge of the dorsal car- apace and a point even with the posterior edge of the orbit. The width of the lateral thoraco-abdominal exten- sor muscle was measured at the junction of the muscle with the dorsal carapace. The CL and MW determina- tions were made between 4 and 8 h after ecdysis to instar II (CLSM) or instar III (CLsm). These time points are re- ferred to in the Results as day 1 , SII or day 1 , SIII, respec- tively. The molt increment (MI) was determined as: [(CLsl,,/CLSii) - 1] x 100. Analysis of variance (F-test) was used to determine statistical significance (P < .05). The density of the epidermal cells was measured from specimens fixed in Carnoy's fluid, rehydrated. and stained with the nuclear fluorochrome bisbenzimide (Hoechst 33258, Sigma Chemical Co., St. Louis. Mis- souri) in phosphate buffered saline (PBS; pH 7.4). The larvae were mounted in PBS or 80% glycerol, with the dorsal carapace up and the long axis of the thorax ori- ented perpendicular to the axis of the slide. All measure- ments were made from an image in which the rostrum pointed towards the top of the field of view. The fluores- cent image of the epidermal region was captured by a Dage-MTI (Michigan City, Michigan) 67M newvicon video camera mounted on a Leitz Dialux photomicro- scope with a PCVISIONplus frame grabber controlled by IMAGEACTIONplus software (both from Imaging Technologies, Inc., Woburn, Massachusetts). A digital rectangle (90 X 109 ^m) was superimposed on the poste- rior dorsal carapace, and the number of nuclei within the rectangle was determined. The cell density was deter- mined on the first and second (and, in some cases, on the third) days of SII, and on the first day of SIII. Because cellular growth leads to expansion in both width and length at ecdysis, the increase in cell number is similar to the potential growth in area of that region of the carapace and therefore approximates the square of the MI in cara- pace length (Freeman, unpubl.). This "growth poten- tial," or predicted MI (% increase), is defined as: [(Dday2/ Dday ,)1/2 - 1] X 100, where D is the density of epidermal cells in SII on the days indicated. Results The feeding regime markedly affected the MI (% in- crease in carapace length) of the instar II larvae (Fig. 1). Larvae fed throughout the molt cycle (control) or on the first two days of the molt cycle (D-l, 2F) demonstrated Mis of 10.6 and 1 1.1%, respectively. These values were significantly greater than those of all other groups. Starved larvae (NO-F) showed a MI of 3.4%, which was significantly lower than those of all other groups. An in- termediate level of growth (5-7%) was observed in larvae fed only on day 1 , 2. or 3 of the molt cycle. There was no significant change in the MCD of starved or partially fed larvae. Many of the larvae that were starved or fed only on day 3 lived for 5 or 6 days without molting. Reduced carapace growth of larvae maintained on a restricted feeding regime was presumably a result of less RF.GUL.ATION OF TISSUE GROWTH 219 Figure 2. The change in density of the epidermal cells in the posterior dorsal carapace during the second and third instars. The nuclei are stained with bisbenzimide and photographed with epi fluorescence optics. A. Carapace epidermal cells in a larva at 6 h after ecdysis to instar II (day 1, SII). The density increased during the first day of the instar, reaching the greatest level by day 2, SII (B). The density decreased when the integument expanded after ecdysis to instar III (C). Bar = 25 growth of the epidermis which secretes it. To determine if starvation or a restricted feeding regime led to reduced growth of the epidermis, the increase in density of the epidermal cells during SII was determined. The cell density (nuclei per 90 X 109 ^m area) in freshly molted SII larvae was 18.3 cells (Figs. 2, 3). In controls and larvae fed on days 1 and 2, the cell density rose to 22-23 cells (Figs. 2, 3) for a predicted MI of 1 1-12%. The densities were greater than those for all groups except those fed on day 1. The density returned to 18 by day 1, SII I. Starved larvae showed the least amount of cell growth ( 1 -2 cells), and the predicted MI (2.7%) was very close to the measured MI (3.4%, Fig. 1). The cell density of starved larvae was significantly different from fed larvae (Control) and larvae fed on day 1, or on days 1 and 2, but not significantly different from those fed on day 2 or day 3. The predicted MI was similar to the mean measured MI (less than one cell difference) for all groups except larvae fed only on day 1 or day 3 (r = 0.91, P = 0.01, for all groups). The cell density of larvae fed on day 1 was not significantly different from fed larvae, but was sig- nificantly greater than larvae fed on day 2 or 3, or starved. Larvae fed on day 1 demonstrated a MI (6.6%) that was well below the predicted MI (9.4%). Conversely, larvae fed on day 3 grew by 5.6%, which was much greater than the predicted value of 3.0%. Not indicated by the value for larvae fed on day 2 (Fig. 3) was the in- crease in cell density in larvae feeding on day 2. The cell density value on day 2 (before feeding) was 19.9, which would give a predicted MI of 4.3%. There was an increase of one cell during the day of feeding. Thus, without feed- ing, these larvae would have shown a growth potential similar to larvae fed on day 3. Since the integument and muscle presumably grow in a coordinated manner, reduced muscle growth would be expected in larvae reared under restricted feeding condi- tions. Muscle width was measured on day 1 of instar II and compared to the width on day 1 of instar III. Muscle growth was greatest in the control larvae and larvae fed on days 1 and 2 (Fig. 4). The growth in these two groups was significantly greater than all other groups. Signifi- cantly less growth was observed in starved larvae than all other groups. Larvae fed only on days 1, 2, or 3 demon- strated intermediate growth levels that were significantly different from the fed and starved groups, although there was no difference among these groups. The growth in muscle width was highly correlated with the MI pre- dicted from epidermal growth (r = 0.84. P = 0.03) and the measured MI (R = 0.92, P = 0.01 ). Discussion This study clearly shows that growth of the integument and epidermal and muscle tissue is modulated by feeding regime or nutritional state. In addition, larvae in re- stricted feeding regimes may demonstrate slightly longer molt cycles, an indication that the low food level affected the molt cycle. The data agree with the findings on this and other species of decapod crustaceans (Knowlton, 1974;Hartnoll, 1982; McConaugha, 1985) and demon- strate, furthermore, that growth can be measured at the cellular level. The high correlation between epidermal growth in the dorsal carapace and carapace length is consistent with previous findings demonstrating that the size of the cuti- cle after ecdysis is a result of the amount of cell growth in 220 J. A. FREEMAN CO I HI U 30 25 20 15 10 j 15 102 Q LU H y Q Sll.1 Con No-F D-1-F D-2-F D-3-F- D-1.2F FEEDING REGIME Figure 3. Effect of the feeding regime during instar II on the growth of the epidermis in instar II larvae. The cell density at early day I was l8cells(SII, I, single bar). The cell density of larvae reared in different feeding regimes was measured on day 2 (open bar; left ordinate). For each group the molt increment predicted by the increase in cell density ([DJjy :/l 8]"3 - 1 x 100. where Dday 2 is the density on day 2) is also shown (diagonal striped bar. right ordinate). Abbreviations as in Figure 1. Each open bar represents the mean and 1 SDof 10-45 larvae. the epidermis during the previous molt cycle (Freeman, 1988, unpubl.). The cell density can he used to predict the MI. The results show a close correlation between the tissue growth and cuticular growth for all groups except those fed on days 1 or 3. The dissimilar Mis measured in larvae fed only on day 1 or 3 cannot be explained by the experiments from this study. Possibly the cell density of larvae fed only on day 1 (measured on day 2) was later reduced by metabolic requirements, such that, at ecdysis, only a 6.6% increase could be realized. Feeding in shrimp larvae on day 1 may be sufficient to reach the point of reserve saturation (An- ger and Dawirs, 1981; Anger, 1984), or a threshold for growth and development (West and Costlow, 1988), but it may not be enough to support the optimal amount of growth. 35 I I 0) o 30 25 IT rirfl -L CON T T NO-F D-1-F D-2-F D-3-F D-1.2-F Feeding Regime Figure 4. Effect of feeding regime during instar II on growth of the lateral thoraco-abdominal muscle. The width of the muscle in larvae in each feeding regime at the beginning of instar II (open bars) is com- pared to the width of the muscle in that group on the first day of instar III (diagonal stnped bars). The difference in the heights of the paired bars represents the amount of growth of the muscle for that group. Abbreviations as in Figure I. Each bar represents the mean and I SDof 56-162 larvae. REGULATION OF TISSUE GROWTH 221 The opposite result was observed in larvae fed on day 3: i.e.. the actual MI was greater than that predicted. This result may be explained by the reduced food available for growth and metabolism, as predicted by a day 2 cell density equivalent to a predicted MI of 3.0%. If the integ- ument was in a weakened state, as described for epider- mal and muscle tissues of starved crab larvae by Anger ( 1 984), then the stretch at ecdysis due to hydrostatic pres- sure may have overwhelmed the resistance of the new cuticle, along with the epidermis and muscle, resulting in a MI greater than that set by the growth potential. A similar enhanced growth, or stretch, is seen in eyestalk- less larvae (Okazaki et a!.. 1989). Subsequent feeding on day 3 may have provided only enough energy reserves to complete the molt. The day 3 feeding regime spans the critical "point of no return," as suggested by Anger and Dawirs (1981). The larvae that molted may have re- ceived food before this point, while those that remained in SII for extended periods before dying may have re- sumed feeding beyond this point. Tissue degradation and nutrient depletion may not have been reversed by feeding at this time. The results from larvae fed on day 2 suggest that recovery from the starved condition is possi- ble if feeding resumes during the middle of the molt cy- cle. This period may be the limit beyond which starva- tion results in tissue degradation and loss of protein (An- ger, 1984;McConaugha, 1985). Preliminary findings suggest that epidermal growth consists of both cell replication and enlargement of di- vided cells. The contribution of each phase to the growth process is not understood. Analysis of the cell cycle changes in the epidermal cells is necessary to find the control points of the growth process. There may be sev- eral control points where nutritional status may be trans- lated into tissue growth. One may be the entrance to mi- tosis, and another may be the Gl-S transition, both of which have been shown to be control points in many cell types (Murray and Kirschner, 1989; Pardee, 1989). Moreover, the growth process may involve entrance of non-cycling cells into the cycling population. In this study, the epidermis and the muscle were ob- served to grow in a coordinated manner, in agreement with earlier studies on muscle growth in crustaceans (Bit- tner and Traut, 1978: Houlihan and El Haj. 1985). Moreover, muscle growth was affected by nutritional stress in a manner similar to that of the epidermis. These findings would argue that a common mechanism con- trols the coordinated growth of both tissues. Conversely, the epidermis may control growth of the muscle, possibly through cell-cell interactions. These mechanisms are currently being examined. Acknowledgments I thank Dr. Robert K. Okazaki for stimulating discus- sions and Ms. Dianne Laurendeau for technical assis- tance. This research was supported by grant no. Rl I- 8996152 from NSF/EPSCoR and the State of Alabama. Literature Cited Anger, K. 1984. Influence of starvation on moult cycle and morpho- genesis of 1 1 van arum-its larvae (Decapoda. Majidae). Hclgol. H7.v.v. Meeresunters. 38: 2 1-33. Anger, K., and R. Dawirs. 1981 . Influence of starvation on the larval development ofHyas araneus (Decapoda. Majidae). Hi-lgol. H'/.v.v Meeresunters. 34: 287-3 1 1 . Bittner, G., and D. L. Traut. 1978. Growth of crustacean muscles and muscle fibers. / Ctmip. Physiol. 124: 277-285. Freeman, J. A. 1986. Epidermal cell proliferation during thoracic de- velopment in larvae of Anemia. J Crust Biol. 6: 37-48. Freeman, J. A. 1988. Cell growth and the molt cycle in Palaemonctcs larvae. Am. Zool. 28: 94A. Freeman, J. A., T. L. West, and J. D. Costlow. 1983. Postlarval growth in juvenile Rhithropanopeus harrisii. Biol. Bull 165: 409- 415. Halcrow, K. 1978. Cell division in the carapace epidermis ofDaplinia magna Straus (Cladocera). Crustaceana 35: 55-63. Hartnoll, R. 1982. Growth. Pp. 1 1 1-196 in The Biology of Crustacea, Vol. 2, L. G. Abele, ed. Academic Press, New York. Houlihan, D. F., and A. J. El Haj. 1985. An analysis of muscle growth. Pp. 1 5-29 in Crustacean Issues 3. Factors in Adult Growth. A. M. Wenner, ed. Balkema Press. Boston. Knowlton, R. E. 1974. Larval development processes and controlling factors in decapod Crustacea, with emphasis on Caridea. Thallasia Jugoslav. 10: 138-158. LeRoux, A. 1 982. Les organes endocrines chez les larves des crustaces eucarides. Intervention dans la croissance au cours de la vie larvaire et des premiers stades juveniles. OceanisS: 505-531. McConaugha, J. R. 1982. Regulation of crustacean morphogenesis in larvae of the mud crab Rhithropanopeus harrisii. J. E.\p. Zool. 223: 155-163. McConaugha, J. R. 1985. Nutrition and larval growth. Pp. 127-154 in Crustacean Issues 2. Larval (jnmih. A. M. Wenner, ed. Balkema Press. Boston. Murray, A. W., and M. W. Kirschner. 1989. Dominoes and clocks: the union of two views of the cell cycle. Science 246: 6 14-621. Okazaki, R. K., J. A. Freeman, and D. M. Laurendeau. 1989. Cell growth and cuticle expansion in eyestalk-ablated Palaemonetes. Am. Zool. 29: 62A. Pardee, A. B. 1989. G, events and regulation of cell proliferation. Science 246: 603-608. Tchernigovtzeff, C. 1965. Multiplication cellulaire et regeneration au cours de cycle d'intermue des crustaces decapodes. Arch. Zoo/. A'.v/) Gen. 106: 377-497. West, T. L., and J. D. Costlow. 1988. Determinants of the larval molting pattern of the crustacean Balanus eburneus Gould (Cir- ripedia: Thoracica). J. Exp. Zool. 248: 33-44. Reference: Bml. Bull. 178: 222-230. (June, 1990) Determination of Alkaline Phosphatase Expression in Endodermal Cell Lineages of an Ascidian Embryo J. R. WHITTAKER Laboratory oj Developmental Genetics. Marine Biological Laboratory, H 'oods Hole, Massachusetts 02543 Abstract, dona intestinalis embryos develop a strong histochemical localization of alkaline phosphatase activ- ity in their known endodermal tissues. Such tissues arise solely from the four vegetal blastomeres at the 8-cell stage and six vegetal blastomeres at the 16-cell stage; these veg- etal cells inherit an endodermal lineage cytoplasm. Pairs of blastomeres from the bilaterally symmetrical 8- and 16-cell stages were isolated and reared as partial em- bryos. Only those partial embryos derived from endo- derm-containing lineages developed a histochemically localized alkaline phosphatase activity. From the results of such restricted developmental autonomy (self-differ- entiation), one can deduce that this enzymic expression of endodermal fate could be specified by events of cy- toplasmic segregation that occur during the early cleav- ages. This conclusion offers additional support to the theory that specification of cell fate in ascidian embryos involves an early differential segregation of histodeter- mining egg cytoplasmic materials. Introduction Elaboration and refinement of cell lineages and the construction of fate maps for certain widely studied ani- mal embryos continue to be important contemporary parts of experimental embryology. Fate maps produced by marking early blastomeres of the embryo indicate what actually happens to each cell or region as it devel- ops, and are an essential component of attempts to inter- pret the causal relations concerned with eventual re- gional specializations in the embryo. Yet fate maps do not indicate when, during the succession of cleavages, cells become irreversibly committed to the pathways of differentiation they later exhibit during histodifferentia- Received 28 August 1989: accepted 28 March 1990. tion. Our one, and still only, radical criterion for discov- ering whether the prospective fate or "determination" of a blastomere has already been settled during early cleav- ages is that, when the blastomere is isolated from its usual cellular environment and associations, some or all of its progeny cells then differentiate only in the fixed or lim- ited directions predicted by a fate map(Lillie, 1929). The underlying hypothesis is the presumption that eventual cell fate is determined by certain localized cytoplasmic agents that become differentially segregated only into certain cells (Whittaker, 1987). This paper is such an in- vestigation of specification in the endodermal cell lin- eages of dona intestinalis. These embryonic cells even- tually become the branchial and digestive tissues of the postmetamorphic juvenile and adult. The first accurate and extensive cell lineages for ascid- ian embryos were described by Conklin (1905). Endo- dermal lineages are segregated at third cleavage (8-cell stage) to the vegetal four cells of the embryo and thereaf- ter become progressively more restricted to certain vege- tal regions of the dividing embryo. Based on general characteristics of cytoplasmic morphology and staining (Conklin. 1905. 191 1), endodermal lineages appear to inherit a particular kind of cytoplasm. Conklin's original designations of the endodermal lineages have been con- firmed recently in studies using injected horseradish per- oxidase (HRP) as a cell lineage marker (Nishida and Sa- toh, 1983, 1985; Nishida, 1987). Quite early in embryogenesis, beginning at the neurula stage, endodermal tissues develop localizations of an es- sentially histotypic alkaline phosphatase; other larval tis- sues lack any significant amount of it (Minganti. 1954a; Whittaker, 1977). In certain species, development of this enzyme is a simple and unequivocal indication of endo- dermal differentiation. Ascidian embryos, which are di- 222 ASCID1AN ALKALINE PHOSPHATASE 223 vision-arrested at various early cleavage stages with cyto- chalasin B, eventually express alkaline phosphatase only in those cells that are known to be of endodermal lineage (Whittaker, 1977; Satoh, 1982). This suggests further that endodermal expression follows a pattern of cy- toplasmic localizations. However, in such cleavage-ar- rested embryos, the enzyme-developing cells are not sep- arated from the influence of their surrounding cells. The present study examines alkaline phosphatase develop- ment in partial embryos originating from blastomere pairs of the bilaterally symmetrical embryo isolated from early cleavage stages. Eventual enzyme expression exclu- sively follows the known endodermal lineages. Such de- velopmental autonomy is likewise consistent with the hypothesis of a differentially segregated cytoplasmic de- terminant. Materials and Methods Organisms Adult dona intestinal is (L.) were collected near Woods Hole, Massachusetts, and maintained on sea ta- bles with continuously flowing seawater and under con- stant light. Phallusia nuiinmillata (Cuvier) was obtained from two sources: from the Gulf of Palermo in Sicily by courtesy of the Institute of Zoology at the University of Palermo, and from the coast of Brittany at Roscoff (France) through the kindness of Dr. Lionel Jaffe. Eggs from two or more animals were removed surgically from the oviducts and fertilized with diluted sperm obtained from the sperm ducts of other adults. Embryos were de- chorionated manually before first cleavage with sharp- ened steel needles and cultured at 18 ± 0.1 °C in sterile Millipore-filtered (0.2 j/m porosity) seawater containing 0.1 mM EDTA (Crowther and Whittaker, 1983). Under these conditions, larvae became fully developed by 18 h from fertilization. Blastomere isolations Dechorionated embryos of the bilaterally symmetrical 8-cell stage of dona were the starting point for isolations of various cell pairs at the 8- and 1 6-cell stages. Blasto- meres were separated with agar-coated glass filament needles. Partial embryos from various isolations were then reared in agar-coated Syracuse watch glasses for pe- riods of development up to 20 h before they were pro- cessed histochemically for an alkaline phosphatase reac- tion. Cleavage inhibition Exposure to cytochalasin B (Aldrich) (2 Mg/ml) pre- vented cell division in embryos (Crowther and Whitta- ker, 1983). Alkaline phosphatase histochemistry The 80% cold ethanol fixative used previously with chorionated ascidian embryos (Whittaker, 1977) gave satisfactory but often variable results when applied to de- chorionated partial embryos. This variability was elimi- nated by employing the glutaraldehyde-formaldehyde fixative devised by Karnovsky (1965) for electron micro- scopic histochemistry; it was used here at 0.5% each, half the suggested aldehyde concentrations. One-hour fixa- tion (5°C) and 20-min wash in the recommended su- crose-containing cacodylate buffer (Karnovsky, 1965) produced excellent and reproducible results with the subsequent alkaline phosphatase reaction. Background staining was insignificant under these conditions. Fixed whole or partial dechorionated dona embryos were reacted for 24 h (at 18°C) in standard Gomori me- dium with /3-glycerophosphate as substrate and the cal- cium phosphate product afterwards visualized by a silver reduction technique (Pearse, 1972). This results in a sta- ble brown deposit of reduced silver at the sites of alkaline phosphatase activity. The stained specimens were dehy- drated in ethanol, cleared in xylene, and mounted in dammar resin. Some staining for alkaline phosphatase activity on lar- vae and dechorionated whole and partial embryos was done with a tetrazolium method using bromochloroin- doxyl phosphate (BCIP) as substrate (McGady, 1970, and as described by Whittaker and Meedel, 1989). Both fixation methods were used with this procedure; similar results were obtained with each. Acetylcholinesterase histochemistry Assays were done on dechorionated whole and partial embryos after using the same aldehyde fixative described above for alkaline phosphatase. The cholinesterase method of Karnovsky and Roots (1964) was applied as described by Meedel and Whittaker (1984). Results Endodermal cell fates predicted by cell lineage studies The most accurate cell lineage relationships for poten- tial endodermal expression have been obtained from cell marking studies with injected HRP (Nishida and Satoh, 1983, 1985;Nishida, 1987). Figure 1 is a lineage diagram of the cell contributions up to the 1 6-cell stage: at the 8- cell stage the four vegetal blastomeres (the bilateral pairs of A4.1 and B4.1) contain endodermal lineages; at the 1 6-cell stage only six of the eight vegetal cells contain en- dodermal lineages. The sensitivity of the HRP detection method has also permitted an identification of the lin- eage origins of certain smaller structures of the embryo designated in Figure 1 as secondary. Figure 2 depicts in 224 J. R. WHITTAKER AB2 - r- a5.3 — r-o4.2-^ 1- a5.4 — I-A3- r- A5.I — LA4.I -\ L A5.2 — Endoderm Trunk I.e. Endoderm Endoderm; i- B5.1 — {B4.1-^ L- B5.2 — Endodermal Notochord Endodermal strand; Trunk v.c. strand h4 ? —\ Endodermal strand 2-CELL 4-CELL 8-CELL 16-CELL Figure 1 . Fate map of endodermal cell lineages in ascidian embryos up to the 16-cell stage, according to Conklin ( 1905), Ortolani ( 1954), and Nishida and Satoh (1983, 1985). Cells from one-half of the bilater- ally symmetrical embryo are indicated. Primary (1°) and secondary (2°) tissues which ultimately develop alkaline phosphutase are underlined. Nomenclature is that of Conklin ( 1905). a middle tailhud stage ( 1 1 h) embryo the various "endo- dermal" regions actually observed by such studies and their respective origins from the bilaterally symmetrical pairs of blastomeres at the 16-cell stage (identified in Fig. 1 ). The regions shaded with diagonal lines in the head part of the embryo diagram (Fig. 2) are the locations of the cells that give rise to the endodermal organs of the post- metamorphic juvenile: a branchial basket and the diges- tive system. These are the fates of the primary endoder- mal cells in Figure 1 . The tail is a strictly larval structure and its tissues, including the endodermal strand (an ex- tension of the main endodermal mass running mid-ven- tral to the notochord), and the notochord itself are de- stroyed at the time of larval metamorphosis. Other tissue areas of the embryo-larva (muscle, neural, and epider- mal) are not shown. Questions about the minor endodermal structures identified in Figures 1 and 2 arise from the cell lineage studies. "Trunk lateral cells" (TLC), which occur in two superficial dorsal wing-like accumulations on either side of the mid-tailbud embryo, are undefined in their fate yet still share a major endodermal lineage as late as the 32- cell stage (Nishida and Satoh, 1985); at the 64-cell stage they separate from endoderm as a separate lineage (Nish- ida, 1987). Two small circular patches of cells, which I have called "trunk ventral cells" (TVC), occur along ei- ther side of the embryo ventral midline at the base of the tail. These have been classified as endodermal by Nishida (1987) because of their location in an endodermal re- gion, but they originate after the 128-cell stage from a lineage (B5.2) that is entirely mesodermal from the 16- cell stage onwards. Finally, two short distal segments of the endodermal strand originate from cells (B5.2 and b5.3 cell pairs) which, after the 16-cell stage, are not oth- erwise endodermal lineages. Alkaline phosphatase expression in tissues of endodermal lineage Because differential alkaline phosphatase expression has been regarded as a histotypic indicator of early differentiation in endodermal tissues of some ascidian species (Minganti, 1954a, and others), occurrence of en- zyme in the so-called secondary larval tissues (Fig. 2) would be a confirmation of their endodermal specifica- tion. The Gomori and BCIP methods of enzyme detec- tion have been applied to 1 1-h (middle-tailbud stage) Ci- ona embryos and also to embryos cleavage-arrested in cytochalasin B at 1 1 h and fixed for reaction at 20-28 h postfertilization. Cleavage-arresting dechorionated 1 1-h embryos has the interesting effect of causing an earlier and more concentrated development of enzyme in cells that might ordinarily divide further. Alkaline phospha- tase does not usually occur strongly in endoderm-de- rived tail tissues until 6-8 h after hatching. Figure 3 shows the BCIP staining of such a cleavage-arrested larva. In previous lineage studies, the middle tailbud stage was used as the reference stage for tissue locations (Nishida, 1987; Nishida and Satoh, 1983, 1985). Cleav- age-arrested 1 1-h embryos (Fig. 3) enable us to make a direct comparison to those results (Fig. 2). Only tissues arising from lineages that share a primary endodermal lineage until after the 16-cell stage are seen to develop alkaline phosphatase. This includes the eight cells at the tip of the notochord, which have such an en- dodermal lineage origin but are not structurally or func- tionally endodermal cells; their lineages first separate from endoderm at the 32-cell stage. The two short termi- nal segments, which are structurally a part of the endo- dermal strand, but which do not share endodermal lin- eages after the earliest cleavages, do not produce alkaline phosphatase. During the first 30 min of BCIP staining, one can see the TLC reacting strongly against the initially lighter staining of the underlying other endodermal cells. The staining time required to reveal the endodermal strand clearly (2-3 h) soon results in sufficient reaction product in the head region to obscure the TLC. During initial staining, or later, one can not see any differential alkaline phosphatase stain in the TVC region. In normal 1 1-h embryos, one finds the same differential staining with BCIP in the TLC, but at that time enzyme has not yet developed in the endodermal strand or notochordal tip cells, as it has in the cleavage-arrested embryos reacted after "hatching." Given their sharing of an endodermal lineage, and their expression of alkaline phosphatase, it seems unlikely that the TLC would be the precursors of ASCIDIAN ALKALINE PHOSPHATASE 225 TLC A5.1 (A5.2) NC B5.1 B5.2 b5.3 A5.2 B5.1 Figure 2. Diagram of" the middle tailhud stage ( 1 1 h) ascidian em- bryo showing the endoderm and possibly endodermally related tissues identified from lineage tracing studies (Nishida and Satoh. 1983, 1985; Nishida, 1987). Lineage origins at the 16-cell stage are given for each defined tissue region. Those tissues that eventually express alkaline phosphatase, including the trunk lateral cells (TLC). the proximal en- dodermal strand (ES), and the distalmost cells of the notochord (NC). are indicated with fine diagonal lines. The (stippled) trunk ventral cells (TVC) develop acetylcholinesterase but not alkaline phosphatase. Figure 3. Dechorionated 1 1-h (middle tailbud) Ciinia intestinalix embryo cleavage-arrested at 1 1 h with cytochalasin B and reacted at 28 h (after fixation) for alkaline phosphatase with the BCIP reagent. Incubation time is 2 h. Bar = 50 ^m. juvenile blood cells (a presumably mesodermal deriva- tive), as suggested by Nishide el al. (1989). The Gomori stain is much less sensitive than the BCIP stain. It does not reveal any enzyme activity in the endo- dermal strand, but shows clearly the TLC staining differentially in the head region (not shown). The two bilateral TVC regions stain significantly for acetylcholin- esterase, a characteristic expression of differentiating muscle and mesenchyme tissue regions (Meedel and Whittaker, 1979). This TVC staining (not shown) occurs in normal 1 1-h embryos as well as 1 1-h cleavage-arrested embryos. By their location, these apparently mesoder- mal tissues may be the primordia of the juvenile heart. Although the inner distal segment of the endodermal strand originates (at the 64-cell stage) from what is other- wise a mesodermal lineage (B5.2), these tissues do not develop an acetylcholinesterase. They thereby differ from the notochordal tip cells by not expressing a vestige of their secondary origin. Design of cell isolation experiments At the 8-cell stage, third cleavage divides the bilaterally symmetrical embryo equatorially across the animal-veg- etal axis into a vegetal quartet of cells containing the en- dodermal lineages and an animal quartet that has no en- dodermal fate (Fig. 4A). Ortolani (1954) used adhering carbon particles to mark the surface areas of the four veg- etal blastomeres which eventually appear in endodermal tissues. Under conditions of appropriate lighting (see be- low) one can actually observe the endodermal regions in- dicated in Figure 4A by the diagonal lines, to contain much more yolky material than the other part of the cell, which remains noticeably clearer; these yolky areas have relatively sharp edges. This confirms Conklin's (1905) observations as well. In this investigation, various blasto- mere pairs have been isolated microsurgically at the 8- and 16-cell stages (Figs. 4B, 4C). Separation of lineage blastomeres The dona 8-cell stage has a very characteristic pattern in lateral aspect (Figs. 4 A, 5) by which the four cell pairs can be identified. Often the pair of polar bodies can be seen resting on the a4.2 cells at the animal pole as dia- grammed in Figure 4A, but the shapes and apparent sizes of the cells are most diagnostic of their lineage. In doing the isolations, overhead lighting (from a quartz halogen fiber optic lamp) is preferred, with a substage mirror set to reflect back some of this illumination. With properly balanced direct/indirect lighting, the vegetal cell pairs (A4.1 and B4. 1), which contain more yolk, appear slightly darker than the others. This facilitates recogni- tion of the A4. 1 cells, which thereby appear darkest. The B4. 1 cells are somewhat flattened in the animal-vegetal direction and seem to be slightly larger in lateral view than the other pairs of cells. Animal and vegetal quartets (half-embryos) were iso- lated by separating the sets as shown in Figure 4B. The cell size and pattern of arrangement in the animal and vegetal quartets (Figs. 6, 7) further simplifies identifica- tion of the various cell pairs at the "8-cell stage" in ob- taining quarter-embryos (Fig. 4C). Similarly, when the A4. 1 and B4. 1 blastomere pairs divide again at the " 1 6- cell" stage, there were characteristic sizes and patterns of cells (Figs. 8, 9), which enabled one to isolate the correct lineage pairs. 226 J. R. WHITTAKER B Figure 4. Diagrams of the surgical operations involved in isolating hlastomeres from the 8-cell stage (A). Isolation of animal and vegetal quartets (B) and quarter-embryos (C). The endodermal territories as mapped hy Ortolani ( 1954) are indicated by diagonal lines. Size and pattern of the cells can be learned initially from Conklin'sf 1905 (diagrams, which are exceptionally accurate. One can also observe the shapes, sizes, and po- sitions of cells (//; situ) when isolated half-embryos divide again in culture. Partial larvae resulting from given blas- tomere pairs have very distinctive morphologic features Figures 5-9. dona intestinalis embryo and isolated blastomeres photographed after brief fixation in the Karnovsky ( 1965) fixative. Fig- ure 5: 8-cell stage in lateral view as in Figure 4A. Figure 6: Animal quartet of cells from 8-cell stage. Figure 7: Vegetal quartet of cells from the 8-cell stage. Figure 8: Isolated A4. 1 cell pair after the next division. Figure 9: Isolated B4.1 cell pair after the next division. The embryo orientation letters are AN (animal), VEG (vegetal), A (anterior), and P (posterior). All magnifications are the same; bar in Figure 7 = 50f/ hisiochemically locali:ed alkaline phosphatase in isolated partial embryos o/Ciona intestinalis B5.1 Blastomeres isolated Number of experiments Embryos examined (positive reaction/total) 8-cell stage B5.2 animal quartet 7 0/155 a4.2 pair 4 0/45 b4.2 pair 4 0/45 vegetal quartet 5 115/115 stomeres A4. 1 pair 6 73/73 live. Fig- B4.l pair 6 107/107 Animal 16-cell stage ells from A5.1 pair 2 42/48 division, embryo A5.2 pair B5. 1 pair 2 3 52/56 60/62 r), and P 50 urn. B5.2 pair 3 1/56 ASCIDIAN ALKALINE PHOSPHATASE 227 c B5.1 B5.2 Figures 10-16. dona intestinalis embryos after 18-20 h of devel- opment, reacted for alkaline phosphatase (Gomori). Bar = 50 ^m. Fig- ure 10: Larva from dechorionated whole embryo. Figure 1 1: Animal The experimental results presented here were done with isolated blastomeres from embryos with completely normal cleavage patterns. In association with the experi- ments of Table I, 5 series containing a total of 1 62 whole dechorionated embryos selected for normal cleavage pat- terns were reared to "hatching" time. Ninety percent of these embryos developed to fully formed "normal" lar- vae. In most respects, dona larvae originating from de- chorionated early embryos are normal, but they lack a properly formed tail fin of the translucent test covering (as previously noted with another species by Cloney and Cavey, 1982). No animal half-embryos developed enzyme (Fig. 1 1 ), nor did any quarter-embryos (a4.2 or b4.2) derived from the animal quartet. Vegetal half-embryos always pro- duced enzyme (Fig. 12), as did the A4. 1 and B4. 1 quar- ter-embryos (Figs. 1 3 and 14) prepared from the vegetal half of the 8-cell stage. Partial embryos developing alka- line phosphatase invariably contained tissue staining over a large area of the resulting partial larva (Figs. 12- 15). Three experimental "control" series were done in which animal and vegetal quartets were isolated and cul- tured only until 4 h after fertilization before they were fixed and reacted. These contained no localized enzyme. Ordinarily, experimental and control embryos were cul- tured for 18-20 h. Partial embryos from A4. 1 and B4. 1 vegetal cells pre- pared after the next cleavage (the "16-cell" stage) also showed alkaline phosphatase development correlated with the segregation of endodermal lineages. Most of the A5.1 and A5.2 '/sth-embryos produced a mass of en- zyme-containing cells. A few embryos did not react. Al- most all of the B5.1 '/sth-embryos developed enzyme (Fig. 15), and essentially none of the B5.2 embryos (Ta- ble I). One B5.2 embryo (out of 56) developed some alka- line phosphatase. Because microsurgical preparation of partial embryos from 16-cell stage blastomere pairs nec- essarily entailed some sequential slight bruising of the cells, the one B5.2 embryo that produced alkaline phos- phatase might reasonably have resulted from a missegre- gation of cytoplasm caused by such trauma. A striking further illustration of the correlation be- tween alkaline phosphatase development and endoder- mal lineage segregation occurs with a cleavage-arrested partial embryo. B4.1 pairs were isolated (Fig. 4C) and, before being treated with cytochalasin B, were permitted to undergo an additional cleavage creating an embryo with B5. 1 and B5.2 daughter pairs (Fig. 9). Almost all of half-embryo. Figure 12: Vegetal half-embryo. Figure 13:A4.1 quarter- embryo. Figure 14: B4. 1 quarter-embryo. Figure 15: B5.1 eighth-em- bryo. Figure 16: Divided B4.1 embryo (as in Fig. 9), cleavage-arrested in cytochalasin B, and showing the B5. 1 and B5.2 cell pairs. 228 J. R. WHITTAKER these embryos (17 out of 18) had staining in both B5.1 cells and none had staining in the B5.2 cells (Fig. 16). This experiment complements work in a previously pub- lished study on expression in cleavage-arrested dona embryos (Whittaker, 1977). When the more sensitive BCIP staining method was used to localize alkaline phosphatase in partial embryos, the same strict lineage expressions could be demon- strated as shown above with the Gomori method. How- ever, separating blastomeres before complete closure of the cytoplasmic bridges between daughter cells some- times caused transfer of very small amounts of cyto- plasm, which resulted subsequently in tiny regions of en- zyme expression. These expressions could not be de- tected with the less-sensitive Gomori technique. Such transfers were avoided by a change in isolation tech- niques. The results will be described in detail elsewhere in another context. Alkaline phosphatase in Phallusia embryos When hatched Phallusia larvae were reacted for alka- line phosphatase uniformly dark Gomori and BCIP staining reaction products occurred in all the tissues, with no indication of localized staining. Similarly, when dechorionated eggs and embryos of early cleavage stages were reacted for enzyme, a same dark reaction product was also found throughout the whole. With each method, this screen of general staining obscured any pos- sibility of seeing a localized specific staining that might otherwise develop in endodermal tissues. These stainings appear to result from the activity of a universally distrib- uted phosphatase enzyme already present in the egg. Unfortunately, Minganti (1954a) failed to note this staining of Phallusia larvae in his survey of several ascid- ian species. His further observation that partial embryos ofP/iulliisiii originating from isolated animal and vegetal quartets of the 8-cell stage both have staining (Minganti, 1954b) proves to be correct, but indicative only of en- zyme already present and not of new alkaline phospha- tase formation in the animal half-embryo during devel- opment. Discussion The classic study by Reverberi and Minganti (1946) on the fate of blastomere pairs isolated at the 8-cell stage showed that anterior and posterior vegetal pairs (A4.1 and B4. 1 ) give rise to quarter-embryos containing some general histological features of organization resembling early gut tissues. Quarter-embryos arising from the ani- mal blastomere pairs (a4.2 and b4.2) did not have this organization, but unfortunately such histologic charac- ters lack the discrimination and sensitivity for evaluating minor expressions of endodermal differentiation. Except for a predominance of yolk granules in cells of endoder- mal lineages and some other lineages (e.g., notochordal) derived from the vegetal half of the egg (Mancuso and Dolcemascolo, 1979), there are no simple cytospecific features of endodermal differentiation even at the ultra- structural level. However, a strong alkaline phosphatase development proves to be a simple, sensitive, and essen- tially histotypic indicator of an early endodermal differ- entiation, at least in some species. An elevated alkaline phosphatase appears to be a universal constituent of the digestive systems of animals (McComb ct at., 1979). As noted in the results, there are ascidians (Phallusia) in which any possible differential development of enzyme is obscured by a uniformly distributed strong alkaline phosphatase activity present throughout development, and originating in the egg before development begins. Results of the present blastomere isolation study indi- cate that partial embryos derived from "endodermal" blastomeres isolated at 8- and 16-cell stages self-differen- tiated extensive patches of cells containing high levels of alkaline phosphatase. Embryos obtained from the non- endodermal lineages did not produce alkaline phospha- tase, at least at any visual level of differential histochemi- cal staining. These findings are in agreement with the fates indicated by previous lineage studies and with ex- pressions of enzyme observed in cleavage-arrested em- bryos (Whittaker, 1977, and Fig. 16). A restriction of fate occurs, therefore, in parallel with the lineage. The endo- dermal lineage map is apparently also a fate map. The theory behind the early specification of cell fate in ascidian embryos and other so-called mosaically devel- oping organisms is the likelihood of differential segrega- tion of specific egg cytoplasmic materials (Lillie, 1929). Ooplasmic rearrangements that occur immediately after fertilization create visible and presumably chemically distinct regional differences in zygote cytoplasm; these regions become segregated into certain cell lineages as the germ divides (Conklin, 1905, 191 1). Cell fate is then ordered by agents or substances (determinants) within these regions of cytoplasmic difference. The present re- sults are consistent with this theory. Some credence can be attached to the theory because muscle lineage fate in ascidians appears to be transferrable to other cells along with myoplasmic cytoplasm (Whittaker, 1987). One might conclude that endodermal alkaline phos- phatase expression is regulated by segregation of an egg cytoplasmic determinant into the major (functional) tis- sues. Because of their early inclusion in an endodermal lineage, the eight distal notochordal cells that express al- kaline phosphatase have probably inherited determinant by segregation. The two distalmost short segments of the endodermal strand are actually not derived from imme- diate endodermal lineages, do not express enzyme, and would seemingly not have received any determinant. ASCID1AN ALKALINE PHOSPHATASE 229 The unresolved nature of egg cytoplasmic determi- nants remains an important issue in embryology (David- son, 1990). In certain cases these agents seem to be masked maternal messenger RNAs for some of the pro- teins involved in a later developmental change. There is indirect evidence from experiments with actinomycin D and other inhibitors of RNA synthesis that the ascidian alkaline phosphatase determinant could be such a pre- formed maternal mRNA (Whittaker, 1977; Bates and JefTery. 1987). Bates and JerTery (1987) have observed by histochem- istry that "activated" but nonnucleate zygote fragments do not elaborate the endodermal alkaline phosphatase after time. Their results were confirmed by more sensi- tive quantitative measurements on similar material (Whittaker and Meedel, 1989). Also, aphidicolin, an in- hibitor of DNA synthesis, has a time-window effect on dona alkaline phosphatase development (Satoh, 1982). While such findings do not establish the necessity of a gene transcription, they indicate a possible involvement of nuclear replication events in releasing the expression of alkaline phosphatase. Nuclear division might be re- lated to a mechanism for processing an inactive mRNA. One possible mRNA processing mechanism to con- sider is translational activation by polyadenylation of a dormant maternal mRNA. There are some examples in the recent literature of translational activation of dor- mant mRNAs being accompanied by elongation of their 3' poly(A) tails (see McGrew et ai, 1989). Huarte el al. ( 1987) describe an mRNA for mouse oocyte tissue plas- minogen activator that accumulates in the cytoplasm during oocyte growth; translational activation of this mRNA occurs at meiosis and is accompanied by in- creased 3'-polyadenylation. Actual meiotic changes may be necessary since the mixing of cytoplasm and nucleo- plasm at germinal vesicle breakdown is not sufficient to initiate the processing. This meiosis-initiated polyadeny- lation is insensitive to inhibition of RNA synthesis. Further speculation about the nature of an alkaline phosphatase determinant would be aided by more direct evidence that there is a differentially segregated egg cy- toplasmic factor, and by some information about the conditions under which it functions. The present investi- gation has established a biological background within which to pursue such questions. A next paper will pres- ent evidence that moving endodermal lineage cytoplasm to nonendodermal lineages results in the acquisition of alkaline phosphatase expression. Acknowledgments This work was supported by Grant HD-21823 from the National Institute of Child Health and Human De- velopment (NIHHS). I thank Robert J. Crowther and Jane L. Loescher for technical assistance and Drs. Giu- seppina Ortolani and Nunzia Farinella-Ferruzza of the University of Palermo for their initial guidance and in- struction. Literature Cited Conklin,E.G. 1905. The organization and lineage of the ascidian egg. J. Acad. Nat. Sci. {Philadelphia) 13: 1-119. Conklin, E. G. 1911. The organization of the egg and the develop- ment of single biastomeres of Phallusia mamillata. J E\p. Zool. 10: 393-407. Bates, W. R., and W. R. Jeffery. 1987. Alkaline phosphatase expres- sion in ascidian egg fragments and andromerogons. Dev. Biol 1 19: 382-389. Cloney, R. A., and M. J. Cavey. 1982. Ascidian larval tunic: extraem- bryonic structures influence morphogenesis. Cell Tissue Res 222: 547_562. Crowther, R. J., and J. R. \\hittaker. 1983. Developmental auton- omy of muscle fine structure in muscle lineage cells of ascidian em- bryos. De v. Biol 96: 1-10. Davidson, E. H. 1990. How embryos work: a comparative view of diverse modes of cell fate specification. Development 108: 365-389. Huarte, J., D. Belin, A. Vassalli, S. Strickland, and J.-D. Vassalli. 1987. Meiotic maturation of mouse oocytes triggers the transla- tion and polyadenylation of dormant tissue-type plasminogen acti- vator mRNA. Genes Dev 1 : 1 20 1 - 1 2 1 1 . Karnovsky, M. J., and L. Roots. 1964. A "direct-coloring" thiocho- line method for cholinesterase. / Histochem. Cytochem. 12: 219- 221. Karnovsky, M. J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol 27: 137A-138A. Lillie, F. R. 1929. Embryonic segregation and its role in the life his- tory. \\'ilhelm Roux's Arch. Enlwicklungsmech. Org 118:499-553. McComb. R. B., G. N. Bowers Jr., and S. Posen. 1979. Alkaline Phos- phatase Plenum Press, New York. 986 pp. McGady, J. 1970. A tetrazolium method for non-specific alkaline phosphatase. Histochemistry23: 180-184. McGrew, L. M., E. Dworkin-Rastl, M. B. Dworkin, and J. D. Richter. 1989. Polyl A (elongation during .Yc'«o/7!/.v oocyte maturation is re- quired for translational recruitment and is mediated by a short se- quence element. Genes Dev 3: 803-8 1 5. Mancuso, V., and G. Dolcemascolo. 1979. Ultrastructural aspects of the endoderm cells of the dona iniexiinalis embryo during the tail lengthening phase. Ada Emhryol. E.\p. 1979: 161-171. Meedel, T. H., and J. R. Whittaker. 1979. Development of acetyl- cholinesterase during embryogenesis of the ascidian dona intc.\ti- nalis.J.Exp. Zool. 210: 1-10. Meedel, T. H., and J. R. VVhittaker. 1984. Lineage segregation and developmental autonomy in expression of functional muscle ace- tylcholinesterase mRNA in the ascidian embryo. Dev. Biol 105: 479-487. Minganti, A. 1954a. Fosfatasi alcaline nello sviluppo delle Ascidie. Puhbl. Sla; Zool. Napoli 25: 9-17. Minganti, A. 1954b. Fosfatasi alcaline nei semiembrioni animali e vegetative di Ascidie. Puhhl Sla:. Zool. Napoli 25: 438-443. Nishida, H. 1987. Cell lineage analysis in ascidian embryos by intra- cellular injection of a tracer enzyme. III. Up to the tissue restricted stage. Dev. Biol. 121:526-541. Nishida, H., and N. Satoh. 1983. Cell lineage analysis in ascidian em- bryos by intracellular injection of a tracer enzyme. I. Up to the eight-cell stage. Dev. Biol 99: 382-394. Nishida, H., and N. Satoh. 1985. Cell lineage analysis in ascidian em- 230 J. R. WHITTAKER bryos by intracellular injection of a tracer enzyme. II. The 16- and 32-cell stages. Dc\- Biul. 1 10: 440-454. Nishide, K., T. Nishikata, and N. Satoh. 1989. A monoclonal anti- body specific to embryonic trunk-lateral cells of the ascidian Halo- cvnlhin rorei:i stains coelomic cells of juvenile and adult basophilic blood cells. Dfv Growth Differ. 31: 595-600. Ortolani, G. 1954. Risultati definitivi sulla distnbuzione dei territori presuntivi degli organi nel germe di Ascidie allo stadio VIII. deter- minuti con !e marche al carbone. Puhbl. Sla:. Zoo/. Napoli 25: 161- 187. Pearse, A. G. E. 1972. Histochemislry. Theoretical and Applied, Vol. 2. 3rd ed. London: Churchill Livingstone, London. 1518 pp. Reverbcri, G., and A. Minganti. 19-16. Fenomeni di evocazione nello sviluppo di Ascidie. Risultati dell'indagine sperimentale sull'uovo di Ascidiella aspersa e di Ascidia malaca allo stadio di otto blastom- eri. Pithhl. Sla:. Zooi Napoli 20: 199-252. Satoh, N. 1982. DNA replication is required for tissue-specific en- zyme development in ascidian embryos. Differentiation 21: 37-40. Whittaker, J. R. 1977. Segregation during cleavage of a factor deter- mining endodermal alkaline phosphatase development in ascidian embryos./ Exp. Zoo! 202: 139-153. \Vhittaker, J. R. 1987. Cell lineages and determinants of cell fate in development. Am. Zoo! 27: 607-622. VVhittaker, J. R., and T. H. Meedel. 1989. Two histospecific enzyme expressions in the same cleavage-arrested one-celled ascidian em- bryos./ Exp. Zoo/. 250: 168-175. Reference: Bin/. Bull. 178: 231-238. (June, 1990) The Effect of Strontium on Embryonic Calcification of Aplysia californica JOSEPH P. BIDWELL1, ALAN KUZIRIAN2, GLENN JONES3, LLOYD NADEAU4, AND LISA GARLAND3 Howard Hughes Medical Institute, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543 Abstract. During embryogenesis of the marine opis- thohranch gastropod Aplysia californica Cooper, 1 863, there is a brief critical time (window) during which stron- tium is essential for the onset of calcification. The present study was undertaken to elucidate the role of this ele- ment in mineralization. Strontium performed no struc- tural function; deformed shells of strontium-deprived animals had normal atomic crystal structure and the ele- ment was excluded during calcification. Calcium deposi- tion and fixation was reduced by approximately 80% in the absence of strontium but was not significantly altered in the presence of sub-optimal concentrations of this metal ion despite dramatic deficits in shell and statolith morphology. This suggests that calcium deficiency per se is not responsible for deficits induced by strontium deprivation. The reduced total calcium may be a second- ary effect resulting from the complete inhibition of pre- cipitation. Strontium did not modulate total alkaline phosphatase activity or total sulfated mucopolysaccha- ride synthesis during embryogenesis, and no morpholog- ical abnormalities of the organic shell were observed. Al- though the role of strontium in embryonic calcification of Aplysia californica remains enigmatic, these data sug- gest that strontium affects a highly discrete regulatory component because these more general indicators of Received 19 July 1989; accepted 26 February 1990. 1 Present address: Endocrine Research Unit, Mayo Clinic and Foun- dation, Rochester, Minnesota 55905. To whom requests for reprints and all correspondence should be addressed. ; Marine Biological Laboratory, Woods Hole, Massachusetts 02543. 1 Woods Hole Oceanographic Institution. 4 Present address: Toxikon, 225 Wildwood Avenue, Woburn, Massa- chusetts 01 801. calcification and differentiation are unaffected by its absence. Introduction Strontium is required for the normal embryonic devel- opment of a variety of marine molluscs including gastro- pods (Bidwell et ai. 1986), bivalves (Gallager el a/., 1989), and cephalopods (Hanlon et a/., 1989). Embryos reared in the absence of strontium lack mineralized statoliths or shell, yet the soft-tissues appear normal. There is a critical window for strontium during embryo- genesis of the opisthobranch gastropod Aplysia californica Cooper, 1863; normal mineralization requires 4 ppm strontium [~45.7 ^M, half the concentration of natural seawater(Bruland, 1983)], lower concentrations ( 1-3 ppm) or exposure to high levels (80 ppm) results in abnormal and incomplete calcification (Bidwell et ai, 1986). The dramatic specificity of the effect of strontium on molluscan embryogenesis affords a unique opportunity to study mineralization, a process only superficially char- acterized for any organism (KrampitzandGraser, 1988). Is the role of strontium primarily structural, as in the SrSO4 tests of the marine protozoa Acantharia cantharia spp. (Anderson, 1981), or is a biochemical mechanism operative? We report here the effects of varying the strontium concentration of artificial seawater on the atomic crystal structure of the embryonic shell and statoliths of Aplysia californica. We also describe the effects of medium stron- tium concentration on whole embryo levels of calcium, strontium, and magnesium throughout embryogenesis. Finally, we present histochemical analyses of strontium's influence on alkaline phosphatase activity and muco- polysaccharide synthesis. 231 232 J. P. B1DWELL ET AL Materials and Methods Bioassay Bioassays were conducted as described previously (Bidwell et ai, 1986). Briefly, a fresh egg mass ofAplysia californica was washed in basal medium, an artificial sea- water consisting of the major salts of natural seawater except SrCl:-6H;O. Stock solutions of SrCl2-6H:O, CaCl:-2H:O, and MgCl:-6H:O were standardized via flame atomic absorption. The egg mass was cut into strands and distributed among treatments. Natural sea- water (NSW) was prefiltered ( ~ 1 /im, Millipore) before use and the pH and salinity recorded (pH ~ 8.0, salinity 32%o). All experiments were conducted at 21-22°C un- der constant illumination. Treatment waters were re- placed with fresh media as noted below. X-ray crystallographic experiments Embryos were reared in one of three treatments in- cluding basal medium plus 3 ppm Sr (strontium. 34.2 fiM),- basal medium plus 80 ppm Sr (0.913 mM) and NSW. To obtain sufficient material for X-ray analysis, approximately 30 g of egg strands were maintained in 1 2 1 of each test medium. Media were aerated constantly and replenished daily. At 1 68 h after oviposition, strands were removed from treatment, rinsed with a 4% solution of ammonium ace- tate (to pH 8 with ammonium hydroxide), stored at -80°C until lyophilized, soaked in 5% reagent sodium hypochlorite (Baker) for 24 h. and the empty shells rinsed and dried with methanol. Adult shells, removed from the mantle tissue of 20 animals, were prepared as described above. Samples were analyzed for crystal structure using X- ray diffraction methods. One-quarter to one-half gram of clean shells was backed with 40 mesh granular zinc and press-mounted onto standard Phillips aluminum sample holders using an applied pressure of 2 tons. Data were obtained with a Phillips Model 3500 XRD with a fine- focus Cu-kcv X-ray tube operated at 40 kV and 20 ma. A theta compensator slit was placed between the sample and the X-ray tube, and a 0.2° receiving slit and graphite crystal monochrometer were placed between the sample and the scintillation detector. The X-ray analysis was controlled using an IBM-AT computer and all data were collected and stored digitally. 5 We express medium ionic concentrations initially in units of parts per million (ppm) and molar (M) but use ppm thereafter. Whole em- bryo concentrations of calcium, strontium and magnesium are ex- pressed in moles/mg ash because mole units are more descriptive when comparing concentrations of different elements (see Bidwell and Spolte. 1985. fora review of this topic). Each sample was scanned from 3 to 60° 20 (29.45-1.54 A d-spacing) in 0.03° steps, counting for 3 s between each step. Calcium, strontium, ami magnesium analysis of whole embryos Egg strands ( 1 cm) from a single egg mass were distrib- uted randomly between the five treatments of basal me- dium (0 ppm Sr), 3 ppm Sr. basal medium plus 8 ppm Sr (91.3 nAl), 80 ppm Sr, and NSW. Three egg strands were placed in each covered petri dish and its replicate. The test media (50 ml/dish) were replaced twice daily. Throughout development, six strands were collected for each treatment, rinsed with the ammonium acetate solu- tion, transferred to microcentrifuge tubes (1.5 ml), plunged into liquid nitrogen, and stored at -80°C. Egg strands were lyophilized, the individual dry weights re- corded, and ashed at 500°C (3 h) for ash weight. The ashed strand was digested to dryness with 50 ^1 of con- centrated nitric acid (EM, Specpure) and stored at 4°C. One hour before analysis the residue was dissolved in 0. 1 N HC1 (EM Specpure)/5 mM LaClr7H2O (Fisher AA grade). A Perkin Elmer 2280 atomic absorption spectropho- tometer equipped with an HGA-400 graphite furnace was used to determine total calcium, magnesium, and strontium [methodology after Delaney (1983)] in whole embryos. Calcium and magnesium were analyzed via flame spectroscopy and strontium was determined with the furnace. The strontium and magnesium distribution coeffi- cients were determined from these data. The distribution coefficient is defined as: K [A/]/[Ca] tissue ~ [A/]/[Ca] medium where M is the mole concentration of strontium or mag- nesium (Dodd. 1967). This ratio is a unitless index for characterizing biochemical and/or crystallographic dis- cernment. A value of one indicates a lack of regulation, below one is evidence for discrimination and above one concentration. In this study the [A/]/[Ca] ratio of the whole embryonic tissue, including the egg strand, was de- termined rather than the tissue or mineral alone because a clean separation of the embryo, shell, statolith, and egg strand with ultrapure reagents was not successful. Alkaline phosphatase experiments Egg strands, from a single egg mass, were maintained either in 2-1 erlenmeyer flasks under constant aeration (45, 1 cm strands/flask) or in the petri dishes (3, 1 cm THE EFFECT OF STRONTIUM ON CALCIFICATION 233 strands/dish). Test media were replenished every 48 h for the 2-1 flasks and twice daily for the petri dishes. Treat- ments included NSW, 0 ppm Sr, 8 ppm Sr, and 8 ppm Sr plus 0.09 ppm beryllium ( 1 0 ^ A/ Be, as BeSO4 • 4H2O). Be is a potent inhibitor of alkaline phosphatase, both in v///-<» (Aldridge, 1950;Chevremontand Firket, 1951) and in vivo (O'Day, 1972). Dose response experiments were conducted with Be (9 X 10~5 to 45 ppm) in the presence of 8 ppm strontium. Histochemistry. Both the histochemical and biochem- ical assays for alkaline phosphatase were developed in the laboratory of Richard Whittaker, Marine Biological Laboratory, and kindly made available to us. Embryos were relaxed, while encapsulated, by the addition of 8% MgCl:-6H;O to the test medium. Egg strands (3-4 mm) were prefixed with 4% formaldehyde (from paraformal- dehyde) in 0.2 jum filtered NSW for 30 min (4°C). The strands were incubated in 10 ml of Tris-buffer solution ( 1 00 nM Tris-Base, 1 00 mA/ NaCl, 5 mA/ MgCl: • 6H:O, pH 9.5) containing 33 jul of nitro blue tetrazolium (Sigma), (50 mg/ml in 70% dimethylformamide) (Sigma), and 33 ^1 of 5-bromo-4-chloro-3-indoyl phosphate p-to- luidine salt (Sigma). (25 mg/ml in 100% dimethylform- amide). The color reaction was monitored over 30-60 min with a dissecting scope (21°C). Fixation was contin- ued with 3% glutaraldehyde/1.5% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) containing 5 mA/ EGTA, 5 mM MgCl2 • 6H:O, and 10% sucrose (4 h to overnight). Whole mounts were prepared by transferring the egg strands from the cacodylate/sucrose buffer to 0. 1 A/ so- dium cacodylate and cutting the capsules to free the em- bryos. Specimens were rinsed with 50%> methanol, dehy- drated with dimethoxypropane (DMP, Muller and Jacks, 1975), further rinsed with 100%- ethanol, cleared with Histoclear (National Diagnostics, Sommerville, New Jersey) and mounted on a glass slide using Diatex (Scientific Products). For tissue sections, egg strands were post-fixed in 1% osmium in 0.1 A/ cacodylate (40-45 min, 2 PC), rinsed 3X with 0.1 A/ cacodylate followed by 50% methanol, and dehydrated in DMP. Following transfer to propyl- ene oxide, the tissues were infiltrated and embedded in Epon/Araldite. The sections were cut (2 ^m), heat mounted on clean, untreated slides, and epoxy extracted with a solution of saturated KOH in 100% ethanol for 10 min. These sections were cleared and mounted to check the specific location of the reaction product. Sections and whole mounts were examined using a Zeiss Universal microscope equipped with a polarizing and differential interference contrast (DIC) optics. Biochemical analysis. Egg strands were transferred to microcentrifuge tubes, plunged into liquid nitrogen, and stored at — 80°C until analysis. Samples were thawed on ice and homogenized for 1 h in lysis buffer (4°C) contain- ing 50 mA/Tris HC1, 1 mA/MgCl:.6H2O, 1 MMZnCl2, 0.025% (w/v) Triton-X, and 0.0625% (w/v) sodium deoxycholate. The homogenate was centrifuged at 25,000 X g for 10 min, 4°C. Alkaline phosphatase activ- ity and total protein of the supernatant were determined colorimetrically via the p-nitrophenol (Sigma) and Brad- ford (Biorad, microassay) methods, respectively. Modi- fications of the alkaline phosphatase method included an extended incubation time for color development (16 h vs. 15 min) and a lower incubation temperature (2 PC vs. 37°C, Whittaker, pers. comm.). Alkaline phosphatase activity was normalized to total protein. SEM analysis. Embryos from Be dose-response exper- iments were processed for scanning electron microscopy (SEM) as described previously (Gallagher et a/.. 1989). Mucopolysaccharide synthesis experiments Egg strands were maintained in 2-1 flasks or petri dishes as described above. Treatments included 0 ppm Sr, 8 ppm Sr. and NSW. Samples were removed from the treatments throughout embryogenesis and analyzed for m ucopolysaccharides. Histochemistry. Following experimental treatment the encapsulated embryos were relaxed with 8% MgCl: • 6H:O and transferred to the complete glutaraldehyde/ paraformaldehyde fixative described above supple- mented with 0.1% ruthenium red. Fixation lasted 2-4 h (20°C) or overnight (4°C) followed by rinsing and storage in 0. 1 A/ cacodylate buffer plus 25%. sucrose. Following routine methods outlined above, the embryos were em- bedded in epoxy resin. Tissue sections were cut, heat mounted on slides, epoxy extracted and treated with the following stains: Aldehyde fuchsin/alcian blue (pH 2.5) in sequence (Spicer and Meyer, 1960); 0.5% alcian blue (pH 2.5) alone, and 1% alcian blue (pH 1.0) (Lev and Spicer, 1964). Statistical analysis Values were log transformed for total calcium, stron- tium, and magnesium concentrations and alkaline phos- phatase-specific activities. A two-way ANOVA was used to compare data from two or more treatments; time and treatment were fixed factors and the significance of the time X treatment interaction was determined. Means and least-square means were compared followed by the Student-Newman-Keuls (SNK) multiple comparison test and pairwise contrasts (adjusted by the Bonferonni method), respectively. The calcium and magnesium con- centrations for the 0 ppm Sr treatment were analyzed 234 J. P. BIDWELL ET AL 8 ppm Sr (NSW) 100- 80- 60 50 40 Degrees 2-Thela 60 50 40 Degrees 2-Theta 30 Figure 1. X-ray diffraction spectra of mineralized shell and statolith from indicated treatments. All material was aragonite and there was no evidence of paracrystalline or amorphous material in the deformed shells (3 and 80 ppm Sr). separately using a one-way ANOVA followed by the SNK. Calculations were performed with SAS. Results A'-rav crystal structure Atomic diffraction spectra of abnormal shells and statoliths from embryos reared in the 3 and 80 ppm Sr media were indistinguishable from spectra of normal tis- sue (Fig. 1 ). All diffraction peaks observed for each sam- ple were identified as belonging to the aragonite form of calcium carbonate. There were no significant shifts in peak position, changes in peak heights, nor evidence of amorphous or paracrystalline structure in the abnormal mineralized tissue. H 'hole embryo concentrations of calcium, strontium. and magnesium Total calcium at the end of embryogenesis ( 192 h) was diminished by approximately 80% in those embryos reared in the absence of strontium as compared to those organisms from strontium-containing media; total cal- cium of embryos from the 3 and 80 ppm Sr treatments were not significantly different from levels of the control organisms (8 ppm Sr, NSW, Fig. 2A). Thus, no correla- tion between total calcium and shell morphology was ev- ident. Profiles for calcium concentration paralleled min- eralization in all media containing strontium (Fig. 2A). At 96 h, a small but statistically significant increase in total calcium was observed coincident with the appear- THE EFFECT OF STRONTIUM ON CALCIFICATION 235 0 ppm Sr 3 ppm Sr 8 ppm Sr NSW 80 ppm Sr CRITICAL WINDOW FOR Sr 24 80 96 120 HOURS FROM OVIPOSITION CRITICAL WINDOW FOR Sr H« «H 72-96 hr § 48 96 (20 HOURS FROM OVIPOSITION l°°l 3 ppm Sr ^^ 8 ppm Sr H NSW E%1 80 ppm Sr 144 192 ance of the shell cap and statolith granules. Total calcium was not observed to increase in animals from the basal medium until 144 h, without evidence of mineralization. Profiles representing total strontium concentration of whole embryos paralleled those for calcium but were proportional to medium levels (Fig. 2B). Total magnesium concentration of whole embryo de- creased during embryogenesis and this decline was atten- uated in the absence of strontium (Fig. 2C). Although small, the decrease in whole embryo magnesium concen- tration over time from the 0 ppm Sr treatment was sig- nificant (P< 0.001). Comparison of the strontium distribution coefficients (¥^r) over time indicated that the discriminatory mecha- nism for this element was not acquired until after the critical window. Ksr (Fig. 3A) for all treatments contain- ing strontium had values close to one before the onset of mineralization, (1.05 ± 0.01, mean ± S.E., combined means of all treatments containing strontium, 24-96 h, n = 12). An abrupt decrease in the values of K^ (0.34 ± 0.03, mean ± S.E., n = 4) was observed at 120 h as mineralization began. The distribution coefficient for synthetic aragonite prepared from artificial seawater is approximately 1.0 at room temperature (Kinsman, 1969). The values of the magnesium distribution coefficient, KMg, for the 0 ppm Sr treatment ranged from 1.16 + 0.02 at 24 h to 0.45 ± 0.07 at 192 h (mean ± S.D., n = 5 and 6, respectively) indicating discrimination of this element (Fig. 3B). This may represent the biochemical compo- nent of the regulatory mechanism for total magnesium because shell formation was absent. ^ C CRITICAL WINDOW FOR Sr h- H 3 24 48 M 0 ppm Sr F°l 3 ppm Sr ^§ 8 ppm Sr H NSW El BOppmSr 80 96 120 HOURS FROM OVIPOSITION Figure 2. Chemical analysis of whole embryos plus egg strand. (A) Total calcium; a small but statistically significant increase was observed at 96 h (total calcium at 24-80 h = 0.39 ± 0.02 MA//mg ash; total cal- cium at 96 h = 0.45 ± 0.05, mean of combined treatments 3, 8. 80 ppm Sr and NSW ± S.D., P < 0.05. There was no significant difference in total calcium levels nor a significant time X treatment interaction be- Alkaline phosphatase activity Positive staining for alkaline phosphatase in whole specimens was coincident with the onset of the critical window (approximately 72 h) and was localized in areas where mineralization was imminent, i.e., the prevelar lobes (statoliths) and the shell field; intensity of the stain was not dependent upon whether the organism had been tween the 3 ppm and 8 ppm profiles or the NSW and 80 ppm profiles. Planned pairwise comparisons between the ASW treatments 3, 8. and 80 ppm Sr revealed thai differences in calcium concentrations were limited to 120 and 144 h (P< 0.001 ). (B) Total strontium; this element was not detected with graphite furnace analysis in organisms from the 0 ppm Sr medium. Strontium samples ( 80 h) were unsuitable for analysis from this particular experiment. (C) Total magnesium; there was no significant difference between the 3 and 8 ppm Sr profiles but a signifi- cant contrast between these two profiles and that for the 80 ppm Sr treatment from 120 h through 192 h (P < 0.001). There was also a significant difference between the 80 ppm Sr and NSW profiles (P = 0.003) but no difference in magnesium levels at 192 h. 236 J. P BIDWELL ET AL. 2.0 r A 1.5 0.5 0.0 3 ppm Sr 8 ppm Sr I 24 HOURS FROM OVIPOSITION 14 12 10 08 06 04 00 HOURS FROM OVIPOSITION Figure 3. Distribution coefficients for (A) strontium and (B) mag- nesium for indicated treatments ( mean ± S.D., n = 3-6). reared in the presence of strontium (0 and 8 ppm Sr, Figs. 4A, B). Staining intensified in both treatments as devel- opment progressed, encompassing the velum, mantle, and the statoliths (8 ppm Sr) or the empty statocyst cavi- ties (0 ppm Sr). Exposure to beryllium (0.009-0.09 ppm), a potent in- hibitor of alkaline phosphatase (Aldridge, 1950; O'Day, 1972), resulted in abnormalities of the embryonic shell and statoliths similar to those observed for strontium- deprived organisms (Fig. 4C). Nevertheless, this metal ion (0.09 ppm) did not attenuate alkaline phosphatase activity as measured biochemically (Fig. 4D), nor was the measured activity diminished in organisms from 0 ppm Sr. Mucopolysacchan.de synthesis The presence of strontium had no effect on mucopoly- saccharide synthesis as indicated by histochemical analy- sis, despite the failure of the organic shell to tan in those animals from the basal medium. Whole specimens from both the 0 and 8 ppm Sr treatments stained darkly with ruthenium red during embryogenesis; the indicator ap- peared evenly dispersed throughout the soft tissue, the organic matrix, and the egg strand. Differential staining with alcian blue and aldehyde fuchin demonstrated the presence of highly sulphated mucopolysaccharides and an absence of the acidic forms; again no differences were detected in embryos from the 0 and 8 ppm Sr treatments. No morphological abnormalities of the organic shell of embryos from the 0 ppm Sr treatment were observed upon examination of thick sections using light micros- copy. Discussion The marine protozoa Acanlharia spp., the only other organisms known to require strontium, use the element for the formation of their SrSO4 tests (Anderson, 198 1 ). Paradoxically, the deficits as a result of strontium depri- vation ofAplysia califarnica are specific for calcification, yet the defect is not mineralogical. We have demon- strated that strontium is discriminated against during mineralization and is not required for the stabilization of the aragonite polymorph in seawater. This element pre- vents the aragonite-to-calcite transition in calcium car- bonate preparations (McLester el al., 1970; Yoshioka el at.. 1986). Because strontium is not part of the shell itself, this element may regulate a biochemical pathway vital to the onset of mineralization. Total embryonic calcium was reduced dramatically (~80%) in organisms reared in the absence of strontium, but calcium increase was not abol- ished. Furthermore, there was no correlation between to- tal calcium levels at the end of embryogenesis and shell morphology in organisms from strontium-containing media. This suggests that calcium deficiency per se is not responsible for deficits induced by strontium depriva- tion. The reduced calcium may be a secondary effect re- sulting from the complete inhibition of precipitation. The present data do not address whether calcium uptake or transport is modulated by strontium and isotope ex- periments will be required to investigate this possibility. Alkaline phosphatase has been implicated as a nucle- ating agent in precalcifying matrices (Vittur el al., 1984; Marks and Popoff, 1988), and, although the onset of al- kaline phosphatase activity was coincident with the criti- cal window, it was not dependent upon the presence of strontium. Beryllium did not diminish this activity, al- though its presence during embryogenesis resulted in de- fects remarkably similar to those observed as a conse- quence of strontium's absence. Beryllium's inhibitory action on alkaline phosphatase is immediately reversible with magnesium in both histo- and biochemical prepara- tions (Raven, 1966; Aldridge, 1950). and therefore mag- THE EFFECT OF STRONTIUM ON CALCIFICATION 237 B v c_> CJ> Q- CO CO Q- CO O r Res In\l Bel Liv 6: 103-160. Sabbadin. A. 1962. Bande intersifonali di pigmento purinico in Bo- iry/lus schlossen ( Ascideacea) e loro determmazione genetica. Boll. 7.001. 29:721-726. Sabbadin, A. 1969. The compound ascidian Bolrrltus scklossen in the field and in the laboratory1. Puhhl Sla:. Zoo/. NapoliJil: 62-72. Sabbadin, A. 1977. Linkage between two loci controlling colour poly- morphism in the colonial ascidian, Botryllus schlosseri. Expenenlia 33: 876-877. Sabbadin. A. 1979. Colonial structure and genetic patterns in ascidi- ans. Pp. 433-444 in Biology aiul Systematic* oj Colonial Organ- isms. G. Larwood and B. R. Rosen, eds. Academic Press. London and New York. Sabbadin, A., and G. Graiiani. 1967. New data on the inheritance of pigments and pigmentation patterns in the colonial ascidian liuiryl- lus selilosscn (Pallas). Ri\: Biol 60: 559-598. Saito, Y., H. Muk. H. and H. \\atanabe. I981a. Studies on Japanese compound styelid ascidians. I. Two new species of Botrvllus from the vicinity of Shimoda. Puhl. Scto Mar Bio/. Lab 26: 347-355. Saito, Y., H. Mukai, and H. \\atanabc. 198lb. Studies on Japanese compound styelid ascidians. II. A new species of the genus Bolrvl- loules and redescription of B. vio/acein Oka. Puhl. Selo Mm Biol Lah 26: 357-368. Saito, Y., and H. VVatanabe. 1982. Colony specificity in the com- pound ascidian. Botryllus scalans Proc .//>/) lead 58: 105-108. Saito, Y., and II. VVatanabe. 1985. Studies on Japanese compound styelid ascidians. IV. Three new species of the genus Bolr\-lloidcs from the vicinity of Shimoda. Puhl. Scto Alar. Biol. Lnh. 30: 227- 240. Savigny, J. C. 1816. Memoncs sur Ics Amman* Sana \\-rlchrcs Paris, part 2. Schlumpberger, J. M., I. L. Weissman, and V. L. Scofield. 1984. Monoclonal antibodies developed against Bolryllus blood cell antigens bind to cells of distinct lineages during embryonic de- velopment. / Exp. Zool. 229: 205-2 1 3. Scofield, V. L., and L. Nagashima. 1983. Morphology and genetics of rejection reactions between oozooids from the tunicate Boinllus jvWo.s.vir/ Biol Bull 165: 733-744. Scofield, V. L., J. M. Schlumpbergcr, L. A. West, and I. I.. \Veissman. 1982. Protochordate allorecognition is controlled by a MHC-like gene system. Nature 295: 499-502. Taneda. Y., Y. Saito, and II. VVatanabe. 1985. Self or non-self dis- crimination in ascidians. Zool. Sci. 2: 433-442. Taneda. Y., and II. VVatanabt. 1982. Studies on colony specificity in the compound ascidian. Bmryllu.s primigenusQ]t&. II. In vivo bioas- say for analyzing the mechanisms of "nonfusion" reaction. Dev. Comp Immunol. 6: 243-252. Tokioka, T. 1953. Aseulian\ ol Salami Bay. Iwanami-shoten, Tokyo. Tokioka, T. 1967. Contributions to Japanese ascidian fauna. XXII. Ascidians from Sado Island. Puhl. Selo Mar. Biol. Lah. 15: 239- 244. Van Name, \V. G. 1945. The North and South American ascidians. Bull. Am. Mus. !*,\it Hist. 84: 219-230. Verrill, A. K. 1871. Descriptions of some imperfectly known and new ascidians from New England. Am. J. Sci. (ser. 3) 1: 21 1-212. \\ atterson, R. L. 1945. Asexual reproduction in the colonial tunicate, Bolrylliis vi7i/r>suv/ (Pallas) Savigny. with special reference to the developmental history of intersiphonal bands of pigment cells. Biol Bull 88:71-103. Reference: Biol. Bull 178: 251-259. (June. 1990) Behavioral and Metabolic Responses to Emersion and Subsequent Reimmersion in the Freshwater Bivalve, Corbicula fluminea ROGER A. BYRNE1, ERICH GNAIGER2, ROBERT F. McMAHON*. AND THOMAS H. DIETZ Department of Zoology and Physiology, Louisiana State University, Baton Rouge, Louisiana 70803, and *Section of Comparative Physiology. Department of Biology, The University of Texas at Arlington. Arlington, Texas. 76019 Abstract. When exposed to air, the freshwater bivalve. Corbicula fluminea, displayed valve movement behav- iors, such as mantle edge exposure, wider gaping "venti- latory" response, and an escape or "burrowing" re- sponse. The proportion of the emersion period spent in these behaviors, relative to valve closure, increased with decreasing temperature. Emersion at 35°C inhibited valve movement behaviors, whereas emersion in a nitro- gen atmosphere stimulated ventilatory activity. High rates of aerial oxygen uptake (M0,) were associated with initial valve opening and ventilatory behaviors, and lower MO, occurred during bouts of mantle edge expo- sure. Heart rate was affected by temperature, but not by mantle edge exposure. Heart rate increased during bur- rowing and ventilatory behaviors suggesting a hydraulic function for hemolymph. Emersed C. fluminea had short bursts of heat production followed by longer peri- ods of lower heat flux when measured by direct calorime- try. The mean heat production rate was 1.11 mW (g dry tissue)"', significantly higher than the mean value for clams exposed in a nitrogen atmosphere, 0.50 mW (g dry tissue)'1. On reimmersion, C. fluminea showed no sig- nificant "oxygen debt" until after three days aerial expo- sure. The bursts of activity, while emersed, may be the Received 3 November 1989; accepted 20 February 1990. 1 Present address: Department of Biosciences, University of Calgary, Calgary. Alberta. Canada T2N IN4. : Present address: Institut Zoophysiologie. Universitat Innsbruck, A- 6020 Innsbruck, Austria. Abbreviations: M0j — oxygen consumption rate; fh — heart beat fre- quency; TW — tapwater; ,q — weight-specific heat flux. result of periodic renewal of oxygen stores followed by immediate oxygen use. Introduction Aerial exposure of marine intertidal bivalves results in a variety of behavioral and metabolic responses (McMa- hon, 1988; Shick et al. 1988). In general, bivalves inhab- iting the shore will either close their valves while emersed and undergo anaerobic metabolism (especially lower shore species such as Mytilus edulis. Cerastoderma glau- cum. Boyden, 1972a; Widdows et al., 1979). or their valves will periodically gape allowing the maintenance of an aerobic metabolism (predominantly higher shore species, Cerastoderma edu/e, Geukensia demissa. Boy- den, 1972a; Widdows et al.. 1979). The intertidal environment is characterized by emer- sion periods that are predictable and of short duration. In contrast, bivalves inhabiting the shallow regions of freshwater lotic and lentic environments are subject to periods of emersion that are highly unpredictable in their duration, timing, and temperature. Freshwater bivalves can withstand periods of aerial exposure ranging from a few days to months, and will consume oxygen while in airfDietz, 1974; McMahon and Williams, 1984). To sur- vive such prolonged emergence, bivalves must balance two opposing requirements: to maintain contact with the atmosphere for gas exchange, while minimizing evapora- tive loss of water. The Asian freshwater clam, Corbicula fluminea (Mtiller), is commonly found in shallow lakes and streams throughout the United States (McMahon, 251 252 R. A. BYRNE ET AL 1982). A recent invader of freshwater, C. Jhiminea has higher tolerances to emersion than its estuarine relatives, but a relatively low tolerance among other freshwater bi- valves (McMahon, 1979; Byrne ct ai. 1988). It displays several behavior patterns when aerially emersed, such as mantle edge exposure and valve gaping, which are associ- ated with aerial oxygen uptake (McMahon, 1979, 1983; McMahon and Williams, 1984). In this study, we examined the effects of temperature and hypoxia on the behavioral responses of C. fluminca to emersion. Exposure of emersed clams to a N2 atmo- sphere allowed us to discriminate between responses to aerial exposure and hypoxia. We examined metabolic re- sponses, including heart rate, aerial oxygen uptake, and heat flux on emersed clams. In addition, the responses to reimmersion after varying periods of aerial exposure were observed. Materials and Methods Animals Specimens of C Jhimincu were collected, either from the Clear Fork of the Trinity River at its outflow from Lake Benbrook, Tarrant Co., Texas, or from the littoral region of the south shore of Toledo Bend Reservoir on the Texas-Louisiana border. Specimens were main- tained, unfed, in aquaria containing either aged tapwater (TW) or artificial pondwater (Dietz and Branton, 1975) at 22-24°C for at least one week prior to use. The animals ranged in size from 19 to 43 mm shell length; 3.8 to 2 1 g total wet weight; and 0. 1 5 to 0.79 g dry tissue mass. Dry tissue mass was about 8% of total wet body mass, but varies with season (Williams and McMahon, 1989). Behavioral measurements The effects of temperature on valve movements of ae- rially exposed specimens ofC.fluminea were determined on 5 clams at each of 3 temperatures, 15, 25, and 35°C. In addition, the effects of exposure to a severely hypoxic (N:) atmosphere (P0, = 1 torr) were examined. Mea- surement of valve movements was made by gluing a monofilament line to a point 1-2 mm from the leading edge of a valve. The opposite valve was attached to a Syr- acuse dish by gently embedding the bivalve, on its side, in modeling clay. The Syracuse dish with its attached bi- valve was placed inside a 45-ml glass jacketed chamber sealed by a rubber stopper. About 1 ml of distilled water was added to the chamber to maintain relative humidity near saturation. The line was threaded through an 18- gauge hypodermic needle passing through the rubber stopper and was attached tautly to the lever of a displace- ment transducer. The amplified output was directed to a strip chart recorder. The lever was counterweighted so that little force was exerted on the line attached to the valve, and the line was coated with silicone grease to pro- vide a gas seal but to also allow movement. Temperature inside the chamber was maintained at 15, 25, or 35 ± 0. 1 °C by means of a circulating water bath connected to the glass jacket of the respiration chamber. A period of temperature equilibration (30-60 min) was allowed before recording. Depending on the treatment tempera- ture, experiments continued for 24-150 h. The major categories of valve movement behavior were identified by simultaneous observation of behaviors and the trac- ings. A hypoxic atmosphere was achieved by flushing the chamber with N: gas at a high rate (300 ml/min) for the first 10 min, and at 50-75 ml/min during the experi- ment. The gas was appropriately temperature equili- brated and humidified before being introduced into the chamber. The P0, in these chambers was not routinely measured but we have recorded about 1 torr after the 10- min flushing period. Heart rate The heart rate of aerially exposed clams was measured simultaneously with valve movements under the three temperature treatments. Measurement of heart beat rate (fh) was accomplished by a modification of the method of Dietz and Tomkins (1980), a non-invasive technique that records the shadow of the beating heart by means of a photocell attached to the outside of the shell. The photocell (silicon selenium; 5X5 mm) was positioned over the heart and affixed by a small piece of modeling clay or glue to the valve. The clam was prepared for valve movement recording, as described above, and the leads from the photocell were threaded through a hole bored in the stopper and then sealed with rubber cement. The photocell current output was amplified with a Keithly microammeter (100 nA full scale), and was input to an amplifier/chart recorder. Light from a fiber optic lamp was directed through the clam from outside the chamber and adjusted above ambient illumination until the re- corder deflection, caused by the movement of the heart, was maximized. Only when the clam was performing burrowing behaviors, were the tracings difficult to inter- pret. Aerial oxygen consumption Aerial oxygen consumption rates (at 25°C) of clams exposed under normoxic conditions, and of clams after an exposure to hypoxic environments, were recorded. The aerial oxygen consumption rate (M0,) was mea- sured by the method of McMahon and Williams (1984). Moreover, valve movements and fh were recorded simul- taneously. Clams were prepared for valve movement and CORHICULA IN AIR AND WATER 253 fh recording as outlined above. Specimens were scrubbed to remove organisms adhering to the shell that might in- terfere with MO determinations. A polarographic oxy- gen sensor (Yellow Springs) was inserted through the rubber stopper sealing the chamber so that its tip was positioned approximately halfway into the chamber. Other openings drilled through the rubber stopper for leads were sealed. After a 30-min equilibration period, simultaneous recordings were made of valve movement, fh, and P0,. Reliable determination of M0l could only be made over a period of <24 h as the sensor fluid would need to be replaced. Although M0, measurements were attempted on fifteen specimens, only recordings of indi- viduals displaying valve movements were used in the analyses. M0, was calculated from the decline in percent oxygen saturation and expressed as ^mol (g dry tis- sue-h) '. Sensor drift was measured in an empty cham- ber and corrections applied to the M 0, calculations. Direct calorimetry Metabolic heat production by clams exposed in air or nitrogen gas was determined. The heat flux (mW) by emersed clams was measured by direct calorimetry, over periods of 24 to 168 h, with the LKB ThermoMetric 2277 Thermal Activity Monitor microcalorimeter (Suurkuusk and Wadso, 1982; Gnaiger, 1983; Gnaiger et al., 1989). Small clams (shell length 1 .9-2.3 cm) were glued to a specially molded plastic platform (10 X 10 X 10 mm). This platform was designed to present the clam lying on its side in the metabolic chamber (the ori- entation used previously), while suspending the clam above a 1-ml reservoir of distilled water placed in the chamber to maintain relative humidity near saturation. A 25-cm3 metabolic chamber was used in all experi- ments. Rates of heat dissipation were recorded by sub- tracting the output of a blank chamber (4 ml distilled water, the approximate thermal equivalent mass of the contents of the experimental chamber) from that of the experimental chamber, and recording the result on a strip-chart recorder. As the time-response curve of this larger chamber was not instantaneous, but approximates a first order exponential function (Gnaiger, 1983), the rates of heat dissipation derived from the experiments were averaged over 10-min periods and corrected to give instantaneous heat flux readings. Heat production of the control chamber (4 ml distilled water replacing the clam) was determined after every two experiments, and an instrument calibration was per- formed every four experiments, or at least once a week. To determine the effects of emersion in a nitrogen atmo- sphere, the chamber was flushed for 30 min with humidi- fied N2 carried in capillary tubing incorporated into the cap of the metabolic chamber containing the experimen- tal animal. Aquatic A/0, on reimmersion One hundred specimens ofC.fluminea, individually marked and weighed (±0.0001 g), were aerially exposed in desiccators above a layer of water maintaining relative humidity at near saturation, at 25°C. Five individuals, picked at random, were removed after 1, 2, 3, 5, and 6 days of emersion and used to determine the aquatic M0l upon reimmersion. These clams were immersed in dechlorinated, aged tapwater, and allowed to open their valves and commence siphoning activity for 5 min. The immediate M0, was determined by placing the clams in- dividually into a sealed, temperature controlled respira- tion chamber (65 ml volume; 25 ± 0. 1"C) filled with aer- ated TW. The clam was supported on a nylon mesh plat- form above a magnetic stirbar. The decline in chamber dissolved oxygen was measured with a pre-equilibrated oxygen sensor (Yellow Springs) connected to a strip- chart recorder. Rates of oxygen consumption were deter- mined on the basis of the first 10% decline in air satura- tion, which was usually accomplished in 5-10 min after the method of McMahon and Russell-Hunter (1977). The clams then were returned to aerated TW. and the aquatic M 0, remeasured after a total reimmersion period of 1 h to detect any temporal changes in respiratory re- sponses of reimmersed individuals. Data analysis Data are expressed as mean ± SEM, and n = the num- ber of animals. Differences were considered significant at P < 0.05 with Student's /-test, or a one-way ANOVA followed by Duncan's Multiple Range tests. Results Behavioral responses to emersion On emersion, specimens ofC.Jhiminea displayed four categories of behavior (Fig. 1 ). The first was the closed condition with valves clamped shut and no tissues ex- posed to the environment. The second condition was mantle edge exposure with the valves parted slightly ( 1- 2 mm) and portions of the leading edge of the mantle exposed along the complete extent of the gape. The man- tle edges were moist or fused with a hardened mucus over the surface, and no opening into the mantle cavity was evident. On many occasions, the mantle was extended over the edge of the valves, exposing more mantle tissue. The other two behavioral categories of valve move- ment on emersion were less common (Fig. 1). After a period of mantle edge exposure, the valves and mantle would part further, forming an opening into the mantle cavity. This position would be maintained for a few min- utes followed by rapid valve closure; the opening and closing of valves could continue for some minutes giving 254 R. A. BYRNE ET AL. CO CD (B > 10 mm Emersion Period Figure I. An amalgamation of several recordings to demonstrate the four categories of valve movement behavior in emersed Corbicula tlumineti I . Valves closed. 2. Valves gaping slightly with little valve adduction, characteristic of mantle edge exposure. 3. Valves gaping wider with small medium frequency adductions, often associated with "ventilatory" movements. 4. Valves widely gaping with high frequency adductions indicative of the "escape" or "burrowing" behavior. the appearance of a form of "ventilation" (category 3 in Fig. 1). The fourth behavior (rare) was a parting of the valves, and an extension of the foot. While the foot was extended, the valves would shut on the foot, then open, and the foot would extend further. This behavior would be repeated until the foot was extended maximally and touching the substratum. This activity resembles the "burrowing" behavior of immersed specimens of C. fliiminen. and is interpreted as an escape behavior. Although the patterns of behavior varied extensively between clams, the general progression of valve move- ment behaviors was similar for all. It began with a period of valve closure which lasted from 8.08 to 1 7.42 h ( 1 1 .94 ± 1.98; n = 4) at 15°C, from 7.20 to 27.55 h at 25°C (15.44 ± 3.57; n = 5), and [when valve movement behav- ior was noted (only 3 of 10 cases)] from 0.33 to 6.52 h at 35°C (3.94 ± 1 .86; n = 3). After the initial period of valve closure, bouts of mantle edge exposure were occasionally interspersed with short periods of ventilatory and, rarely, burrowing behaviors. The duration of this period was variable, but ranged from 36.16 to 203.59 h (126.31 ± 35.66; n = 4) at 15°C. from 64.78 to 93.38 h at 25°C (82.71 ± 6.19; n = 5), and 9.00 and 27.55 h at 35°C (n = 2). Following the period of valve movement, the valves remained closed to the end of the experiment, or death. We determined the percentage of time spent by each clam in each of the behavioral categories with the times spent in ventilatory and burrowing behaviors combined. These values were averaged for each temperature treat- ment (Table I). An analyses of variance on the trans- formed values (arcsine of square root of the percentage as a proportion) showed that temperature had a significant effect on the relative time spent in the behavioral catego- ries. The most striking was the inhibition of valve move- ments at 35°C: only 20% of clams exposed at 35°C dis- played any valve movement behavior. Clams exposed at 15°C spent significantly (P < 0.05) less time closed and more time in the mantle edge exposure behavior than those clams emersed at 25°C. Clams exposed to hypoxia in a N2 atmosphere (25°C) after they began valve movements, spent 40.3 ± 10.1% (n = 3) of the time with mantle edges exposed, and 57.0 ± 7.6% (n = 3) in either ventilatory or burrowing behav- iors. After a period of severe hypoxia, and while ventila- tory movements were still occurring, the chamber was flushed with humidified air. The result was a significant change in the pattern of valve movement behavior; i.e., clams exposed their mantles 84.6 ± 1.0% (n = 3) of the time and spent 12.1 ±4.2%(n = 3) of the time in ventila- tory behaviors. Heart rate while emersed The heart rate ofC.Jluniinea was highly variable, and this was true also of emersed specimens of C. fluminea; i.e., for an individual clam in one experiment, the highest values could be more than twice the lowest. Temperature had a significant effect on mean fh (Table II) with an ap- proximate Q,o of 2. As we were interested in the effects of valve movement behaviors on fh, we determined the heart rate 5 min before the valve movement began, the rate at the onset of valve movements, and fh at 5 min after valve movement began. Because of individual vari- ability in fh, the change in fh was expressed as the frac- tional change relative to the fh during the first 5 min of the valve movement (Table III). At 15 and 25°C. the oc- currence of mantle edge exposure behavior (Fig. 1, cate- gory 2) resulted in no significant change in fh. The onset of the ventilatory behaviors (Fig. 1, categories 3 and 4) resulted in no change in fh; however, there was a signifi- cant 35%. drop in fh 5 min after the onset of the ventila- tory behavior at 15°C. At 25°C, 5 min after the onset of ventilation, fh declined 13% (Table III). As valve move- Table I The I'/Av/.s "I temperature on valve movement behaviors in emersed Corbicula fluminea Temperature Valves closed Mantle edge exposed Ventilatory and burrowing behaviors 15 25 35 29.5 ±5.9 A 51.2 ±4.2 B 90.5 ± 8.0 C 65. 8 ±5. 5 A 43.5 ± 3.8 B 9.1 + 7.7C 4.7 ± 1.7 A 5.3 ± 0.9 A 0.4 + 0.3 B Dissimilar letters after values indicate significant differences between temperature treatments (one-way ANOVA; Duncan's Multiple Range Test; P < 0.05; arcsine of square root transformation). Values are the mean ± SEM percentage of the emersion period spent in each behav- ioral category; ventilatory and burrowing activity were combined (n = 5 for each temperature). CORBICULA IN AIR AND WATER 255 " . TO 0) 0 20 16 12 80 S ~o a. 60 40 3 2&3 2 01234 Exposure Duration (h) Figure 2. An example of valve movements (upper panel) with the same activity pattern notations as in Figure I , simultaneous recordings of heart beat rate (middle panel) and aerial oxygen uptake (M0,; lower panel) in emersed Corbicula fluminea. The initial high rate of oxygen consumption is associated with valve opening and may be the result of oxygen depleted mantle cavity air mixing with the air in the respiration chamber. Heart rate increases during major valve movements. ment behavior was rare at 35°C, no effects on fh were measured. Aerial M0, Of 15 attempts to record simultaneously M0l. valve movements, and fh in aerially exposed clams, measur- able M0, was recorded for only three animals. Figure 2 shows an example of O2 uptake with the concurrent valve movement and fh recordings. The pattern here, and in the other recordings, was an initial high rate of O2 up- take coincident with the initial 2-3 min of valve move- ment, followed by a reduction in oxygen uptake during the period of mantle edge exposure. Further ventilatory movements were associated with elevated rates of oxy- gen depletion. Oxygen consumption during mantle edge exposure, although low, was measurably greater than when the valves were closed. Oxygen consumption rates integrated over periods of valve movement were 11.1 to 77. 5 ^molO:(g dry tissue -hr' (40.4 ± 19. 6; n = 3), with most of the oxygen uptake occurring in short bouts. When the valves were shut, M0, ranged from 0 to 0.5 ^mol O: (g dry tissue -h) '. When ventilatory move- ments were initiated, the heart rate increased briefly then declined, even though valve movements continued. Direct calorimetry The pattern of weight-specific heat flux (,q; mW-g"1) in emersed specimens of C. fluminea consisted of periods of steady heat flux interspersed with short bursts of rela- tively high ,q (Fig. 3 A; Table IV). The peak rates of heat dissipation were between 1.7 and 9 times the average "basal" aerial rate (heat flux between peaks); but the du- ration of these peaks was less than one hour, and clams had an average of one peak every 7.50 ± 0.89 h (n = 12) of emersion. The time of emersion (in seconds) times the average tq calculated from the continuous recordings over the entire period of emersion [mW (g dry tissue)"1 = mJ (g dry tissue -s)"'] yields the total energy expendi- ture over the period of aerial exposure. Clams exposed to hypoxia did not display the bursts of peak activity noted in clams exposed in normoxia (Fig. 3B). The mean ,q of nitrogen-emersed clams was not significantly different from the "basal" level of nor- moxic emersed clams (Table IV), but was significantly lower than the mean ,q (= average energy expenditure) of normoxic emersed clams. Aquatic MO, on reimmcrsiim There was an increase in aquatic M0, related to expo- sure time ( ANOVA F = 1 2.66; P < 0.00 1 ). However, the effects of aerial exposure were not immediate (Fig. 4). Initial rates of oxygen consumption were not signifi- cantly different from control values even after three days of aerial exposure (Duncan's Multiple Range). This sug- gests that the mussels were not accumulating a signifi- cant oxygen debt during three days of emersion. Initial oxygen consumption rose significantly (P < 0.05) from a control value of 61.7 ± 5.7 /^mol (g dry tissue -h)'1 to 135.0 ± 18.2 jiimol (g dry tissue -h)~' after 5 days emer- sion (Fig. 4). MO, continued to rise to 186.1 ± 8.9 /umol (g dry tissue -h)1 after 6 days of exposure. There were no significant differences between initial and 1 -h M0, values at any time. Table II Effect oflemperalure on frequency of heart beat [t],; beats- min '), in emersed Corbicula fluminea Temperature (°C) 15 35 Heart rate 8.4 ±0.8 (3) A 14.7 ±1.8 (6) B 35. 8 ±6.4 (3) C QIC 1-75 2A4 Dissimilar letters after the values indicate significant differences (one-way ANOVA; Duncan's Multiple Range; P < 0.05). Values for individual clams were averaged, and the grand mean ± SEM (n) for each temperature is presented. 256 R. A. BYRNE ET AL Table III Change in heart heat frequency (/,,. heal v • niiir ') associated with valve movements in emersed Corhicula tluminea Fractional change in th Mantle edge exposed Ventilatory and burrowing behavior Temperature n 5 min before 5 min after n 5 min before 5 min after 15°C 25°C 5 14 0. 1 1 ± 0.05 -0.02 ± 0.02 0.10 ±0.12 0.01 ±0.03 10 9 0.19 + 0.06 0.14 ±0.03 -0.35 ± 0.07* -0.13 ±0.02* The asterisks designate values within a temperature or behavior category significantly different from one another (P < 0.05 ). Values are fractional changes in fh (+SEM) from 5 min before the onset of the behavior, compared to the fh at the beginning of the behavior, and the fractional change of the fj, 5 min after the behavior had commenced, compared to the th at the onset of the behavior. A positive value indicates an increase in lh. By using the oxycaloric equivalent of -450 kJ/mol O: (Gnaiger el a/., 1989), heat production can be estimated from the M0, of aquatic clams, and is approximately 27 .— 03 0) 6 2- 1- 0 4- 3 0 0 10 20 30 40 B Nitrogen emersed 5 10 15 20 Exposure Duration (h) 25 Figure 3. A. Example of the pattern of energy flux for an emersed Corhicula llummcii as measured by direct calorimetry. Note the bursts of higher rates of heat dissipation between periods of much lower activ- ity. B. Part of a record of rates of heat dissipation in an emersed speci- men of C ' fluminea exposed in a normoxic and anoxic atmosphere. No burst activity was noted in nitrogen exposed clams. J (g dry tissue -h) l [=7.5 mW(g dry tissue) ']. Convert- ing energy flux expressed in units of J-s ' (= mW) to units of J-h ', the absolute peak values of aerial heat flux were around 22 J (g dry tissue -h) ',or80% of the aquatic rate. The average peak heat dissipation rate was 9.4 J (g dry tissue-hr' (34% of the aquatic rate), the overall mean heat flux was 4.0 J (g dry tissue -h)'1 (15%) and the basal rate was 2.3 J (g dry tissue • h) ' (9%). Discussion Corbicula fluminea displayed a suite of behavioral re- sponses to emersion: mantle edge exposure; valve venti- latory behavior and burrowing response. These behav- ioral responses occupied a larger proportion of the total emersion period than had been estimated previously. McMahon and Williams ( 1 984) reported a value for the proportion of time exposing mantle edge at 1 1.5% in C. fluminea emersed at 20°C. In the present study, valve Table IV Mean valuer of rates of heat dissipation (mW-g ') in emersed Corbicula fluminea mW.g Normoxic emersed Mean peak rate Mean basal rate Mean heat flux Mean heat flux 2.55 ±0.29 (12)* 0.65 ±0.12 (12) 1.1 1 ±0.13(12)* 0.50 + 0.10(3) The asterisks indicate significant differences between mean rates of heat flux of nitrogen emersed clams and the aerially emersed bivalves (P < 0.05). Peak values are maximum rates sustained during bursts of activity. Mean basal values are average rates of heat dissipation mea- sured during periods of no burst activity. Mean heat flux is the average of all values and approximates the mean energy expenditure of emersed clams. Values are expressed as the mean + SEM and the number of animals is given in parentheses. CORU1CUI.A IN AIR AND WATER 257 200 160 120 O 80 40 — i — — i — — i — — i — ~n — — i — ~n 0123456 Emersion Period (days) Figure 4. Rates of oxygen consumption (M0,) of Corhiciilu Ili/nii- iicn on reimmersion after periods of aerial exposure. Circles represent initial values, within 5-15 min of the initiation of siphoning activity upon reimmersion. Squares are values measured one hour after siphon- ing activity commenced. The unconnected points represent corre- sponding initial and 1 h control M0, measurements in tapwater accli- mated clams. Bars are standard errors of the means and n = 5 animals (breach point. movements in emersed clams occupied 70% and 49% of the exposure period at 15 and 25°C, respectively. A sim- ilar pattern of valve movement behaviors while emersed has been reported for the high estuarine mangrove cor- biculid, Polymesoda erosa. In P. erosa, ventilation of the mantle cavity air space occurred at irregular intervals, interspersed with periods of mantle edge exposure and valve closure (McMahon, 1988). These behaviors in C. jluminea seem to be associated with aerial oxygen uptake (this study; McMahon and Williams, 1 984). However, the highest rates of aerial oxy- gen consumption took place during the first few minutes of valve opening in what was referred to as ventilatory behavior, with lower rates during mantle edge exposure. This is consistent with the hypothesis of a periodically renewed, mantle cavity oxygen store (McMahon and Williams, 1984; Pamatmat, 1984). When the valves first open, the oxygen depleted mantle cavity gas mixes with the air, causing a sudden decline in atmospheric P0l in the respiration chamber. Aerial oxygen uptake has been reported for several freshwater bivalves. The unionid clam, Ligumia subro- slrata was reported to have aerial oxygen uptake rates of between 21-23%- of the aquatic rate (Dietz, 1974), and the sphaeriid, Sphaerium occidentale, have similar rates of aerial oxygen uptake (Collins, 1967). Heming and co- workers (1988) reported that the freshwater mussel, Margaritifera margaritifera, periodically gaped in air with the result that dissolved oxygen levels in the mantle fluid were maintained at approximately half those of im- mersed bivalves. Among the intertidal and estuarine bivalves. Lent (1968) suggested that air-gaping in Geiikemia demissa was an adaptation for air breathing. Non-gaping inter- tidal bivalves vary from having no measurable aerial O2 uptake to oxygen consumption rates of 4-17%. of the aquatic rate (Widdows el til.. 1979). In contrast, the gap- ing bivalve species have an aerial M0l similar to that in water, or have aerial rates of between 28% and 79% of the aquatic rate (Boyden, 1972a; Booth and Mangum, 1978; Widdows el a/., 1979). In general, higher shore bi- valve species have higher rates of aerial O2 consumption associated with valve gaping, whereas lower shore clams, emersed for short periods, tend to remain closed and consequently have lower aerial M0, (McMahon, 1988). Emersion of C. jluminea in a nitrogen atmosphere seemed to stimulate a wider gape response, indicative of ventilatory activity, and the higher level of activity was diminished on return to a normoxic environment. This observation suggests that clam ventilatory behaviors have a respiratory function and may maintain some level of aerobic metabolism while emersed. In addition, a ven- tilatory loss of CO2 is also clearly associated with mantle edge exposure (Byrne, 1988) as well as with O2 uptake, as demonstrated here. The ability to dissipate metabolic CO2 and maintenance of acid-base balance may be as important as O2 uptake in the ability of C. Jluminea to tolerate emersion. Emersed C. Jluminea also displayed bursts of heat pro- duction interspersed among periods of lower "basal" or quiescent activity. Freshwater bivalves have endogenous rhythms of activity and changes in oxygen consumption (McCorkle et a/.. 1979). Our calorimetry chamber did not allow concurrent measurements of valve movement. However, emersed specimens of marine bivalves have valve gaping patterns associated with aerial oxygen up- take and elevated heat dissipation rates that are qualita- tively similar (Pamatmat, 1984; Widdows and Shick, 1985). The bursts of heat production we observed could have been associated with valve closure or foot move- ments. However, the duration of valve movement is usu- ally of short duration, but the peak ,q activities we mea- sured was of greater duration making this possibility un- likely. A likely explanation is periodic ventilation and gas exchange during valve gaping episodes resulting in a short-term elevation of aerobic metabolism. Recharging spent phosphagen and ATP stores during short periods of aerobic metabolism would result in increased heat dis- sipation rates. After valve closure the O2 availability would be decreasing, even if relatively quiescent, and clams would gradually become anaerobic. Before incur- ring a significant oxygen debt, however, another bout of valve opening and ventilation would occur. Perhaps the 258 R. A. BYRNE ET AL. gradual depression of metabolic rate is due to a decrease in body fluid pH (deZwaan 1983). Peak values of aerial heat dissipation in C. fluminea approached 80% of the aquatic rate. The mean peak rate of heat dissipation was 34% and the overall mean was 15% of aquatic rates in this study. These data correspond well to the aerial O: consumption rate being about 20%. of the aquatic rate in other freshwater bivalves (Collins, 1967;Dietz, 1 974) and C Jltinn/iea (McMahon and Wil- liams, 1984). In a N: atmosphere, emersed C. JJuniinea did not display bursts of heat production but maintained a low level of ,q similar to the quiescent periods during normoxic exposure. This suggests that the observed bursts of heat flux in normoxia are the result of aerial respiration. In most cases where bivalves gape while exposed, there is a continuation of heart beat during emersion. Changes in heart rate during emersion are variable, from essen- tially no change of fh during emersion in some species, to a distinct bradycardia on emersion in other species ( Boy- den, 1972b; Coleman and Trueman, 1971). There ap- pears to be no consistent direct effect of valve movement on heart beat frequency; some bivalves show occasional changes in fh associated with valve movements and oth- ers display a suppression of heart activity (Trueman and Lowe, 1971; Coleman, 1976; Dietz and Tomkins. 1980). Although valve gaping is associated with a respiratory function in emersed bivalves, the importance of circulat- ing blood in the delivery of oxygen to tissues is not cer- tain. Mussel blood typically has no oxygen carrier, and Booth and Mangum (1978) found that only 14%. of O2 in the blood ofModiolus dcniixsux was delivered to tissues, leading them to conclude that the circulatory system was not important in this regard. We noted that increases in heart rate in emersed C.fluminea were associated mainly with ventilatory and burrowing behaviors. Foot and muscular movements are facilitated by hydraulic pres- sure and increases in heart rate may be associated with a redistribution of hemolymph among blood sinuses. Al- ternatively, when clams periodically have bursts in meta- bolic activity while emersed, the increased fh may be in response to the momentary increases in perfusion re- quirements. When returned to water after three days of emergence, C. Jluminea had aquatic oxygen consumption rates that were elevated when compared to pre-emersion rates. The increased M0, was evident within 5-10 min ofreimmer- sion and remained elevated 1 h later. Repayment of an "oxygen debt," characterized by an elevated M0,, is commonly encountered in marine bivalves reimmersed after periods of aerial exposure (McMahon, 1988; Shick el al., 1988). Frequently, the size of the oxygen debt is proportional to the duration of the exposure period and is repaid during the first hour of reimmersion (Bayne el a/.. 1976; deVooys and deZwaan, 1978; Widdows el a/., 1979; Widdows and Shick, 1985). In contrast, oxygen consumption rate in resubmerged C. fluminea is not a direct function of exposure time and, indeed, M0, rates did not rise significantly above pre-emersion values until after three days emersion. MOj also did not decline sig- nificantly after one hour of reimmersion. Both observa- tions suggest that the elevated M0, observed after three days of emersion was not a typical oxygen debt repay- ment in C. fluminea. Rather, over moderate periods of emersion. C. Jhnninea appears to maintain a sufficient level of aerobic metabolism to be able to avoid depen- dence on anaerobic metabolism. This corresponds di- rectly with the maintenance of ventilatory and valve movements during the early stages of emergence. Ele- vated oxygen consumption after three days of emergence may be associated with long-term catabolic and anabolic demands resulting from the emersion stress. Corbicula fluminea has evolved an additional suite of respiratory and behavioral adaptations, compared to P. erosa, an estuarine member of the family Corbiculidae (McMahon, 1988). Although valve gaping and limited emergence tolerance time are similar to high intertidal clams, the novel responses of C.fluminea include the ex- posure of mantle edges alternating with short bouts of valve gaping and ventilatory behavior. These responses would allow rapid exchange of mantle cavity gasses, yet minimize evaporative water loss. After three days of emergence. C.fluminea shows evidence of increased O2 demand on reimmersion. and mortality increases. Al- though C. flumincadoes not have the emersion tolerance of the more ancient families of freshwater bivalves (unio- nids, sphaeriids), it is a successful inhabitant of lakes and streams. Its short-term physiological mechanisms, and high reproductive capacity allow this species to be suc- cessful in the variable freshwater habitats. Acknowledgments The research was supported by a Sigma Xi Grant-in- aid of Research to R.A.B., the University of Texas at Ar- lington Research Grants to R.F.M. and NSF grant DCB 87-0 1 504 to T.H.D. E.G. was partially supported by the LSU Department of Zoology and Physiology Visiting Scientist Program and Fonds zur Forderung der wissen- schaftlichen Forschung in Osterreich, project J0011. This study was part of a dissertation submitted by R. A.B. to the Graduate School of Louisiana State University and A&M College in partial fulfillment of the Ph.D. de- gree. Literature Cited Bayne, B. L., C. J. Bayne, T. C. Carefool, and R. J. Thompson. 1976. The physiological ecology of Afylilti.i califormanus Conrad CORBICL'LA IN AIR AND WATER 259 2. Adaptations to low oxygen tension and air exposure. Oecologia (Berl.) 22: 229-250. Booth, C. K.. and C. P. Mangum. 1978. Oxygen uptake and transport in the lamellibraneh mollusc Modiolus demissus. Physiol. /.ool 51: 17-32. Boyden, C. R. I972a. 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Metabolic heat flow patterns in the intertidal mussel Ischadium ( = Modiolus = Geukensia) demissum demissum during aerial and underwater respiration. Int. Revue des Gesamten Hydrobwl. 69: 263-275. Snick, J. M., J. VViddows, and E. Gnaiger. 1988. Calorimetnc studies of behavior, metabolism and energetics of sessile intertidal animals. Am. Zool. 28: 161-181. Suurkuusk, J., and I. Wadso. 1982. A multiple channel modular mi- crocalorimeter. Chiin. Scrip/a 20: 155-163. Trueman, E. R., and G. A. Lowe. 1971. The effect of temperature and littoral exposure on the heart rate of a bivalve mollusc, Isognomon alatus, in tropical conditions. Comp. Biochem. Physiol 38A: 555- 564. deVooys, C. G. N., and A. deZwann. 1978. The rate of oxygen con- sumption and ammonia excretion by Mytilus edulis after various periods of exposure in air. Comp. Biochem. Physiol. 60A: 343-347. W iddows, J., B. L. Bayne, D. R. Livingstone, R. I. E. Newell, and P. Donkin. 1979. Physiological and biochemical responses of bivalve molluscs to exposure to air. Comp Biochem Physiol. 62A: 301- 308. W iddows, J., and J. M. Shick. 1985. Physiological responses ofMyli- lus edulis and Cardium edule to aerial exposure. Mar Biol. 85: 2 1 7- 232. Williams, C. J., and R. F. McMahon. 1989. Annual variations in tis- sue condition of the Asian freshwater bivalve. Corbicula fluminea, in terms of dry weight, ash weight, carbon and nitrogen biomass and its relationship to downstream dispersal. Can. J. Zool. 67: 82- 90. deZwaan, A. 1983. Carbohydrate catabolism in bivalves. Pp. 137- 175 in The Mollusca. Vol. 1: Metabolic Biochemistry and Molecu- lar Biomechanics. P. W. Hochachka ed. Academic Press, San Diego. Reference: Biol. Bull. 178: 260-266. (June. 1990) Potentiation of Hypoosmotic Cellular Volume Regulation in the Quahog, Mercenaria mercenaria, by 5-hydroxytryptamine, FMRFamide, and Phorbol Esters* LEWIS E. DEATON Department of Biology, University of Southwestern Louisiana, Lafayette. Louisiana 70504 and The Whitney Laboratory, I 'niveisity of Florida, 9505 Ocean Shore Blvd., St. Augustine, Florida 32086 Abstract. Ventricles isolated from clams (Mercenaria niereenaria] that had been acclimated to 1000 mOsm seawater (SW) release amino acids when incubated in 500 mOsm SW. Taurine, glycine, and alanine account for nearly all of the released amino acids, and total about 37 jtmol/g dry tissue weight during a 2-h incubation. The release of amino acids is increased to 69 ^mol/g by the addition of 10 6 AI 5-hydroxytryptamine (5HT) to the hypoosmotic SW, and to 83 nmol/g by the addition of 10~6 M FMRFamide to the medium. The potentiation of the release by 5HT is blocked by methysergide. The amino acid release is increased by two phorbol esters — phorbol 1 2, 1 3-diacetate and phorbol 12-acetate. 13-my- ristate — to 97 and 83 ^mol/g, respectively. Forskolin and other cyclic 3',5' adenosine monophosphate agonists have no effect on the release of amino acids in hypoos- motic SW. Phorbol esters, 5HT. and FMRFamide have no effect on the release of amino acids from ventricles incubated in 1000 mOsm SW. Ventricles, first isolated from clams acclimated to 1000 mOsm SW, and then transferred to 500 mOsm SW. increase in wet weight by 20-25%. The increase is maintained for 30 min, and the tissues return their original weight in the ensuing 30 min. The addition of 5HT, FMRFamide, or phorbol esters to the hypoosmotic SW decreases the time necessary for the tissues to return to pre-transfer weights. These results im- plicate protein kinase C in the responses of bivalve tis- sues to hypoosmotic media, and suggest that these re- Received 2 January 1990; accepted 19 March 1990. * This is contribution number 29 1 from the Tallahassee, Sopchoppy and Gulf Coast Marine Biological Association. sponses may be modified by neuronal or neurohumoral control. Introduction In osmoconforming marine bivalves, the restoration of cellular volume in response to changes in the ambient salinity is accomplished by the adjustment of cytoplas- mic concentrations of ions and amino acids (Gilles, 1979; Pierce, 1982). The cells of these animals release amino acids when exposed to a hypoosmotic medium, thereby reducing the osmotic gradient between the me- dium and the cytoplasm (Pierce and Greenberg, 1972; Gainey, 1978; Amende and Pierce, 1980). The extirpation of particular ganglia in bivalves has been reported to affect the water balance of the ani- mals (Lubet and Pujol, 1963; Nagabushanam. 1964; Durchon, 1 967). In both Crassostrea virginica and Myti- his galloprovincialis, putative neurosecretory cells lost their granular inclusions when the animals were exposed to hypoosmotic media (Lubet and Pujol, 1963; Nagabu- shanam, 1964). In theopisthobranchAplysiacalifomica, the electrical activity of cell R-15 in the abdominal gan- glion is depressed by exposure of the whole animal to dilute seawater (Bablanian and Treistman. 1983). Ninety minutes after homogenates of R- 1 5 were injected into an intact A. californicu. the animal's wet weight in- creased by 5% (Kupfermann and Weiss, 1976). The gain in weight induced by the homogenate occurred even in a 5% hyperosmotic medium, in which the animals would be expected to lose water. Hyperpolarization of R- 1 5 in intact animals causes large increases in the free amino acid content of the blood (Bablanian and Treistman. 260 VOLUME REGULATION IN MERCENARIA 261 1985). These observations, while far from conclusive, suggest that neurosecretory products might be involved in the regulation of osmotic balance in molluscs. This paper describes the effects of known molluscan neurotransmitters and neurohormones on the hypoos- motic volume regulation of isolated ventricles of the clam Alercenariu mercenarui. In addition, the effects of cyclic 3'5' adenosine monophosphate (c-AMP) and pro- tein kinase C agonists on volume regulation have been investigated. The neuropeptide FMRFamide, 5-hy- droxytryptamine, and phorbol esters, but not c-AMP ag- onists, potentiate hypoosmotic volume regulation. Materials and Methods Animals and media Mercenaria mercenariavfere collected from several lo- cations in the Intracoastal Waterway in St. Johns County, Florida, or obtained from the Marine Biological Laboratory, Woods Hole, Massachusetts. The animals were maintained, unfed, in either running seawater (SW) (940-1000 mOsm), or in aerated aquaria containing nat- ural SW (1000 mOsm). In all cases, the test media were compounded from vacuum-filtered (0.45 p.m) natural SW. The 500 mOsm SW was made by dilution of filtered SW with deionized water. Concentrated solutions of Pharmaceuticals were pre- pared as follows: forskolin was dissolved in ethanol; phorbol. phorbol 12,13-diacetate, and phorbol 12-ace- tate, 13-myristate were dissolved in dimethylformamide; all others were dissolved in deionized water. The neural substances tested included the molluscan neuropeptides phenylalanyl-methionyl-arginyl-phenyl- alanylamide (FMRFamide), phenylalanyl-leucinyl-argi- nyl-phenylalanylamide (FLRFamide), para-glutamyl- aspartyl - phenylalanyl - leucinyl - arginyl - phenylalanyl - amide (pQDFLRFamide), small cardioactive peptide B (SCPB), acetylcholine (Ach), and serotonin (5HT). The 5HT receptor blocker methysergide (UML 491 ) was also used. These drugs were added to either 1000 mOsm or 500 mOsm SW to obtain the desired concentration. Sol- vent carriers other than water were added to 1 000 mOsm or 500 mOsm SW in the same amounts necessary to achieve the desired drug concentrations; such media were used to control for the effect of solvent. Analysis of the tissue free amino acid pool Ventricles were removed from animals that had been acclimated to 1000 mOsm SW, and lyophilized. The dried tissues were weighed and homogenized in a small volume of 80% ethanol ( 1 ml/5 mg dry tissue). The ho- mogenates were centrifuged in a microcentrifuge and 20 n\ of the supernatant fluid removed, evaporated to dry- ness in an oven (60°C), dissolved in 0.02 N HC1, and ana- lyzed for amino acids with an amino acid analyzer (Hi- tachi 835, Na" citrate). Measurement oj release of amino acids Ventricles were dissected from clams, placed in a large volume of 1000 mOsm filtered SW, and incubated for an hour. The ventricles were then placed in 2 ml of either 1000 mOsm or 500 mOsm SW. Preliminary studies of the time course of the release of amino acids from ventri- cles exposed to 500 mOsm SW showed that the levels of amino acid accumulated in the bath became maximal within 90-120 min. Consequently, a 2-h incubation time was chosen for subsequent experiments. At the end of the incubation period, a 500-^1 sample of the bath fluid was removed, acidified with 5 ^1 of 0.2 N HC1, and analyzed for amino acid content on an amino acid analyzer (Hitachi 835). A protein hydroly- sate program was used; quantitation was provided by analyses of a standard mixture of amino acids (Pierce Chemical). Preliminary experiments showed that ala- nine, glycine, and taurine accounted for over 95% of the total amino acid release; the analysis program was there- fore truncated to analyze only those amino acids eluting before cysteine. The amino acid levels are reported in this paper as the sum of alanine, glycine, and taurine. and are reported as /umol/g dry weight. In experiments involving the phorbol esters and the c-AMP agonists, the tissues were incubated in 1000 mOsm SW containing the desired concentration of the agent for 30 min, and were then transferred either to 1000 mOsm or 500 mOsm SW containing the same concentration of these agents. Measurement of changes in wet weight The ventricles were removed from clams and incu- bated in 1000 mOsm SW for an hour. The ventricles were blotted repeatedly on tissue paper until they left no bead of water when touched to a glass plate, and then weighed on an analytical balance (Sartorius 2463). The ventricles were then transferred to test tubes containing 2 ml of the test medium and weighed at intervals. This procedure produced repeatable weights for 3-4 weigh- ings, but with further blotting and weighing, the tissue adhered to the blotting paper and a substantial amount (20-30% wet weight) was lost. Statistical treatment of the data Changes in wet weight were expressed as percentages of the original weight of the ventricle; these values were transformed by the arcsin transformation and means and standard deviations were computed. Differences in the mean changes in weight between control and treated 262 L. E. DEATON Table I Iniinoiicul content oj ventricles from Mercenaria mercenaria acclinuilt'd to 1000 mOsm seawater Aminoacid Content (fjmol/gdry weight) Taunne 415.8 ±11.9 Aspartic acid 12.0 + 4.1 Glutamic acid 32.1 ± 5.1 Glycine 27.9 ± 5.5 Alanine 30.9 ± 6.7 Arginine 8.2 ± 0.8 Others 8.0 ± 2.5 Total 534.4 ±29.7 ventricles were assessed by Student's t test. Student's t test was also used to evaluate differences among the means of treatment groups in the dose-response experi- ments. The data for the release of amino acids by ventri- cles were analyzed by a one-way analysis of variance. Differences between treatment groups and the appropri- ate solvent control groups were assessed by a priori F tests. Results The amino acid content of ventricles from Mercenaria mercenaria is summarized in Table I. Taurine, glutamic acid, glycine, and alanine make up 95% of the total pool. Aspartate and arginine, as well as very small amounts (5 ^mol/g or less) of proline, threonine, and serine also contribute to the total pool. Traces of the other neutral and basic amino acids were detected in some, but not all, tissues. Taurine, glycine, and alanine account for about 80, 10. and 5%, respectively, of the total net loss of amino acids from isolated Mercenaria ventricles incubated in either 500 mOsm SW or 1000 mOsm SW. The effects of molluscan neurotransmitters and neurohormones on the release of the three amino acids from ventricles ex- posed to dilute medium are shown in Figure 1 . Ventricles incubated in 500 mOsm SW release 37. 1 ^mol/g in 2 h. The addition of 10~6 M 5-hydroxytryptamine (5HT) to the dilute medium increases the net release of amino acids by 87%; the increase is significant (F, ,,5 = 5.7; P < 0.05). The molluscan neuropeptides FMRFamide. FLRFamide, and pQDFLRFamide, in concentrations of 10~6 M significantly increase the release of amino acids by 1 10% or more, but neither SCPB nor acetylcholine have any effect (Fig. 1 ). The effect of 5HT on the amino acid release is blocked by the 5HT receptor blocker methysergide (UML); UML alone has no effect on the release of amino acids (Fig. 2). Dose-response curves for the effects of 5HT and 140 120 100 80- 60- 40- 20- 0 Cont 5HT FMRF pQD FLRF SCP Ach Figure 1. The effects of molluscan neural products on the release of amino acids from Mercenaria ventricles transferred from 1000 mOsm seawater to 500 mOsm seawater. Each bar represents the mean of 10- ventricles; error bars are 1 SD. Treatments: Cont = controls; 5HT = 5-hydroxytryptamine; FMRF = FMRFamide; FLRF = FLRFamide; pQD = pQDFLRFamide; SCP = SCPB; Ach = acetylcholine. The con- centration of each agent was 10~6 M Each point is the mean ± SD, n = 9. Asterisks indicate treatments significantly different from controls; * = /><0.05;** = /3<0.01;*** = />< 0.001. FMRFamide on the release of amino acids by ventricles in hypoosmotic seawater are shown in Figure 3. The difference in amino acids released between control ven- tricles and those exposed to 5HT and FMRFamide are significant at concentrations of 10""' M (for 5HT, t = 5.78. P < 0.001; for FMRFamide, t = 2.57, P< 0.05) and above. Concentrations of 5-HT greater than 10~8M elicit no further significant increase in the release of amino acids. The amino acid releases elicited by concen- trations of FMRFamide from 10"'° to 10~6 M are not 80- 60- 40- 20- 0 Cont 5HT 5HT + UML UML Figure 2. The effect of methysergide and 5-hydroxytryptamine on the release of amino acids from Mercenaria ventricles transferred from 1000 mOsm seawater to 500 mOsm seawater. Each bar represents the mean of five ventricles; error bars are 1 SD. Treatments: Cont = con- trols: 5HT = 5-hydroxytryptamine (10"6 A/); 5HT + UML = 5-hy- droxytryptamine ( 10 " M land methysergide ( 10 ~5 A/); UML = methy- sergide ( 10 5 .I/). The asterisks indicate treatments significantly differ- ent from controls: *** = P < 0.00 1 . VOLUME REGULATION IN MERCENAR1 I 263 l^U- 1 DU- 140- 100- ^,{ \ — T •^ T £ S* * horbol(10~7 A/); PDA = phorbol 12,13-diacetate(10~7 M); PMA = phorbol 12-acetate,13-myristate (10 7 A/). Asterisks indicate treatments that are significantly different from controls: * = P < 0.05; ** = P<0.01:*** = P<0.001. significantly different, but the difference between tissues exposed to 10~'° and 10~5 M is significant (t = 3.22, P <0.01). The effects of several cyclic 3',5'adenosine monophos- phate (cAMP) agonists on the release of amino acids in hypoosmotic seawater are shown in Figure 4. None of these agents affect the amino acid release. The cyclic gua- nosine monophosphate agonist 8-bromo-cyclic GMP also has no effect on the release of amino acids from ven- tricles in 500 mOsm SW (data not shown). In contrast, two phorbol esters potentiate the amino acid release, while 4-j5 phorbol has no effect (Fig. 5). The effective es- 80 • • 60-- 40-- 20-- 0 Cont ETON Forsk IBMX 8Br-cAMP Figure 4. The effects of adenosine 3-'5'cyclic monophosphate ago- nists on the release of amino acids from Mercenaria ventricles trans- ferred from 1000 mOsm seawater to 500 mOsm SW. Each bar repre- sents the mean of 10 ventricles; error bars are 1 SD. Treatments: Cont = controls; ETOH = ethanol (0.1%); Forsk = forskolin (10 - A/); IBMX = 3-isobutyl-l-methylxanthine (10~3 A/); 8Br-cAMP = 8-bro- mo-adenosine 3'-5'cyclic monophosphate ( 10 3 M). ters, phorbol 12,1 3-diacetate and phorbol 1 2-acetate, 1 3- myristate, increase the release of amino acids by 140 and 106%, respectively; the comparisons were made to ven- tricles incubated in hypoosmotic seawater containing di- methylformamide ( 1 .4 mA/). The effects of 5HT, selected molluscan neuropeptides, and the phorbol esters on the release of amino acids from ventricles incubated in isosmotic seawater for 2 h are shown in Figure 6. The release of amino acids from ven- tricles in isosmotic SW is considerably lower than that from tissues exposed to hypoosmotic SW. None of the molluscan neural products affects the release of amino acids from tissues in isosmotic medium. The release of amino acids in the presence of phorbol esters and 4-0 phorbol is not different from that of ventricles incubated in 1000 mOsm SW containing dimethylformamide ( 1.4 mAf). The release of amino acids from ventricles incu- bated in dimethylformamide is significantly higher than that of control tissues incubated in 1000 mOsm SW. The changes in wet weight experienced by ventricles incubated in hypoosmotic seawater containing 5HT, FMRFamide, and phorbol esters are shown in Figure 7. Ventricles transferred from 1000 mOsm SW to 500 mOsm SW gained about 20-30% in wet weight within 10 min; this gain is maintained for 30 min, and then gradually decreases to zero over the following 30 min. The addition of FMRFamide (10 6 M) to the bathing medium reduces the time required by the tissues to regu- late volume (Fig. 7a). The changes in wet weight of tis- sues incubated in media containing FMRFamide are sig- nificantly lower than those of control tissues at both 30 (t = 10.1, P < 0.00 1) and 60 min (t = 5.69, P < 0.001) after transfer. 264 L. E. DEATON 16 14 12 10 8 6-- 4.. 2- 1 0 Cont 5HT FMRF pQD FLRF DMF 4|3 PDA PMA Figure 6. The effects of various agents on the release ot'amino acids from Mercenaria ventricles transferred to isosmotic seawater (1000 mOsm). Each bar represents the mean of live ventricles; error bars are 1 SD. Treatments: Cont = controls; 5HT = 5-hydroxytryptamine ( 10~6 A/); FMRF = FMRFamide ( 10 " A/); pQD = pQDFLRFamide ( 10'" M): FLRF = FLRFamide (10"" .M): DMF = dimethylformamide (1.4 ml/); 4/3 = 4-beta-phorbol ( 10~7 A/): PDA = phorbol 12.13-diacetate (10~7A/):PMA = phorbol 1 2-acetate, 1 3-myristate ( 10 7 A/). The wet weights of ventricles incubated in 500 mOsm SW containing 10 6 M 5HT are lower than control tis- sues at 10, 30, and 60 min (t = 9.72; P< 0.001; t = 10.7, P < 0.001; t = 3.17, P < 0.01; respectively) (Figs. 7a, b). Ventricles incubated in 500 mOsm SW containing forskolin ( 10 5 M) gain significantly more weight than controls in the first 10 min (t = 63.2. P < 0.001), but there is no difference in wet weight gain 30 and 60 min after transfer (Figs. 7a, b). Phorbol 1 2-acetate. 1 3-myristate (10~7 M) reduces the changes in weight relative to control tissues at 10, 30, and 60 min (t = 38.3, P < 0.00 1 ; t = 1 7.9. P < 0.00 1 ; t = 6.55, P < 0.001, respectively) following transfer from 1000 mOsm SW to 500 mOsm SW (Fig. 7c). Tissues trans- ferred from 1000 mOsm SW to 500 mOsm SW contain- ing phorbol 12.13-diacetate show significantly higher (t = 124.4. P < 0.001 ) weight gain than control tissues 10 min after transfer, and significantly lower (t = 7.83, P < 0.001) weight gain 30 min after transfer. There is no significant difference between these tissues and controls 60 min after transfer (Figs. 7a. c). The changes in wet weight of ventricles transferred from 1000 mOsm SW to isosmotic SW containing FMRFamide. 5HT. and phorbol esters are shown in Fig- ure 8. There is no significant change in wet weight in con- trol tissues following transfer, nor did any of the treat- ments effect significant differences in weight change rela- tive to control tissues. Discussion The release of amino acids from Mercenaria cardio- myocytes is potentiated by 5HT and by the molluscan neuropeptide FMRFamide and its naturally occurring analogs. These agents also cause a reduction in the time required for ventricles exposed to hypoosmotic media to O 20- 10- o g -10 -20 40 30- 20- 10- o -10- -20 -20 O 15 30 Time (min) 45 60 O 15 30 Time (min) 45 60 30 Time (min) 45 60 Figure 7. The effects of various agents on the time course of changes in wet weight of Mercenaria ventricles transferred from 1000 mOsm seawater to 500 mOsm SW. Each point is the mean of 10 ventri- cles; error bars are 1 SD. Treatments are indicated as follows: 7a — solid circles = controls, open circles = FMRFamide ( 10 " A/); 7b — solid cir- cles = 5-hydroxytryptamine (10" A/), open circles = forskolin (I0~5 A/); 7c— solid circles = phorbol 12.13-diacetate ( 10~7 A/), open circles = phorbol 1 2-acetate. 1 3-myristate (10~7 A/ 1. VOLUME REGULATION IN MERCENAR1A 265 10 5- -5- -10 15 30 Time (min) 45 60 -10 15 30 Time (min) 45 60 Figure 8. The effects of various agents on the time course of changes in wet weight ofMercenaria ventricles transferred to isosmotic seawater ( 1000 mOsm). Each point is the mean of 10 ventricles; error bars are 1 SD. Treatments are indicated as follows: 8a — solid circles = controls; 8b — solid circles = FMRFamide (10~6 A/), open circles = 5-hydroxytryptamine (10~6 M): 8c — solid circles = phorbol 12,13- diacetate (1(T7 A/), open circles = phorbol 1 2-acetate, 1 3-myristate (IQ-7M). volume regulate. The effect of 5HT on amino acid re- lease is mediated by 5HT receptors, since this effect is blocked bv UML. The effective concentrations for the potentiation of amino acid release by 5HT and FMRF- amide are in the nanomolar range; the concentration of FMRFamide in the hemolymph of the clam Macrocal- lista nimbosa is also in this range (Nagle, 1982). Both 5HT and FMRFamide stimulate the mechanical activity of isolated Mercenaria ventricles (Price and Greenberg, 1980); the cardioexcitatory effects and the potentiation of the volume regulatory response in hypo- osmotic media might be due to similar intracellular mechanisms. However, the release of amino acids from ventricles in isosmotic seawater is not affected by either 5HT or FMRFamide. Thus, the chain of events responsi- ble for the cardioexcitatory effects cannot be identical to that responsible for the increase in the release of amino acids and the decrease in the time necessary for the vol- ume regulatory response of the tissues. Previous studies suggest that stimulation of the me- chanical activity ofMercenaria ventricles by 5HT and FMRFamide involves an increased sequestration of Ca++ ions by the sarcoplasmic reticulum effected by an increase in the intracellular level of c-AMP (Higgins. 1974; Higgins and Greenberg, 1974; Higgins el a/.. 1978), but the cardioexcitatory effects of 5HT and FMRFamide cannot be completely explained by this mechanism (Paciotti and Higgins, 1985; Deaton and Gray, 1989). The failure of forskolin to affect the release of acids suggests that c-AMP is not involved in the poten- tiation of this process by 5HT and FMRFamide. Phorbol esters stimulate protein kinase C, which is also stimulated by diacylglycerol, one component of the phosphoinositol cellular signal transduction system (Ni- shizuka, 1984; Berridge, 1986). Phorbol esters potentiate the volume regulatory response and increase the release of amino acids of ventricles exposed to dilute media. The phorbol esters only effect increases in the release of amino acids from tissues exposed to hypoosmotic media. The biologically inert compound. 4-/3 phorbol, also has no effect on amino acid release. The regulatory volume decrease of red blood cells from the clam Noetia ponder- osa is potentiated by PMA, which appears to affect cy- toplasmic K+ levels (Pierce et ai, 1989). Phorbol esters also potentiate the release of amino acids from elasmo- branch erythrocytes incubated in hypoosmotic media (Leite and Goldstein, 1987), and mimic the effect of os- motic shrinking on the ion exchangers responsible for adjustment to hyperosmotic stress by cultured lympho- cytes (Grinstein et ai, 1986). There is, however, no in- crease in the IP, levels in skate red blood cells exposed to hypoosmotic medium (McConnell and Goldstein, 1988). These results suggest that protein kinase C is in- volved in volume regulation; but the role, if any, of IP,, is not clear. Incubation of ventricles from the mussel Geukensia demissa in isosmotic medium containing 0.54 mM KC1. 266 L. E. DEATON which depolarizes the cells by about 60 mV (Wilkens, 1972), increases the release of amino acids from 15 to 21 ^mol/g dry wt (Pierce and Greenberg, 1976). These observations raise the possibility that the effects of 5-HT, FMRFamide. and phorbol esters on the release of amino acids might be due simply to depolarization of the cells. This seems unlikely for two reasons. First, there was no increase in the amino acid release from ventricles treated with 5-HT, FMRFamide, or phorbol esters in isosmotic seawater. Second, large doses ( 10 6 A/) of 5-HT have lit- tle effect on the membrane potential of either /< w , O JZ Q. tn o CT O -o c Figure 2. Octopamine- and forskolin-stimulated phosphorylation of the 1 22 kD protein in Limulus lateral eye slices in vitro as determined by the back phosphorylation procedure. Isolated tissues were incubated for 10 min in saline (Control), or saline containing 2 fiAt octopamine (OCT), or 10 jiA/forskolin (FOR) prior to analysis using the back phos- phorylation procedure. This figure presents the results of one experi- ment. Shown are the autoradiograph, the amount of '2PO4. and the calculated relative level of unlabelled PO4 (endogenous phosphoryla- tion) associated with the 1 22 kD protein in treated samples containing the same amount of 122 kD protein. The amount of protein as deter- mined by fast green staining on the nitrocellulose blot. The results were similar in four replicates. back phosphorylation procedure (6 of 8, or 75% of the experiments). At least two factors may contribute to this increased reproducibility. (1) The 122 kD protein may have been extracted more efficiently by the homogeniza- tion procedure used in the present experiments (homog- enization using a glass-glass homogenizer) as compared to the sonication procedure used previously. (2) Changes in the level of phosphorylation of the 122 kD protein may have been obscured in the conventional experi- ments due to high background radioactivity in the auto- radiographs (See Figure la in Edwards and Battelle, 1987). The 1 22 kD protein was also consistently (8 of 8 exper- iments) phosphorylated in lateral eye slices incubated with forskolin. a nonspecific adenylate cyclase stimula- tor (Fig. 2). Changes in the level of 122 kD protein phosphorylation in vivo in response to: (a) Endogenous efferent activity at night. To test our hypothesis that the 1 22 kD protein becomes phosphory- 272 S. C. EDWARDS ET AL lated at night in response to endogenous activation of the retinal efferent neurons, we compared the level of phos- phorylation of the 122 kD protein in extracts of lateral eyes that had received endogenous efferent nerve input with those that had been deprived of this input by Iran- section of the LON (Barlow, 1983). Because efferent ac- tivity and an increase in the sensitivity of the eye, as mea- sured by an increase in ERG amplitude, are strongly cor- related (Barlow, 1983), we monitored the ERG activity of the intact eye to determine when the efferent fibers became active. In the experiment shown in Figure 3, the ERG ampli- tude began to increase above the daytime level at approx- imately 17:00 and continued to increase until it reached a maximum, sustained level 30 min to 1 h later ( 18:00). At 22:00, both eyes were quickly removed from the ani- mal, processed for SDS-PAGE, and then analyzed for the relative level of endogenous phosphorylation of the 122 kD protein. In this experiment we measured a 34.7% in- crease in the level of endogenous phosphorylation of the 122 kD protein in the eye receiving efferent input com- pared to the control eye that had been deprived of effer- ent nerve activity (See Table IA; animal 1, assay 1). Three separate assays of material from the same animal gave a mean increase (± one SEM 4) of 36 ± 8.4%. Sim- ilar results were obtained with two other animals (Ta- ble IA). Similar results were also obtained in an additional ex- periment in which the eyes of the animal were removed just before the onset of subjective day. In that experi- ment, the level of endogenous phosphorylation of the 122 kD protein in the eye receiving efferent input was 42% greater than that in the deprived eye. This implies that the level of endogenous phosphorylation of the 122 kD protein remains elevated in response to efferent activ- ity throughout the subjective night. (h) Optic nerve stimulation during the clay. The effer- ent input to the lateral eye can be activated during the subjective day by electrically stimulating the LON; this also results in an increase in the sensitivity of the eye (Barlow 1983). Therefore, we examined whether electri- cal stimulation of efferent axons during the day resulted in enhanced phosphorylation of the 122 kD protein in the lateral eye. The animals were prepared, and one lateral optic nerve was stimulated as described in Materials and Methods. The ERG amplitude began to increase within 10 to 30 min after the onset of stimulation, and contin- ued to increase until it reached a maximum level approx- imately 40 to 50 min after the onset of the stimulation (Fig. 4). The ERG amplitude of the unstimulated, sham operated eye was unchanged. When the amplitude of the ERG of the stimulated eye reached a stable, maximum level, both the stimulated eve and the unstimulated eve were removed, processed for SDS-PAGE, and the rela- tive level of 1 22 kD phosphorylation in the two eyes was determined. In the experiment shown in Figure 4, the level of endogenous phosphorylation of the 122 kD pro- tein increased 64.7% in the eye that received electrically stimulated efferent input compared to the intact, day- time, unstimulated eye (See Table IB; animal 2, assay 1 ). The average increase (± one SEM) measured in four separate assays of material from the same animal was 42. 1 ± 2.4%., and in similar experiments performed with 2 other animals, this increase in endogenous phosphory- lation measured 47.2 ± 1 1.2% and 14.5 ± 4.0% (Table IB). Therefore, the increase in endogenous phosphoryla- tion we measured in electrically stimulated eyes during the day was in the same range as what we observed with endogenous efferent input. Distribution of the 122 kD protein in Limulus nervous tisane The 122 kD protein appears to be restricted to those tissues in Limulus involved in visual processes (Fig. 5). It is quantitatively a major protein in the lateral eye, the ventral eye, the lateral and median optic nerves, and the lamina. It is usually observed in the median eye (4 of 6 animals), but its relative abundance in this tissue is vari- able. It is not a major protein constituent in the medulla, the central body region, or other portions of the brain, more posterior portions of the central nervous system, or the leg nerve. Furthermore, it was not detected in the cardiac ganglion, a tissue in which OCT receptors may also be present (Watson and Augustine, 1982; Groome and Watson, 1987). Discussion In this study we present strong evidence that efferent nerve activity stimulates the phosphorylation of a 122 kD protein in Limulus lateral eye. Efferent nerve activity could also modulate the amount of 122 kD protein in Limulus eyes, but because our assays were done on ali- quots of LE extracts that contained the same amount of 122 kD protein, they reveal changes in the level of phos- phorylation of the protein and not changes in its absolute amount. The activity of the efferent nerves that project to Limu- lus eyes is driven by a circadian clock in the central ner- vous system, and efferent nerve activity causes an in- crease in the sensitivity of the lateral, ventral, and me- dian eyes to light during the night (Barlow. 1983; Kass and Renninger, 1988). We show that enhanced phos- phorylation of the 122 kD protein in lateral eyes in vivo correlates with the increased sensitivity of the lateral eye at night. We infer that the phosphorylation of this pro- tein is regulated in a circadian manner, and we speculate EFFERENT-STIMULATED PROTEIN PHOSPHORVLATION IN /./l/C/.r.V I VI S Electroretinogram 273 Time of Day 16^00 18:00 I I Protein Stain Autoradiograph -122 kD- E a. 500- 80 o a 1000 (M CM 1500- ° 2000- O 2500 Q. 3000- LON LON Cut Intact LON LON Cut Intact -100 90 a_ a (M CM Figure 3. The 1 22 kD protein is phosphorylated in vivo in the Limuliis lateral eye in response to activa- tion of the efferent neurons by the circadian clock. This figure shows the results of a single assay (Table IA; animal 1, assay 1). Early in the day, the LON was cut just anterior to one of the eyes to block efferent input to that eye. Input to the other eye was left intact. The animal was then placed into the dark in a standard apparatus for recording the ERG of the intact eye ( Barlow, 1 983). In the record shown, the ERG amplitudes were recorded every 10 min. Before 16:20, the small ERG amplitudes were partially obscured by voltage noise, probably arising from muscle contractions. After 16:20, the ERG amplitudes were clearly visible, and they began to increase after 17:10 as a result of efferent nerve activity. The ERG amplitudes (light sensitivity) stabilized at an elevated level by 19:00. At 22:00, both eyes were removed from the animal and immediately immersed in liquid N:. Each eye was then briefly placed in ice cold saline so that the cornea could be removed, the tissue proteins were solubilized, and the amount of endogenous phosphorylation associated with the 1 22 kD protein from each eye was determined by the back phosphorylation procedure. The lower portion of this figure shows (left to right): the pattern of proteins visualized by silver stain, (aliquots of solubilized preparations of both eyes contained about the same amount of 122 kD protein); an autoradiograph of a nitrocellulose blot containing larger (4 • ) aliquots of these samples, subjected to the back phosphorylation procedure; and the amount of 3:PO4 associated with the 122 kD protein from both eyes. The relative amount of endogenous phosphorylation in response to efferent stimulation was deter- mined from the difference in the amount of labeled phosphate incorporated into the protein from efferent- stimulated and unstimulatedeyes. The amount of labeled phosphate incorporated into the 122 kD protein from the unstimulated eye was expressed as 100%. The increase in endogenous phosphorylation observed in this assay was 34.7%; the mean increase (± one SEM) determined from three separate assays of material from the same animal was 36.0 ± 8.4%. Experiments with two other animals produced similar results: the results of all of these assays are presented in Table IA. 274 S. C. EDWARDS ET AL Table I Percent increase in lite endogenous phosphorylation of the 122 kD protein following: A. Endogenous efferent nerve activity3 Assay number*1 Animal 1. 2 3. Mean ± SENT 1. 34.7 31.4 32.8 33.0 ± 1.2 2. 28.9 26.4 52.6 36.0 ± 10.0 3. 36.1 48.7 44.7 43.2 ± 4.6 B. Electrically stimulated efferent nerve activity"1 Assay number*1 Animal 1. •> 3. 4. Mean ±SEMl 1. 40.3 41.3 38.7 41.1 42.1 ± 2.4 2. 64.7 33.8 43.2 47. 2 ± 11.2 3 18.7 16.6 8.1 14.5+ 4.0 a The experiment was performed on three separate animals exactly as described in the legend to Figure 3. b For each animal studied, the relative level of phosphorylation of the 122 kD protein in homogenates of the unstimulated eye. and the eye receiving efferent input, were compared at least three separate times using the back phosphorylation procedure. c Mean ± one SEM of three or four separate assays of the same set of homogenales. d The experiment was performed on three separate animals exactly as described in the legend to Figure 4. that the phosphorylation of this protein contributes to some aspects of the structural, physiological, or bio- chemical changes that occur in Linuilns eyes in response to efferent nerve activity. Direct correlation helm-en efferent nerve activity and the phosphorylation of the 122 kD protein The direct relationship between efferent nerve activity and the phosphorylation of the 122 kD protein is estab- lished by the combined results of the experiments done during the night and during the day. The observation that the 122 kD protein was relatively more phosphory- lated at night in eyes receiving endogenous efferent nerve input, compared to eyes deprived of input, strongly sug- gests that enhanced phosphorylation of this protein is due to endogenous efferent nerve activity, but it does not eliminate the possibility that other factors, which may be present at night and not during the day, might also be required for the phosphorylation of the protein. How- ever, results of the experiments in which we electrically stimulated the efferent axons during the day demon- strated that all factors necessary for the lateral eye to re- spond to efferent input with enhanced phosphorylation of the 1 22 kD protein are also present during the day. Tissue distribution of the 122 kD protein The 122 kD protein is a quantitatively major protein component of many of the tissues of the Limulus visual system. Its enrichment in the ventral eye, which consists predominately of photoreceptor cells (Clark el al, 1 969), argues that it is enriched in photoreceptor cells. Further- more, the protein is very likely distributed throughout the photoreceptor cell, because it is found in both cell body- and axon-enriched portions of the ventral eye (Ed- wards and Battelle, 1987). But the protein may not be found exclusively in photoreceptor cells; much of the volume of the LON, which also contains a large amount of the 122 kD protein relative to other proteins, is com- posed of the large diameter axons of eccentric cells (Fah- renbach, 1971). It is also found in a high concentration in the first optic ganglia, or laminae, which contain the terminals of photoreceptor and eccentric cells from the lateral eye (Chamberlain and Barlow, 1980). The 122kD protein may also be present in non-neuronal cells of the lateral eye. The absence of a conspicuous 122 kD protein band in the medulla was surprising at first, because this tissue is innervated by cells from each of the eyes (Chamberlain and Barlow. 1980). This observation may be explained by a recent finding, which suggests that all photorecep- tors from the lateral compound eye terminate in the lam- ina and do not innervate the medulla (Caiman el al., 1 990). Thus, compared to the lamina, photoreceptor ter- minals in the medulla occupy a relatively low percentage of the total volume of the tissue. The 1 22 kD protein may be distributed throughout the cells that contain it, but our evidence indicates that it is modified by phosphorylation in the somata of these cells and not in their axons. Previously we showed that activa- tion of cAMP PK stimulated the phosphorylation of the 122 kD protein //; vitro in portions of the ventral eye en- riched in intact photoreceptor cell bodies (Edwards and Battelle, 1987); here we showed that the protein becomes phosphorylated in slices of the lateral eye in vitro and in the intact lateral eye in vivo. By contrast, in the LON, the 122 kD protein appears not to be a normal substrate of phosphorylation by cAMP PK. It becomes phosphory- lated by activation of cAMP PK in broken cell prepara- tions of the LON, but it is not phosphorylated in the LON /'/; vivo, at least during the day (See Legend to Fig. 1 ). nor does it become phosphorylated in intact LON or axons of ventral photoreceptors in response to activation of cAMP PK (Edwards and Battelle, 1987). Conse- quently we believe that, in the optic nerves, the 122 kD substrate protein is physically separated from the cAMPPK. The 122 kD protein is quantitatively the major sub- strate for cAMP PK in broken cell preparations of the Time of Day Loteral Optic Nerve Intact 14:00 I I Light Electroretinogram 15:00 ^M^WUHMM^ Lateral Optic Nerve Stimulated Protein Stain Autoradiograph — 122KD — Stimulated o cL O ^c 00 (M C? CL IOO 200- 400- 600- 800- IOOO- I200 LON LON Intact Cut t Stimulated Figure 4. The 1 22 kD protein is phosphorylated in vivo in the lateral eye in response to electrical stimulation of the efferent axons. This figure shows the results of a single assay (Table IB; animal 2, assay I ). Early in the day, the LON was cut just anterior to one of the eyes. After the animal was placed in the apparatus to record ERG activity, the cut end of the LON that remained with the eye was placed into a suction electrode. The animal was placed in the dark for 2 to 3 h. then the axons of the efferent neurons present in the LON were stimulated continuously with 1 5 V pulses (4 pps, 2 ms pulse duration) for 9 min (indicated by the arrow); the stimulation was then turned off for I min while the ERG amplitude was monitored. This sequence was repeated until the ERG amplitude reached a maximum, at which time both the stimulated and unstimulated eyes were removed and treated as described in Figure 3. As in Figure 3, the ERG amplitudes of both eyes were initially obscurred by voltage noise. In the lower record, the ERG amplitudes began to increase after the second interval of electrical stimulation. The ERG amplitudes remained small in the upper (control) record. The lower portion of this figure shows: the protein pattern visualized by silver stain (aliquots of solubi- lized preparations of both the stimulated and nonstimulated eyes contained about the same amount of 1 22 kD protein); an autoradiograph of a nitrocellulose blot containing larger ( I0x) aliquots of these samples, subjected to the back phosphorylation procedure; the amount of 3:PO4 associated with the 1 22 kD protein; and the calculated amount of endogenous phosphory'ation of the protein in both samples. In the assay shown, we measured a 64.7% increase in endogenous phosphorylation of the 1 22 kD protein in the stimu- lated lateral eye of this animal; the average increase (± one SEM), determinated from separate assays of aliquots of the same extract, was 47.2 ± 1 1.2%. Experiments with two other animals produced similar results. The results of all assays are presented in Table IB. 275 276 S. C. EDWARDS ET AL. 122 kD —- MON LAM MED CBR LE LON VE Protocerebrum Figure 5. The \22 kD protein is a prominent protein component in Linniliis eyes and optic nerves, and in the first optic ganglion. It does not appear prominently in other parts of the Limulux nervous system. Tissues were homogenized in I • I.acmmli ( 19701SDS buffer. After the amount of protein in each sample was determined by a modified Lowry procedure (Peterson. 1977), aliquots containing 1 jig of total tissue protein for each sample were analyzed by SDS-PAGE and silver staining. Abbreviations: lateral eye (LE); lateral optic nerve (LON); ventral eye including P- and A-fractions ( VE); median eye (ME); median optic nerve (MON); regions of the protocerebrum — first optic ganglion (lamina — LAM), medulla (MED), cen- tral body region (CBR), remaining portions (R); circumesophageal nng (CER); abdominal nerve cord (AN'C); peripheral leg nerve (PLN); cardiac ganglion (CG). lamina as well (data not shown). Because we have not yet examined whether it is phosphorylated in the intact tissue in response to agents that increase intracellular cAMP, the significance of these results are presently un- known. A model lor the regulation of visual function by efferent innervution Our current model for how efferent nerve activity modulates the function of Linuilits eyes is presented in Figure 6. The results described in the present study pro- vide support for some aspects of this model. The circadian nature of the efferent nerve input to Limuhis eyes is well established (reviewed in Barlow. 1983), and there is convincing evidence that OCT is a neurotransmitter in the efferent axons. OCT is synthe- sized and stored in the efferent axons that project to the ventral and lateral eyes (Battelle et a/.. 1982; Evans et al.. 1983), and it is released from these axons in a Ca:+- dependent manner //; vitro in response to veratridine (Battelle and Evans, 1986) or depolarization with high extracellular potassium (Battelle and Evans. 1984). OCT mimics many of the physiological effects of endogenous efferent innervation when applied to the lateral eye in situ (Kass and Barlow, 1984), or to in vitro preparations of either the lateral (Kass et at.. 1988; Renninger et al.. 1989), or ventral eye (Kass and Renninger, 1988). Clo- EFFERENT-STIMULATED PROTEIN PHOSPHORYLATION IN UMULUS EYES 277 Limu/us brain Figure 6. A model for the regulation of visual function hy efferent innervation. ( I ) At night, a circadian clock in the central nervous sys- tem activates the efferent libers innervating Limulus eyes, causing the release of the efferent neurotransmitter octopamine (OCT). (2) The in- teraction of OCT with OCT-specific receptors on the photorecoptors, and perhaps other cell types in the lateral eye, increases the activity of adenylate cyclase, thereby elevating intracellular cyclic AMP levels. (3) This results in the activation of cyclic AMP-dependent protein kinase which phosphor, lates the 122 kD protein. We propose that this phos- phoprotein is involved in the anatomical, biochemical, and physiologi- cal processes responsible for increasing the sensitivity of the eye. zapine, an OCT-receptor blocker (Dougan and Wade, 1978; Evans, 198 1 ), blocks both the physiological effects of exogenously added OCT and those generated by en- dogenous efferent activity when it is applied to the lateral eye in situ (Kass and Barlow, 1984). Direct evidence for the release of OCT from efferent fibers in response to electrical stimulation or endogenous activation by the circadian clock //; vivo (Fig. 6, #1) is lacking, but as de- scribed below, the results of this study are consistent with this idea. In the Introduction, we describe the results of many studies that suggest that many of the physiological effects of efferent activity in Limuhts eyes are mimicked by cAMP. Thus, it is predicted that the release of neuro- transmitter from efferent terminals will stimulate an in- crease in cAMP in Limuhis eyes (Fig. 6, #2). Here we showed that efferent nerve input enhanced the phos- phorylation of a major substrate for cAMP PK, the 122 kD protein (Edwards and Battelle, 1987; this study), at sites specific for purified cAMP PK. Because the 122 kD protein is not a substrate for Ca+2/calmodulin protein kinase (Weibe el cil.. 1989) or protein kinase C (Weibe, Caiman, and Battelle, unpub. obs.), our results provide strong indirect evidence that efferent nerve input in- creases the intracellular concentration of cAMP in the lateral eye. OCT, acting apparently through an OCT-specific re- ceptor, stimulates a rise in intracellular cAMP in prepa- rations of ventral and lateral eyes in vitro (Kaupp et at.. 1982; Battelle and Wishart, unpub. obs.). Thus, our cur- rent results are also consistent with the idea that OCT is released in response to efferent fiber activity /// vivo (Fig. 6, #1). However, we cannot exclude the possibility that the 122 kD protein is phosphorylated in response to an- other, unidentified neurotransmitter that is released from efferent terminals and acts at a receptor coupled to adenylate cyclase. Several observations lead us to predict that the 1 22 kD protein is involved in some aspect of the efferent-stimu- lated changes in retinal function (Fig. 6, #3). Its phos- phorylation is stimulated by efferent innervation, and correlates with efferent stimulated changes in visual sen- sitivity. It is a major substrate for cAMP PK in Limuhis eyes, and cAMP is believed to mediate many of the effects of efferent innervation on retinal function. Fur- ther analysis of the role of this protein in efferent-stimu- lated changes in visual function requires a detailed char- acterization of the protein and its cellular distribution. Is the 122 kD protein the only protein phosphorylated in response to efferent innervation? The 122 kD protein was the only detectable substrate for OCT stimulated phosphorylation in intact retinal cells //; vitro (Edwards and Battelle, 1987), and in the ex- periment shown in Figure 3 of this study, the 122 kD protein was the only one that showed a detectable change in phosphorylation that correlates with efferent input in vivo. However, we wish to emphasize that, in the present study, conditions were optimized specifically to examine changes in the level of phosphorylation of the 122 kD protein, and changes in the phosphorylation of quantita- tively more minor protein components may have been missed. Studies with broken cell preparations revealed other potential substrates for cAMP-dependent phos- phorylation (Edwards and Battelle, 1987), but it is un- clear whether these are relevant substrates in intact cells. Because the effects of efferent nerve activity, OCT, and cAMP on retinal function in Limuhis are many and di- verse, the 122 kD protein is unlikely to be the only pro- tein that becomes modified. But the 122 kD protein clearly is a major protein substrate in Limulus eyes, and it is phosphorylated, and presumably regulated, by cAMP-, OCT-, and efferent innervation. Acknowledgments We thank Lynn Milstead and James Netherton for as- sisting in producing the figures. This work was supported by basic research grants from the National Science Foun- dation (BNS 86-07660 and BNS 89-09052 to B-A.B.), and undergraduate research training grants from the Na- tional Science Foundation (BBS 87-12402) and the Grass Foundation (to E.M.W.). Contributions of the Authors S. C. Edwards was principally responsible for designing, conducting, interpreting, and describing this 278 S. C. EDWARDS ET AL. study, A. W. Andrews performed the back phosphoryla- tion assays; G. H. Renninger provided expertise critical to the performance of the electrophysiological assays; E. M. Wiebe set up the electronics required for monitor- ing retinal sensitivity and examined the distribution of the 122 kD protein. B-A. Battelle oversaw all aspects of the study, was heavily involved in its design and the in- terpretation of the data, and, together with G. H. Ren- ninger, contributed significantly to the generation of the manuscript. Literature Cited Barlow, R. B., Jr. 1983. Orcadian rhythms in the L/HW/H.V visual sys- tem. J. Ncurosci 3: 856-870. Barlow, R. B., Jr., S. J. Bolanowski Jr., and L. M. Brachman. 1977. Efferent optic nerve fibers mediate circadian rhythms in the Linittln.1 eye. Science 197: 86-89. Battelle, B-A., and J. A. Evans. 1984. Octopamine release from cen- trifugal fibers of the Limulus peripheral visual system. J. Neuro- chem. 42:11-79. Battelle, B-A., and J. A. Evans. 1986. Veratridine-stimulated release of amine conjugates from centrifugal fibers in the Limulus periph- eral visual system. / Ncurochcm 46: 1464-1472. Battelle, B-A., J. A. Evans, and S. C. Chamberlain. 1982. Efferent fibers to Limiilun eyes synthesize and release octopamine. Science 216: 1250-1 252. Beavo, J. A., I". J. Beehtel, and E. G. Krebs. 1974. Preparation of homogeneous cAMP-dependent protein kinase(s) and its subunits from rabbit skeletal muscle. Melli En:ymol. 38: 299-308. Caiman, B. G., M. A. Lauerman, A. C. Wishart, M. Schmidt, and B-A. Battelle. 1990. A monoclonal antibody to photoreceptor cells in Limulus Invent Ophlhalmol 1fis.Sci.(suppl.):31'.2%6. Chamberlain, S. C'., and R. B. Barlow Jr. 1980. Neuroanatomy of the visual atferents in the horseshoe crab (Limulus polyphemux). J Comp N enrol. 192:387-400. Clark, A. W., R. Millecchia, and A. Mauro. 1969. The ventral photo- receptor cells of Limulus I. The microanatomy. 7 lien. Phvsiol 54: 289-304. Dougan, D. F. H., and D. N. Wade. 1978. Octopamine receptors and their structural specificity. Clin A'v/> Pharmacot Pfivxiol 5:341- 349. Edwards, S. C., and B-A. Battelle. 1987. Octopamine- and cAMP- stimulated phosphory lation of a protein in Limulus ventral and lat- eral eyes. ./ Neuroxci 7:2811-2820. Evans, J. A., S. C. Chamberlain, and B-A. Battelle. 1983. Autoradio- graphic localization of newly-synthesized octopamine to retinal efferents in the Limulus visual system. J Comp Ncurol 219: 369- 383. Evans, P. I). 1981. Multiple receptor types for octopamine in the lo- cust../ Physio/ (Z. = 40- 0 ' ' ' ' ' ^ 2350 re rr 20- |ll II II Ih 2300 3220 2340 2360 2380 240 m/z Figure 4. Positive ion FAB mass spectrum of the purified, mildly oxidized peak. A. Scan up to 3000 nominal mass. In the inset the x-axis is unchanged, but the y-axis is expanded 10-fold. The masses shown are rounded to the nearest integer. The two labeled ion clusters are the singly and doubly protonated molecular ions. B. The mass region around 2342, expanded to show the singly protonated molecular ion cluster. The theoretical ion distribution expected for a compound with the elemental composition found is shown in the inset. ilar to an ANP isolated from eel heart (2 amino acid differences), and both fish peptides are quite similar to a peptide isolated from porcine brain (3 differences within the ring; Fig. 5). The complete absence of a C-terminal "tail" is a unique feature not previously reported for any natri- uretic peptide. The FABms data establish that the pep- tide sequenced had no tail, but we cannot completely rule out the possibility that a tail was lost by proteolysis during purification. Such degradation is unlikely, how- ever, because the retention time of the immunoreactive peak remains the same when extracts are prepared in other ways (data not shown), and Y. Takei (pers. comm.) has sequenced an eel brain ANP-like peptide that also has no tail. Still, this question will not be settled until the cDNA encoding the precursor has been isolated. The C-terminal tail seems to be irrelevant to either the relaxing activity of the peptide or its immunoreactivity. Thus, synthetic killifish peptide and eel ANP (which has a C-terminal tail) are equipotent in relaxing toadfish aor- 284 D. A. PRICE ET AL G H N R 5 K S S S G C F G PJC F G LJK L D R I G S G|K L D R I G S M Y S G L G C S G L G C Ki 1 1 if ish brain N S R K eel heart SPKTMRDS G C F G R R L 0 R ! G S L S G L G C N V L R R Y pig BNP 7SPKMMHKS G C F G R R L D R I G S L S G L G C N V L R K Y dog BNP* 7SPKMVQGS G C F G I'lh M D R I Ills S S G L G C K V L R R H human BNP* M M R D S G C F G R R I D R 1 G S I S G MJG C N G S R K N chicken heart -NSKHAHSSS C F G fl' 0 R I G A V sfiJ1 L G c D G L R L F rat BNP* -NSKMAHSSS C F G «EI« D R I G A V sm L G c D G L R 0 F rat isoANP S L R R S S CFG G R M D R 1 G •'• <.' S G L G C N S F R Y human ANP S L R R S S C F G G R 1 D R I G A 0 S G L G C N S F R Y rodent ANP S S D C F G S R I D R I G A Q SGJH|GC G R R F frog heart Figure 5. The amino acid sequence of the Fiiihtulitx brain ANP- like peptide compared to other ANP peptides. The residues in these other peptides that are identical to those in Fiindulus are boxed. The one-letter abbreviations for the amino acids are used. 'Predicted from cDNA sequence. ?Exact length of predominant peptide is unknown. -These peptides are longer than shown. o m CD 100 -r= 80- 60- 40- 20- 0001 ---• Rot ANP •A Eel ANP — ° Fundulus ANP 001 O.I pmoles/tube 10 Figure 7. A comparison of the immunoreactivity of rat ANP (1- 28), eel ANP, and Fundulus brain ANP-like peptide using an RIA with an antiserum to rat ANP and with iodinated eel ANP as trace. tic rings (Fig. 6). Moreover, the new peptide is as immu- noreactive as rat ANP in an RIA which employs the eel ANP as trace. The latter result is surprising since the RIA antiserum was raised to the rat peptide. Finally, we con- clude that the apparent functional unimportance of the C-terminal tail is consistent with the dissimilarity of its sequence from one peptide to another (Fig. 5). In mammals, the levels of ANP-like immunoreactivity in the heart are orders of magnitude higher than those of any other tissue, but such a tissue distribution is not a general characteristic of fish. For example, Galli ct at. ( 1988) measured roughly equal levels of immunoreactiv- ity in the brains and hearts of several species of teleosts using the same antiserum employed in our experiments. 100- .1 80 •{ 60^ | 40- o 20 -I 0 --• Rat ANP -* Eel ANP - — o Fundulus ANP 12 10 8 7 Log Concentration (M) Figure 6. A comparison of the relaxing activity on rings of toadfish (Optmmix hcia) ventral aorta, of rat ANP (1-28), eel ANP, and FunJu- lus brain ANP-like peptide. Because this antiserum reacts about equally well with the l-'iimluliis and eel peptides (Fig. 7), and because the eel peptide was isolated from heart, and the Fundulus from brain, the simplest interpretation of the data of Galli el a/. ( 1988) is that heart and brain have roughly equal lev- els of peptide. Still, the immunoreactivity in the eel heart is (as in mammals) mostly in a high molecular weight form (Takei ct ai. 1990), and this form may not be as immunoreactive as the smaller molecules that have been isolated and synthesized, and which we have used as standards. Fish plasma contains ANP-like immunoreactivity, and the levels decrease with adaptation to reduced salin- ity (Galli ct ill.. 1988; Evans el til., 1989). But neither the identity of the circulating peptide, nor its source, is known with certainty. In rats, even though the heart con- tains much more immunoreactivity than the brain, changes in hypothalamic and pituitary secretion of ANP markedly affect the blood levels of ANP (Baldissera el ai. 1 989). Therefore, in species of teleosts like Fundulus. where levels of natriuretic peptide are about equal in brain and heart, the brain may be the major source of circulating peptide. Acknowledgments We would like to thank M. I. Phillips and B. Kimura for providing antiserum and for help in starting up the RIA for ANP; P. Lin and co-workers for providing so many Fundulus brains; Y. Takei and his collaborators at the Protein Research Foundation of Japan for giving us some eel ANP and being so helpful in sharing data on fish ANP-like peptides. We would also like to thank L. Milstead for preparing the figures and M. J. Greenberg for helpful criticism of the manuscript. This work was THE STRUCTURE OF A FISH BRAIN ANP 285 partially supported by grants from NIH (HL28440 to D. A.P.), and NSF (PCM-830262 1 to D.H.E.). Literature Cited Aburaya, M . N. Minamino, J. I him. k. kangawa, and II. Matsuu. 1 989a. Distnhution and molecular forms ol'hrain natriuretic pep- tide in the central nervous system, heart and peripheral tissue of rat. Biochem. Biophys. Res. Commun 165: 880-887. Aburaya, M.. N. Minamino, k. kangawa, k. Tanaka, and M. Matsuo. 1989b. Distribution and molecular forms of brain natriuretic pep- tide in porcine heart and blood. Biochem. Biophys. Rex Commun. 165:872-879. Baldissera, S., J. W. Menani, L. F. Sotero Dos Santos, A. L. V. Fava- retto, J. Gutkowska, M. Q. A. Turrin, S. M. McCann, and J. An- tunes-Rodrigues. 1989. Role of the hypothalamus in the control of atrial natriuretic peptide release. Proc. Natl. Acad. Sci. USA 86: 9621-9625. Bulloch, A. G. M., D. A. Price, A. D. Murphy, T. D. Lee, and H. N. Bowes. 1988. FMRFamide peptide in Helisoma: identification and physiological actions at a peripheral synapse. ./ Neuroxci. 8: 3459_3469. DeBold, A. L1., H. B. Bornstein. A. T. Veress, and H. Sonnenberg. 1981. A rapid and potent natnuretic response to intravenous in- jection of atrial myocardial extract in rats. Life Sci. 28: 89-94. Evans, D. H., E. Chipouras, and J. A. Payne. 1989. Immunoreactive atriopeptin in plasma of fishes: its potential role in gill hemodynam- ics. Am. J Physiol. 257: R939-R945. Fly nn, T. G., A. Brar, L. Tremblay, I. Sarda, C. Lyons, and D. B. Jen- nings. 1 989. Isolation and characterization of iso-rANP, a new na- triuretic peptide from rat atria. Biochem. Biophys. Rex Commun. 161:830-837. Galli, S. M., D. H. Evans, B. Kimura, and M. I. Phillips. 1988. Changes in plasma and brain levels of atrial natriuretic pep- tide in fish adapting to fresh water and sea water. FASEBJ. 2: A524. kangawa, k., Y. Tawaragi, S. Oikawa, A. Mizuno, Y. Sakuragawa, H. Nakazato, A. Fukuda, N. Minamino and H. Matsuo. 1984. Identi- fication of rat gamma atrial natriuretic polypeptide and character- ization of the cDN A encoding its precursor. Nature 312: 152-155. kojima, M., N. Minamino, k. kangawa, and II. Malsuo. 1989. Cloning and sequence analysis of cDNA encoding a precur- sor for rat brain natriuretic peptide. Biochem, Biophys. Res. Com- mun 159: 1420-1426. Lewicki, J. A., B. Greenberg, M. Yamanaka, G. Vlasuk, M. Brewer, D. Gardner, J. Baxter, L. k. Johnson, and J. C. Fiddes. 1986. Cloning, sequence analysis, and processing of the rat and human atrial natnuretic peptide precursors. Fed. Proc. 45: 2086- 2090. Misono, k.S., H.Fukumi, R.T. Grammer, and T. Inagami. 1984. Rat atrial natriuretic factor: complete amino acid sequence and disul- fide linkage essential for biological activity. Biochem. Biophys. Res. Commun. 119:524-529. Miyata, A., N. Minamino, K. kangawa, and H. Matsuo. 1988. Identification of a 29-amino acid natriuretic peptide in chicken heart. Biochem. Biophys. Res. Commun. 155: 1330-1337. Price, D. A. 1982. The FMRFamide-like peptide of Helix aspersa. Comp. Biochem Physiol. 72C: 325-328. Sakata, J., k. kangawa, and H. Matsuo. 1988. Identification of new atrial natriuretic peptides in frog heart. Biochem. Biophys. Res. Commun. 155: 1338-1345. Seilhamer, J. J., A. Arfsten, J. A. Miller, P. Lundquist, R. M.Scarbor- ough, J. A. Lewicki, and J. G. Porter. 1989. Human and canine gene homologs of porcine brain natriuretic peptide. Biochem. Bio- phys. Res Commun 165:650-658. Sudoh, T., k. kangawa, N. Minamino, and II. Matsuo. 1988. A new natriuretic peptide in porcine brain. Nature 332: 78-81. Sudoh, T., k. Maekawa, M. kojima, N. Minamino, k. kangawa, and H. Matsuo. 1989. Cloning and sequence analysis of cDNA encod- ing a precursor for human brain natriuretic peptide. Biochem. Bio- phys. Res Commun 159: 1427-1434. Takei, Y., A. Takahashi, T. X. YYatanabe, k. Nakajima, and S. Sakaki- bara. 1989. Amino acid sequence and relative biological activity of eel atrial natriuretic peptide. Biochem. Biophys. Res Commun 164: 537-543. Takei, Y., H. Tamaki, and k. Ando. 1990. Identification and partial characterization of immunoreactive and bioactive atnal natriuretic peptide in eel hearts. J. Comp Physiol (in press). Reference: Bioi Bull. 178: 286-294. (June, 1990) Adaptations to the Deep-Sea Oxygen Minimum Layer: Oxygen Binding by the Hemocyanin of the Bathypelagic Mysid, Gnathophausia ingens Dohrn N. K. SANDERS AND J. J. CHILDRESS Oceanic Biology Group. Marine Science Institute and Department oj Biological Sciences, University of California. Santa Barbara, California 93106 Abstract. The bathypelagic mysid, Gnathophausia in- gens Dohrn, lives aerohically at oxygen partial pressures as low as 6 torr in the oxygen minimum layer off south- ern California. This study is concerned with the O2 bind- ing properties of this mysid's hemocyanin and the func- tion of the pigment in O: uptake at low P0,. The effect of temperature on in vivo hemolymph pH (ApH/AT -0.018) was measured from 2.5 to 12.5°C. Hemocya- nin concentration was estimated to be 24 mg/1, corre- sponding to an O2 binding capacity of about 0.3 mmol O2/l. Freezing of hemolymph samples significantly de- creased the affinity and cooperativity of HcO2 binding, necessitating the use of fresh hemolymph. The HcO2 affinity was high (PM, of 1 .4 torr at 5.5°C, pH 7.87), allow- ing the loading of O2 even at 6 torr. The cooperativity of HcO2 binding was also high (n<,,, = 3.5 at 5.5°C, pH 7.87); presumably allowing the pigment to function effectively as an O2 transporter within the small PO: difference be- tween the environment and the tissues. Temperature differences within the environmental range (2- 10°C) had no significant effect on the oxygen affinity (AH = -6.7 kJ/mol, pH 7.7) or on the cooperativity of O2 binding. A large Bohr shift (A log P5u/ApH = -0.80 to -0.81) was present at all temperatures. L-lactate produced moderate increases in HcO: affinity (A log P50/A log [lactate] -0.13 at pH 7.9) and in cooperativity. Regional and ontogenetic comparisons suggest that regional and onto- genetic differences in HcO: affinity occur in this species. This mysid has a hemocyanin of unusually high O2 affinity and cooperativity of O2 binding for a crustacean living at low temperatures, and this appears to be an ad- aptation for oxygen loading and transport at the cold, Received 3 1 July 1989; accepted 29 March 1990. low oxygen conditions in deep-sea oxygen minimum layers. The reduced temperature sensitivity of HcO2 affinity may also be an adaptation to low oxygen. Introduction Zones of minimum oxygen are found at intermediate depths in most of the world's oceans and, although the oxygen partial pressure in some of these "oxygen mini- mum layers" is only a few torr, populations of pelagic metazoans exist there (Schmidt, 1925; Sewell and Page, 1948; Banse, 1964). These oxygen minimum layers are pelagic habitats with stable conditions of continuously low oxygen and low temperature at intermediate depths (400-1000 m depth) over vast areas. Previous studies have shown that most of the pelagic crustaceans living off California, where P0, at the oxygen minimum is 6 torr, are able to do so aerobically by being unusually effective at extracting O2 from water (Childress, 1968, 1971, 1975). This remarkable ability has been inten- sively studied in the lophogastrid mysid Gnathophausia ingens Dohrn (Childress. 1968, 1971; Belman and Chil- dress, 1976). Gnathophausia ingens is the largest entirely pelagic crustacean, and has a circumglobal distribution between 30°N and 30°S latitudes. The mature female (instar 13, estimated duration of 530 days) produces and carries a single brood at depths greater than 800 m (Childress and Price. 1978. 1983). The first two free-living instars(3and 4, estimated durations of 95 days each) live at depths as shallow as 1 50-200 m. However, for much of its life (in- stars 5 to 10, "intermediate instars." estimated durations from 168 to 207 days each), G. ingens occurs at depths of about 400-800 m, corresponding to the depth range 286 HrO, BINDING IN GNATHOPHAL'SIA 287 OXYGEN PARTIAL PRESSURE (torr) 0 50 100 150 200 NIGHT DAY 1000 10 20 TEMPERATURE (°C) Figure 1 . The day and night depth distribution patterns ofGnalhu- phuusia ingens shown with environmental temperatures and O: partial pressures off southern California. of the oxygen minimum layer oft" southern California (Fig. 1,6-20 torr O2, 4-7°C) (Childress and Price, 1978, 1983). This species has limited anaerobic capacity, and is able to live aerobically at the lowest P0, it encounters off southern California (Childress, 1968; 1971), although it may use anaerobic metabolism briefly to support high activity levels at the lowest O: levels. Its ability to regulate its oxygen consumption to P0, values as low as 3 torr is due to its ability to maintain a high ventilatory flow (up to 8 body volumes min~'), and simultaneously to re- move a large fraction (50-80%) of the oxygen in the in- haled water (Childress, 1971). These abilities are made possible by the highly developed gills and circulatory sys- tem (Belman and Childress, 1976). Belman and Chil- dress ( 1 976) also showed that a high affinity, high cooper- ativity respiratory protein must be present to provide sufficient hemolymph oxygen carrying capacity and un- loading at the very low P0, values at which these mysids live, although at the time of their studies no respiratory protein had been found in the order Mysidacea. A preliminary report demonstrated the presence, in Gnathophausia ingens, of a hemocyanin having a high affinity for oxygen at 20°C (Freel. 1978), but the proper- ties of this hemocyanin were not measured at environ- mentally appropriate temperatures or pH levels. The re- port by Freel is the only publication on a hemocyanin in the entire order Mysidacea. The high affinity at high temperature reported by Freel appears anomalous; i.e.. the O; affinity of hemocyanin normally increases greatly at low temperatures, so how could the hemocyanin be functional at the much lower environmental tempera- ture? In fact, shallow-dwelling crustaceans living at lower temperatures have hemocyanins with lower Oi affinities as well as lower cooperativities, presumably to maintain a sufficient unloading of O: to their tissues (Redmond, 1968; Mangum, 1982; Mauro and Mangum 1982a). In addition, the temperature sensitivity of O: binding by he- mocyanin is often greater at lower temperatures (Mauro and Mangum, 1982b; Bridges, 1986; Burnett el i»i i '/I ( alilix'nui and Hawaii, and from broodingG. ingens females trom off California Ion California Hawaii Brooding females Na* 525.2+ 13.1(3) 5 16.3 ± 13.1 (3) 512.8 ± 16.9(3) K+ 23. 3 ± 2.1 (3) 21.8 ± 1.3(3) 20.9 ± 1.4(3) Ca2+ 11.6± 3.1(3) 6.6 ± 0.8(3) 7.2 ± 1.2(3) Mg2+ 12.5 ± 1.1(3) 15.4+ 1.2(3) 12.8 ± 1.5(3) sev- 10.1 ± 1.7(4) 4.5 ± 0.9(3) 8.7 ± 2.1(3) er 532.8 ± 16.3(4) 525.6 ± 14.4(3) 530.8 ±2 1.2 (3) Values were determined by ion chromatography and are reported as means ± 1 standard deviation, followed by the number of observations in parentheses. and Hawaii had a hemocyanin concentration of 24 mg/ ml (pooled samples of 5 individuals at each site), while the hemocyanin concentration in hemolymph of brood- ing females from California was lower ( 1 6 mg/ml, 5 indi- viduals pooled). The O2 carrying capacities of the hemo- cyanin in the hemolymphs were estimated at 0.32 and 0.21 mmol/l, respectively. Hemolymph ion concentra- tions were typical for a marine crustacean (Mangum, 1 983a) and differed little among California, Hawaii, and brooding female G. ingens (Table I). Temperature/pH relationship in vivo The regression line calculated from //; vivo pH mea- surements versus experimental temperature (2.5-12°C) is: pH = 7.95-0.018 (T°C), r = 0.82, n = 35. The short term in vivo pH change due to temperature in the hemo- lymph of intermediate instar Gnathophausia ingens (ApH/AT = -0.018) was similar to the change due to temperature in the neutral pH of water (ApH/AT -0.017, Reeves, 1977). This value is also similar to in vivo hemolymph pH changes measured in other crusta- ceans over physiological temperature ranges (McMahon and Burggren, 1981; Morris et ai. 1985, 1988; Morris and Bridges, 1989). Effects of pH and temperature on oxygen binding hy hemocyanin The effects of pH and temperature (2- 1 5°C) on oxygen binding by hemocyanin in dialyzed, never frozen hemo- lymph samples from specimens of Gnathophausia in- gens captured off California are reported in Figure 2. The effect of pH on hemocyanin oxygen affinity was large (A log P5,,/ApH = -0.80, 2 to 10°C and -0.81 at 15°C). There was no significant etfect of temperature on HcO: affinity over the 2- 1 5°C temperature range, as shown by a comparison of the y-intercepts of the regression lines relating log P50 to pH at the different temperatures (AN- COVA). Temperature also had no significant effect on cooperativity from 5 to 15°C (ANCOVA), but the slope of the relationship between n50 and pH at 2°C was sig- nificantly different from those at the higher tempera- tures. The temperature sensitivity of HcO2 binding was analyzed by van't Horf plots (Fig. 3). The data for these plots were obtained by estimating P5,, values at three con- stant values of pH from the regression analyses (A log P50/ApH) of data reported in Figure 2. The low AH val- ues at constant pH (AH = -6.7 kJ/mol, pH 7.7, 2-10°C) emphasize the lack of temperature sensitivity in this spe- cies in the physiological temperature range (Fig. 3). Effects ofL-lactate on oxygen binding by hemocyanin The effects of L-lactate at 5°C on HcO2 binding in dia- lyzed hemolymph (never frozen) of Gnathophausia in- gens from California are shown in Figure 4. The slopes of the log P5(, versus pH regressions for 0.09 and 14.32 mmol/l L-lactate were not significantly different, but the elevations were (P< 0.005, ANCOVA). Thus lactate sig- nificantly increases the affinity of this hemocyanin for O2 (A log Pso/A log [lactate] = -0.17, 5.0°C, pH 7.9). The corresponding regression line at 1.23 mmol/l L-lactate fell in between the other two, but its slope was signifi- cantly different from those of the other two (P < 0.025). Analysis of covariance showed that cooperativity of oxy- o in a ai o Figure 2. The effects of pH and temperature on HcO2 binding in dialyzed (never frozen) hemolymph samples from intermediate instar Gnathophausia ingens from California. Regression equations for oxy- gen affinity of hemocyanin: 2°C. Log P50 = 6.04 - 0.80 pH. r = 0.96; 5°C. Log P50 = 6.50 - 0.81 pH. r = 0.94: 10°C, Log P50 = 6.47 - 0.80 pH, r = 0.98; 15°C, Log P50 = 6.59 - 0.81 pH. r = 0.98. Regressions for cooperativity (lines for2°Cand 5°C): 2°C, nso = -14.83 + 2.47 pH, r = 0.96: 5°C. n,0 = -0.89 + 0.56 pH, r2 = 0.70; 10°C, n50 = -3.22 + 0.88 pH. r = 0.98; I5°C, n50 = -5.50+ 1.14 pH, r = 0.76. 290 N. K. SANDERS AND J. J. CHILDRESS Temperature ( C) in o. O) o 0.2 0.0 1/T*10'°(K) Figure 3. The effect of temperature (2-15'C) on HcO: binding of Gnalhopliiiiisia ingens at three constant values of pH (7.4. 7.7, 8.0). Hemolymph samples were taken from intermediate instars of O' ingens from California. Numbers in brackets are AH values calculated from Log P50/pH data (Fig. 1 ) at the indicated pH over the 2-HVC tempera- ture range. Points plotted are interpolations from the data in Figure 2. gen binding by hemocyanin was significantly increased (P < 0.05) in the presence of L-lactate (Fig. 4). Effects of freezing and regional differences on oxygen binding by hemocyanin When compared with hemolymph samples that had not been frozen, samples from California Gnathophau- 1.0 0.6 k. t 0.2 S Q. O -0.2 O -0.6 4 O in c 7.2 7.4 7.6 7.8 PH 8.4 Figure 4. The effects of L-lactate on HcCK binding at 5°C of inter- mediate instars of Gnathophausia ingens from California. Regression equations for HcO; affinity: 0.09 mmol T1 L-lactate, Log Ps0 = 6.50 - 0.81 pH, r = 0.94; 1.23 L-lactate, Log P50 = 3.42 - 0.62 pH, r = 0.89; 14.32 L-lactate, Log P50 = 5.70 - 0.75 pH. r = 0.99. Regres- sion equations for cooperativity (lines plotted for 0.09 and 14.32 mmol/1 L-lactate): 0.09 mmol 1~' L-lactate. nso = -0.83 + 0.55 pH, r = 0.82: 1.23 L-lactate, n50 = -2.15 + 0.81 pH, r = 0.79; 14.32 L- lactate, nM = -3.88 + 0.81 pH, r = 0.99. Ol o o m C 08 06 04 02 00 -02 -04 3 2 1 70 76 pH 78 Figure 5. HcO: affinity and cooperativity at 5°C in fresh and frozen hemolymph from intermediate instar and brooding female Giuilho- phuiixia tn.vi'ii.'i from California, and from intermediate instar Hawai- ian G pelagic mysid (.inulhophau- sui inxen*. Biol. Bull 150: 15-37. Bridges, C. R. 1986. A comparative study of the respiratory proper- ties and physiological function of haemocyanin in two burrowing and two non-burrowing crustaceans. Comp. Biochem. Phvsiol. 83A: 261-270. Booth, C. K., B. R. McMahon. and A. VV. Finder. 1982. Oxygen up- take and the potentiating effects of increased hemolymph lactate on oxygen transport during exercise in the blue crab. Callinectes sapidus. J Comp. Phvsiol. 148: 1 1 1-121. Burnett, I.. E., D. A. Scholnick, and C. P. Mangum. 1988. Tempera- ture sensitivity of molluscan and arthropod hemocyanins. Biol Bull. 174: 153-162. Childress, J. J. 1968. Oxygen minimum layer: vertical distribution and respiration of the mysid Gnathophausia ingens. Science 160: 1242-1243. Childress, J. J. 1971. Respiratory adaptations to the oxygen mini- mum layer in the bathypelagic mysid Gnathophausia ingens. Biol. Bull. 141: 109-121. Childress, J. J. 1975. The respiratory rates of midwater crustaceans as a function of depth of occurrence and relation to the oxygen min- imum layer off Southern California. Comp. Biochem. Phvsiol. 50: 787-799. Childress, J. J., and M. Nygaard. 1974. The chemical composition and buoyancy of midwater crustaceans as a function of depth of occurrence off southern California. Mar. Biol. 27: 225-238. Childress, J. J., and M. H. Price. 1978. Growth rate of the bathype- lagic crustacean Gnathophausia ingens (Mysidacea: Lophogastri- dae). I. Dimensional growth and population structure. Mar. Biol. 50: 47-62. Childress, J. J. and M. H. Price. 1983. Growth rate of the bathype- lagic crustacean Gnathophausia ingens (Mysidacea: Lophogastri- dae). II. Accumulation of material and energy. Mar. Biol. 76: 165- 177. Childress, J. J., A. J. Arp, and C. R. Fisher. 1984. Metabolic and blood characteristics of the hydrothermal vent tube-worm Ril'iiapa- chyptila. Mar. Biol. 83: 109-124. Childress, J. J., A. T. Barnes, L. B. Quetin, and B. H. Robison. 1978. Thermally protecting cod ends for the recovery of living deep-sea animals. Deep-Sea Res. 25: 4 1 9-422. Cowles, D. L. 1987. Factors affecting the aerobic metabolism of mid- water crustaceans. Ph.D. dissertation. University of California. Santa Barbara. 228 pp. Freel, R. \V. 1978. Oxygen affinity of the hemolymph of the mesope- lagic mysidacean Gnathophausia ingens. J. Exp. Zoo/. 204: 267- 273. McMahon, B. R., and \V. \V. Burggren. 1981. Acid-base balance fol- lowing temperature acclimation in land crabs. J. Exp. Zool. 218: 45-52. McMahon, B. R., and J. L. \Vilkens. 1983. Ventilation, perfusion, and oxygen uptake. Pp. 290-372 in The Biology of the Crustacea. I "<)/. S: Internal Anatomy and Physiological Regulation, L. H. Man- tel, ed. Academic Press. New York. Mangum, C. P. 1980. Respiratory function of the hemocyanins. Am. Zool. 20:19-38. Mangum, C. P. 1982. On the relationship between P50 and the mode of gas exchange in tropical crustaceans. Pac. Sci. 36:403-410. Mangum, C. P. 1983a. Oxygen transport in the blood. Pp. 373-429 in The Bioli >gy < >l I he C 'nnuiceu. 1 'ol. .V Internal . I natoiny and Phys- iological Regulation. L. H. Mantel, ed. Academic Press. New York. Mangum. C. P. 1983b. The effect of hypoxia on hemocyanin-oxygen binding in the horseshoe crab Limulus polyphemus. Molec. Phvsiol. 3:217-224. Mangum, C. P., and J. S. Rainer. 1988. The relationship between subunit composition and O: binding of blue crab hemocyanin. Biol. Bull. 174:77-82. Mauro, N. A., and C. P. Mangum. I982a. The role of blood in the temperature dependence of oxidative metabolism in decapod crus- taceans. I. Intraspecitic responses to seasonal differences in temper- ature. J. Exp. Zool. 219: 179-188. 294 N. K. SANDERS AND J. J. CHILDRESS Mauro, N. A., and C. P. Mangum. 1982b. The role of blood in the temperature dependence of oxidative metabolism in deeapod crus- taceans. II. Interspecific adaptations to latitudinal changes. J. Exp. /ool. 219: 189-195. Morris, S. M., and A. C. Taylor. 1983. Diurnal and seasonal varia- tion in physico-chemical conditions within intertida! rock pools. Estuar. Coast SlielfSd. 17: 151-167. Morris, S. M., and A. C. Taylor. 1985. The respiratory response of the intertidal prawn Palaemon clcf-anx (Rathke) to hypoxia and hy- peroxia. Comp Bioi'hem. Phyuol. 8IA: 633-639. Morris, S. M., A. C. Taylor, C. R. Bridges, and M. K. Grieshaber. 1985. Respiratory properties of the haemolymph of the intertidal prawn Palaemon elegans (Rathke). J. Exp. /no/. 233: 175-186. Morris, S. M., P. Greenaway, and B. R. McMahon. 1988. Oxygen and carbon dioxide transport by the haemocyanin of an amphibi- ous crab, Holthuisana Irtinxverxu. J Comp. Physiol. 157B: 873- 882. Nickerson, K. W., and K. E. van Ilolde. 1971. A comparison of mol- luscan and arthropod hemocyanin. I. Circular dichroism and ab- sorption spectra. Comp Hiocliem. Pltyxiol. 39B: 855-872. Redmond, .1. R. 1968. The respiratory function of hemocyanin. Pp. 5-23 in Biochemistry and Physiology of Hemocyanins, G. Ghiretti, ed. Academic Press, New York. Reeves, R. B. 1977. The interaction of body temperature and acid- base balance in ectothermic vertebrates. Ann. Rev. Physio/. 39: 559- 586. Sanders, N. K. 1989. Functional properties of hemocyanins from deep-sea crustaceans. PhD thesis. University of California. Santa Barbara. 209 pp. Sanders, N. K., and J. J. Childress. 1988. Ion replacement as a buoy- ancy mechanism in a pelagic deep-sea crustacean. J. Exp. Bio/ 138: 333-343. Sanders, N. K., A. J. Arp, and J. J. Childress. 1988. Oxygen binding characteristics of the hemocyanins of two deep-sea hydrothermal vent crustaceans. Respir. Phymoi 71: 57-68. Schmidt, J. 1925. On the contents of oxygen in the ocean on both sides of Panama. Snwi-61: 592-593. Sewell, R. B. S., and L. Fage. 1948. Minimum oxygen layer in the ocean. Nature 162: 949-95 1 . Taylor, E. VV. 1982. Control and co-ordination of ventilation and cir- culation in crustaceans: responses to hypoxia and exercise. J. Exp. Biol. 100:289-319. Reference: Binl. Bull 178: 295-299. (June. 1990) Jelly Layer Formation in Penaeoidean Shrimp Eggs WALLIS H. CLARK JR.1, ASHLEY I. YUDIN2, JOHN W. LYNN3, FRED J. GRIFFIN1, AND MURALIDHARAN C. PILLAI1 ' Bodega Marine Laboratory, University of California at Davis, Bodega Bay, California 94923; 2 Department of Reproductive Physiology, University of California at Davis, Davis, California 95616; and* Department of Zoology and Physiology, Louisiana State University, Baton Rouge, Louisiana 70803 Penaeoid eggs undergo dramatic cortical rearrange- ments and extracellular matrix (ECM) alterations in re- sponse to activation. Prior to activation, the outermost ECM is a fibrous vitelline envelope ( 1 , 2, 3). Beneath the vitelline envelope invaginations of the oolemma form extracellular crypts in the egg surface that project into the cortex of the egg (1,2, 3). These crypts contain jelly precursor (JP), previously termed "jelly-like substance" (4) or cortical specializations or rods ( 1, 2, 5). The pres- ence of JP in both mature ovarian oocytes, and in eggs at spawning, was first reported by Hudinaga (4), but the limitations of light microscopy led him to conclude in- correctly, that the "jelly-like substance" was intraoo- cytic, a misconception that was perpetuated in subse- quent reports (6, 7). Work in our laboratory has clearly demonstrated that JP was housed in extracellular crypts that extended into the cortical cytoplasm (1, 2, 5). We referred to the release of JP during egg activation as a cortical reaction and contrasted it with the exocytosis of cortical vesicles in other systems (1,2). Although techni- cally correct, since cortical rearrangements accompany JP release, this terminology has apparently caused some confusion. Bradfield et at. (8) have cloned cDNA for a major ovarian polypeptide which they clearly localize to JP in the crypts of ovarian oocytes, but which they refer to as a "cortical granule polypeptide." In the present note, we first document the extraoocytic nature of JP in ovarian oocytes of the penaeoid shrimp Sicyonia in- gentis, and then trace the formation of the jelly layer from JP during egg activation. Lastly a synopsis is pre- Received 12 March 1 990; accepted 19 March 1990. Abbreviations: Jelly Precursor (JP). Extracellular Matrix (ECM). sented, in diagrammatic form, of the ECM changes that result from egg activation in penaeoid shrimp. The extracellular crypts that contain JP appear in pen- aeoid oocytes during the latter stages of oogenesis (5, 9, 10). Oocytes of 5. ingentis in this stage possess a centrally located germinal vesicle, a large accumulation of yolk in the form of spherical bodies dispersed throughout the ooplasm, and crypts radially arranged around the pe- riphery (Fig. 1A). Figure IB reveals the fine structural relationship among the oocyte, a crypt, and a follicle cell of an ovarian oocyte at the completion of crypt forma- tion. An invagination in the oolemma forms each crypt (Fig. IB). Although the mechanisms by which such in- vaginations are formed have not yet been demonstrated, the crypts at this stage of oogenesis are clearly extracellu- lar, as has been demonstrated for oocytes ofPenqeus a:- tecus (5), P. setiferus (5), and P. japonicus ( 10). Laterally and basally the crypts are delineated by the oolemma, whereas apically the crypts are not bounded by the oole- mma. Instead, the crypts are overlain by the vitelline en- velope, the outermost egg investment prior to activation ( 1, 2, 3, 1 1 ). At this stage, near the completion of oogen- esis, the oocyte and its investments are still surrounded by a layer of follicle cells (Fig. 1 B). The crypts of S. ingentis eggs contain highly organized, tightly packed "bottle-brush" structures (substructural elements of the jelly precursor) similar to the "feathery" substructural elements described for the cortical rods or specializations of oocytes in P. aitecus and P. setiferus (5). In P. aztecus, the "feathery" material constitutes JP that is 25-30% carbohydrate and 70-75% protein (12). "Bottle-brush" structures are also associated with oo- cytes of the non-penaeoid decapods, Homarus america- nus and H. gamarus (13, 14)). In //. americanus. the 295 296 W. H. CLARK JR. ET AL Figure 1 . Ovarian oocytes of.V/i •yniiiu int;cnli\ near the completion of oogenesis. Ovaries of S were dissected and subsequently fixed and processed according to procedures described by Clark ct al. (2). A. Light micrograph depicting an oocyte with a germinal vesicle (GV) and radially arranged extracellular crypts (arrow heads). B. Transmission electron micrograph of an extracellular crypt (C) containing jelly precursor (JP). The crypt is separated from the cortical cytoplasm by the oolemma (O) and delimited from the follicle cell layer (FC) by a vitelline envelope ( VE). Y = yolk platelet. Figures 2-5. Jelly precursor (JP| extrusion and jelly layer formation in Sicyimia ingenlis eggs. Female 51. ingcnlis were induced to spawn and eggs collected as described by Pillai el til (18). Eggs were fixed at times described below and processed for light microscopy (A) and transmission electron microscopy (B) as described by Pillai and Clark (3). Eggs spawned into fixative (time "0") still possess JP housed in extracel- lular crypts (Fig. 2A, arrowheads). In Figure 2B the time "0" egg and its crypts (C) are enveloped by the vitelline envelope (VE). JP. containing "bottle-brush" structures arranged in parallel aggregates, is sepa- rated from the cortical cytoplasm by the oolemma (O). Granular material (G), destined to become the surface coat (see ref. 3) lies just beneath the vitelline envelope. Yolk platelets (Y'), of two densities, are present in the cortical cytoplasm. At 3-5 min post-spawning, JP extrusion is well underway (Fig. 3A. arrowheads). Although released from the crypts, JP remains predominantly in "bottle-brush" form (Fig. 3B). By 10 min post-spawning, a corona has formed around the egg (Fig. 4A) that consists of a flocculent matrix containing dispersed "bottle-brush" structures! Fig. 4B, arrowheads); Y' = yolk platelets. By 15 min. a fully formed jelly layer (JL) is present (Fig. 5A) that is composed entirely of the flocculent matrix (Fig. 5B). In addition, a surface coat (SC) has formed closely apposed to the oolemma. SHRIMP EGG JELLY FORMATION 297 > — '^ JL 298 W. H. CLARK JR. ET AL Figure 6. Chronological order of ECM alterations during egg activation. Penaeoid oocytes nearingthe completion ol'oogenesis are enveloped in an acellular vitelline envelope and possess extracellular crypts that contain jelly precursor (JP). The oocytes do not possess cortical vesicles within their cortical cytoplasm (Fig. 6A). At the time of spawning, the JP within the crypts is separated from the surrounding environment by only the vitelline envelope (Fig. 6B). Upon exposure to seawater. the crypts become reduced in size which results in the expulsion of the JP, the lifting and eventual loss of the vitelline envelope, and the establishment of a corona (composed of a flocculent matrix and bottle-brush structures) around the egg (Fig. 6C). Continued transformation or breakdown of the "bottle-brush" structures results in a flocculent jelly layer. By the time the jelly layer has formed, the crypts have vanished (Fig. 6D). Two populations of cortical vesicles (dense vesicles and ring vesicles) arise in the cortical cytoplasm (Fig. 6E). The dense vesicles first undergo exocytosis (Fig. 6F) and give rise to the thin hatching envelope (Fig. fiG). This is closely followed by the exocytosis of the ring vesicles (Fig. 6H). The coalescence of the material from the ring vesicles with the thin hatching envelope results in the formation of a fully formed envelope (Fig. 61). "bottle-brush" structures (reported to be of follicular ori- gin) are not contained within crypts, but are rather dis- tributed randomly in the extracellular matrix and con- tribute to the endochorion of the ovarian oocyte (14). Although the origin of the "bottle-brush" structures of .S. ingcntis oocytes has not yet been documented, prelimi- nary information suggests that they are of oocytic origin (unpub. obs.). Prior to spawning, 5. itigentix oocytes undergo germi- nal vesicle breakdown and ovulation (9). Examination of an egg spawned into fixative (time "0") reveals that neither ovulation nor spawning has caused the expulsion of JP (Fig. 2A). At the fine structural level, the vitelline envelope is evident, overlying the egg and crypts (Fig. 2B). In addition, granular material destined to form the "surface coat" is also closely associated with the oo- lemma (see also ref. 3). The "bottle-brush" structures in the time "0" egg remain in parallel arrays, apparently attached in groups by their lateral projections. Yolk platelets, although most prevalent deeper in the egg. are SHRIMP EGG JELLY FORMATION 299 present in the cortical cytoplasm; these are not to be con- fused with cortical vesicles. In S. ingentis eggs, cortical vesicles are not present at the time of spawning, rather they arise approximately 25-30 min after the initiation of egg activation (3, 15). Contact with seawater, at spawning, initiates egg acti- vation, which includes the release of JP and the establish- ment of a jelly layer around the egg (3, 11, 16). By 5 min post-spawning, the release of the JP and the coincident decrease in the depth of the crypts are evident (Fig. 3A, B). At approximately 10 min post-spawning, the crypts have disappeared and the JP has formed a corona around the egg (Fig. 4A). The "bottle-brush" structures are dis- persed by this time, appear to be reduced in number, and are now embedded in a flocculent matrix (Fig. 4B). By 15 min post-spawning, a jelly layer has formed around the egg (Fig. 5 A); this layer is composed entirely of a flocculent matrix with no apparent "bottle-brush" struc- tures remaining (Fig. 5B). In addition, a fibrous surface coat has formed from the granular material that had been closely associated with the oolemma prior to egg activa- tion (see Fig. 2B and also ref. 3). The hatching envelope of S. ingentis eggs forms about 40-45 min post-spawning and is the result of the sequen- tial exocytosis of two populations of cortical vesicles that appear after the initiation of egg activation (3, 16). Not only do the exocytosis of cortical vesicles and the resul- tant development of the hatching envelope occur after jelly layer formation, but these events also appear to be independent of the presence of the jelly layer. Eggs that have been stripped of the jelly precursor, and thus do not form a jelly layer, develop hatching envelopes that appear identical to those of normal eggs (3 ). Morphologi- cal evidence suggests that the surface coat serves as a tem- plate on which the hatching envelope forms (3). Egg activation in the Penaeoidea initiates a series of cortical changes that coincide with the loss of one ECM (the vitelline envelope) and the formation of three other ECMs (the jelly layer, surface coat, and hatching enve- lope) (see Fig. 6). The time course of these alterations may differ from one genus, or species, to the next, but the events are consistent for all penaeoids studied to date (1, 2, 3, 11, 15, 17). The jelly layer in penaeoid eggs ap- pears after the onset of egg activation and is formed from JP that is housed in extracellular crypts prior to activa- tion. The hatching envelope is elaborated subsequent to jelly layer formation and is the result of the exocytosis of two populations of cortical vesicles that arise in the cor- tex of the egg after initiation of activation. Acknowledgments. This work was supported in part by NOAA, National Sea Grant College Program, Depart- ment of Commerce, under grant number NA85AA-D- SG140 project number R/A-61 through the California Sea Grant College Program, grant number NA85AA-D- SG141 project number R/SA-1-PD through the Louisi- ana Sea Grant College Program, and USDA Competitive Grant number 87 CRCR- 1 -25 1 4. Literature Cited 1. Clark, W.H., Jr., and J.W. Lynn. 1977. A Mg++ dependent cor- tical reaction in the eggs of penaeid shrimp. J. E\p. Zoo/. 200: 1 77- 183. 2. Clark, W. H., Jr., J. W. Lynn, A. I. Yudin, and H. O. Persyn. 1980. Morphology of the cortical reaction in the eggs of Penaetis a:tecus. Bioi Bull. 158: 175-186. 3. Pillai, M. C., and W. H. Clark Jr. 1988. Hatching envelope for- mation in shrimp (Sicyonia ingentis) ova: origin and sequential exocytosis of cortical vesicles. Tissue & Cell 20: 941-952. 4. Hudinaga, M. 1942. Reproduction, development and rearing of Penaeusjaponicus. Jpn. J. Zoo/. 10: 305-393. 5. Duronslet, M. J., A. I. Yudin, R. S. Wheeler, and W. H. Clark Jr. 1975. Light and fine structural studies of natural and artificially induced egg growth of penaeid shrimp. Proc. 6th Ann. Workshop, World Maricul. Soc. 6: 105-122. 6. King, J. E. 1948. A study of the reproductive organs of the com- mon marine shrimp. Penueiix se/ijerus (Linnaeus). Biol. Bull. 94: 244-262. 7. Cummins, W. C. 1961. Maturation and spawning of the pink shrimp, Penaeus durontm Burkenroad. Trans. Am. Fish. Soc. 90: 462-468. 8. Bradfield, J. Y., R. L. Berlin, S. M. Rankin, and L. L. Keeley. 1989. Cloned cDNA and antibody for an ovarian cortical granule polypeptide of the shrimp Penaeus vanamei. Biol. Bull. Ill: 344- 349. 9. Anderson, S. L., E. S. Chang, and W. H. Clark Jr. 1984. Timing of post vitellogenic ovarian changes in the ridgeback prawn Sicyo- nia ingentis (Penaeidae) determined by ovarian biopsy. Aquacul- t lire 42: 257 -27 1. 10. Yano, I. 1988. Oocyte development in the kuruma prawn Pen- aeusjaponicus. Mar. Biol 99: 547-553. 11. Clark, W. H., Jr., A. I. Yudin, F. J. Griffin, and K. Shigekawa. 1984. The control of gamete activation and fertilization in the marine Penaeidae. Sicyonia ingentis. Adv. Invert. Reprod. 3: 459- 471. 12. Lynn, J. W., and W. H. Clark Jr. 1987. Physiological and bio- chemical investigations of the egg jelly release in Penaeus aztecus. Biol. Bull. 173:451-460. 13. Talbot, P. 1981. The ovary of the lobster, Homants americanus, I. Architecture of the mature ovary. J. Ultrastruct. Res. 76: 235- 248. 14. Talbot, P., and M. Goudeau. 1988. A complex cortical reaction leads to formation of the fertilization envelope in the lobster, Ho- inarus. Gam. Res 19: 1-18. 15. Pillai, M.C., and W.H.Clark Jr. 1990. Development of cortical vesicles in Sicyonia ingentis ova: their heterogeneity and role in elaboration of the hatching envelope. Mol. Reprod. Dev 26: (in press). 16. Pillai, M.C., and W.H.Clark Jr. 1987. Oocyte activation in the marine shnmp, Sicyonia ingenlis. J. E.\p. Zoo/. 244: 325-329. 17. Glass, P. S., J. W. Lynn, and J. D. Green. 1989. Extracellular assembly of the hatching envelope around Trachypenaeus similis eggs in low sodium seawater. ./ Cell Biol 109: 1 28a. 18. Pillai, M. C., F. J. Griffin, and W. H. Clark Jr. 1988. Induced spawning of the decapod crustacean Sicyonia ingentis. Biol Bull 174: 181-185. Reference: Biol Bull. 178: 300-304. (June, 1990) Revival of Dobell's "Chromidia" Hypothesis: Chromatin Bodies in the Amoebomastigote Paratetramitus jugosus LYNN MARGULIS, MICHAEL ENZIEN*. AND HEATHER I. MCKHANN** Department of Botany. University of Massachusetts, Amherst, Massachusetts 01003 Multiple fission of a mature Paratetramitus jugosus (approx. 10 ^m long) resulted in the production of many small, roughly spherical (2-7 ^m in diameter) amoebae (1). Our observations of live material and examination of over two hundred micrographs lead us to suggest that DNA-containing membrane-bounded chromatin bodies bud amitotically from the nucleus. DAPI-stained bodies of these were observed in the cytoplasm of amoebae, mastigotes, and cysts, and at least some of these chroma- tin bodies seemed to be released into the medium. This interpretation revives for P. jitgosits the "chromidia hy- pothesis" of Dobell (2). Our data, consistent with the de- scriptions of Dobell (2), Hogue (3), and Wherry (4), indi- cate that encysting amoebae may reproduce by chro- midia. Dobell's original chromidia concept was limited to amoebae. Others claimed for it far-reaching conse- quences: "chromidia" were touted as an explanation for embryogenesis and histogenesis of metazoa. Although there is no evidence for chromidia in animals, outright rejection of Dobell's chromidia hypothesis sensu stricto as an amitotic multiple fission process in amoebae is un- justified. Paratetramitus jugosus from microbial mats grows rapidly (5); a small inoculum of this amoebomastigote can lead to a confluent culture populating a petri plate within 2-3 days (6). Small DNA-positive bodies and tiny amoebae are ubiquitous in all P. jugosus cultures ( 1 ). Chromatin bodies, recognized in electron micrographs (EMs) of Paratetramitus jugosus and in the medium, are Received 15 September 1 989; accepted 12 March 1990. * Current address: Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215. ** Current address: Department of Biology. University of California at Los Angeles. Los Angeles. CA 90024. not present in cytoplasmic buds ( 1 ; Fig. 1 A), but they are clearly present in the cytoplasm (Fig. 1A, B). After we failed to obtain evidence for sufficiently rapid reproduc- tion by mitosis, budding, or other known reproductive mode, we noticed that chromatin bodies are present in vacuoles ( 1 ). Nearly all amoebae are conspicuously vacu- olated but, in EMs, water-pumping and food-digesting cytoplasmic vacuoles cannot be distinguished. Cytoplas- mic vacuoles generally contain either the chromatin bod- ies or decomposing bacterial food; both may be present in a single vacuole. The intracellular chromatin bodies themselves show varying morphologies from extremely compact forms to highly vacuolated structures (Fig. 1). Chromatin bodies seem to bud from nuclei in close prox- imity to vacuoles (Fig. 2). They may pass through heavily vacuolated cytoplasm or be released directly into the me- dium (Fig. 1 1 in ref. 1). The tendency to vacuolate is clearly intrinsic to the organism since heavily vacuolated amoebae with degraded cytoplasm (e.g.. Fig. 1 B) are seen in the same thin sections as well-preserved, entirely in- tact P. jugosus cells. Nuclear budding, vacuolation, re- lease of chromatin bodies, and digestion of bacteria ap- parently occur in the mastigote as well as the amoeba stage (note in Fig. 1 1 of ref. 1, the presence of the unduli- podium, signifying the mastigote stage; the figure was originally misidentified as an amoeba). The "chromatin bodies" strikingly resemble structures described early in the century by several investigators. Are they "chro- midia"? "Chromidia," by definition, are nucleus-derived bod- ies composed of the same material as chromatin (i.e.. nu- cleoprotein complexes comprising eukaryotic chromo- somes) and capable of producing new cells — or at least new nuclei. Chromidia were defined by Dobell (2) in a description of the life history of Araehnula impatiens (Cienkowski): 300 "CHROM1DIA" HYPOTHESIS REVIVED 301 Figure 1 . A. Heavily vacuolated cytoplasm containing at least eight chromatin bodies (c) distinguishable from mitochondria (m). Bud or fragment of amoeba (b) (Bar = 1 /jm; neg. 1117). B. "Degrading cyto- plasm or cystic residue" in which at least three chromatin bodies (c) can be seen (Bar = 1 ^m: neg. 2048). "Numerous refringent granules can be seen . . . they vary in size, but are mostly of extreme minuteness. Many of them stain deeply with chromatin stains, and for this rea- son— and for another which will be apparent when I have described other stages in the life cycle — I shall call them chromidia." (2, p. 322) In reviewing this and other work. Professor E. B. Wilson of Columbia University, writing in the most influential biology text of the early century, concluded that chro- midia are "small granules or larger irregular clumps of chromatin or a related substance, scattered through the protoplasm without forming a single individualized body [nucleus]." (7. p. 33) Characterized in part by their staining reactions and re- sistance to "peptic digestion" (proteolytic enzymes, ref. 7, p. 25), chromidial granules were asserted to multiply by division. "Both chromidia and mitochondria formerly belonged to that miscellaneous assemblage of granules known as 'mi- crosomes'. Up to a rather late period the two were often confused and even now considerable uncertainy exists concerning their identification. Theoretically an essential distinction lies in the fact that chromidia are of nuclear origin and composed of 'chromatin' while mitochondria are considered by nearly all recent students of the subject as strictly cytoplasmic; but in practice the determination of the origin of these bodies is not an easy task." (7, p. 700) Two well-known German zoologists then conferred great meaning on "chromidia." R. Hertwig first sug- gested, and R. Goldschmidt then developed, an elabo- rate "chromidial hypothesis," which was reviewed by Wilson (7) (see Wilson for original references). ". . . differentiation ... is largely brought about by a pe- riodic emission of chromatin from the nucleus . . ." (7. p. 703) "The chromidia hypothesis underwent a sudden expan- sion with the works especially of Goldschmidt, Popoffand still later of Buchner and of Schaxel by whom an attempt was made to extend it to the Metazoa and to elaborate Figure 2. Mature amoeba with at least five vacuoles (v); the mem- branes of at least one are in direct contact with the nucleus (n). (Bar = 1 pm; neg. 3633). 302 L. MARGULIS ET AL. a general theory of the chromidia. Goldschmidt (1904) described in the epithelial, muscular, glandular and con- nective-tissue-cells a 'chromidial apparatus' consisting of basophilic granules and fibrillar formations which were assumed, mainly because of a general similarity of stain- ing-reactions, to be extruded basichromatin destined to play a particular role in the trophic functions of the cell (p. 726); and Goldschmidt accepted the problem of a similar origin of many other well-known cytoplasmic structures, including the mitochondria, the yolk-nucleus, nebenkern, pseudochromosomes, reticular apparatus, ergastoplasm and cytomicrosomes. These conclusions were extended to the germ-cells by Wassilieff ( 1907), Popoff (1907), and Bucher ( 1 909. 1910), all of whom concluded that the cy- toplasmic granules and filaments aggregated near one pole of the nucleus . . . are chromidia extruded from the nucleus. . ." (7, pp. 702-703) Most careful observers rejected the attempts of Gold- schmidt and others to relate chromidia to theoretical ex- planations of embryonic development. The case was considered closed by Dogiel and most other protozoolo- gistsby 1965. Chromidia are no longer considered as chromatin inclu- sions, emerging from the nucleus into the cytoplasm and capable of reproducing new nuclei in the plasma. All the examples of this kind of nucleus formation have been shown to be based on inaccurate and erroneous observa- tions by investigators during the first two decades of the twentieth century. Therefore, the theory of chromidia has been abandoned and, to avoid misunderstanding, the term 'chromidia' should not be used. (8, p. 28) We, however, find the original "chromidia hypothe- sis" for reproduction of amoebae to be an attractive ex- planation for the rapid growth of P. jugosus populations (1). Furthermore, Dobell's (2) study of amoeba repro- duction accurately describes the salient features of the P. jugosus life history. Dobell's light-micrograph-based drawings ofArachnnla (especially Fig. 343 a, c, g and f; also reproduced in ref. 7, p. 702) are uncannily similar to ours, although Paratetramitus jugosus is much smaller and seldom contains more than a single vesicular nucleus. "Dobell's recent studies of this form show that in its ordi- nary. . . condition it contains a variable number of vesic- ular nuclei. During encystment the nuclei are said to give off numerous chromidia to the cytoplasm and finally wholly disappear as such, now being represented only by the scattered chromidial granules (Fig. 343). The cell thereupon breaks up to form a brood (10-20) of small daughter cells, containing chromidia which give rise to a number of vesicular nuclei each of which seems to arise by growth (and multiplication?) of a single granule. (7, pp. 701-702) Wilson cautiously concludes that increase in number and size of chromidia over the life cycle ". . . has never been sufficiently confirmed by later ob- servers, either in bacteria or other Protista; nevertheless there has been a rather general, more or less tacit, assump- tion that the chromidia are endowed with such powers." (7, p. 702) The expanded version of the chromidia hypothesis was justifiably abandoned; chromidial reproduction has not been shown in animals and cannot serve as an expla- nation of metazoan differentiation. Yet Dobell's original concept was perhaps prematurely rejected for protoc- tists. At any stage, one or more chromatin bodies may be present in amoeba cytoplasm. In healthy Paratetramitus jugosus, chromatin bodies are apparently released through parental vacuoles, whereas in degenerating amoebae, they may be released from heavily vacuolated parental cytoplasm as it disintegrates, yielding what Do- bell called a "cystic residue" (2, p. 333; Fig. IB, 3). We suggest that the chromatin bodies we are observing are tiny young "chromidia." which contain membranes, ri- bosomes, and a thin layer of cytoplasm (Fig. 1 A; 3A, C); these upon release develop into small amoebae (2-3 ^m) which then grow into the typical "limax" amoebae with a vesicular nucleus. Life histories comparable to Dobell's Arachnula were seen in live material for two "vahlkampfiid amoebae," I 'ahlkampfia calkensi (3) and I 'ahlkampfia sp. No. I (4). The figures of Wherry (3. 4, 7 and 9) and Hogue (25-27 and 3 1 ) depict remarkably well what we observe in most P. jugosus amoebae. Chromidial reproduction stages in vahlkampfiid amoebae (from the Oakland, California, water supply, ref. 4; or symbiotic in oysters from Woods Hole, Massachusetts, ref. 3) were clearly depicted. Hence, Dobell's (2) A. impatiens is only one of several amoebae with "chromidial stages." Arachnula, now clas- sified with the Granuloreticulosa, is still poorly known; although presumed to be a "naked foraminiferan" (9), it may be related to vahlkampfiid amoebomastigotes. Chromidia and multiple fission products leading to a 10-20 offspring "brood," amply detailed by Dobell, re- quires further investigation. Our examination of live ma- terial and over 200 micrographs (phase contrast, Nomar- ski differential interference contrast, scanning and trans- mission electron micrographs) of Baja California, Cuban, and American Type Culture Collection Paratet- ramitus jugosus cultures lead us to agree with Dobell (2), Hogue (3) and Wherry (4), all of whom claimed that chromidia are normal propagules in the life history of certain mastigote amoebae. Now that protoctists are excluded from the animal kingdom, which is defined by its blastular embryos, "protozoa" as a phylum no longer exists. We propose reviving the possibility that Paratetramitus jugosus and "CHROMIDIA" HYPOTHESIS REVIVED 303 Figure 3. Vacuolated cytoplasm of Paratetramitus jugosus; vacuoles may contain either chromatin bodies (c) or bacterial remains (b). A. Swollen vacuole contains coated chromatin body (c); four other vacuoles (v) probably contain bacterial food remnants. Mitochondria (m), not in vacuoles, are distinguish- able from chromatin bodies. The [9(2) + 2] axoneme of two undulipodia (u), here and in B, suggest these organisms are mastigotesfneg. 1 120). B. Bacterial spore (S) in one vacuole and remains (b) in another with a chromatin body (c) apparently in the process of release (neg. 2155). C. Bacterial remains (b) and mem- brane-coated chromatin body in large peripheral vacuoles (v) (neg. 1261). All bars = 1 nm. other protoctists (such as certain vahlkampfiids and Ara- chniila) produce and release chromidia. That multiple fission, which usually occurs within cysts, can take place entirely independently of encysta- tion was amply demonstrated in Entamocba histolytica by Cleveland and Saunders (10); their "chromatoid bod- ies," thought to be phosphorus reserves, deserve, like ch- romidia, re-evaluation in a modern context. Multiple fission, which has long been known in other protoctists. notably suctorian and chonotrich ciliates (11, 12), was recently seen in large Trichosphaerium-tike marine amoebae which feed on brown algae (e.g., Sargassum: ref. 13). These studies with live amoebae demonstrate that electron microscopy and molecular biology enor- mously aid in the interpretation of live material but can never supplant it. Anoxia may select for rapid propagule production; chromidia formation seems to be more prevalent than standard amoeba promitosis under conditions of low ambient oxygen as suggested by Wherry (4). Observa- tions of live amoebomastigotes, grown under controlled oxygen concentrations, must be further correlated with their fine structure morphology. Chromidia must be iso- lated and cloned for study by molecular biological tech- niques. The qualitative and quantitative relationship of the DNA of chromatin bodies to nuclear DNA must be determined. The nature of the membranes and ribo- somes accompanying the DNA of chromatin bodies must be characterized by biochemical techniques. Only after such difficult tasks are accomplished can the exis- tence of chromidia be entirely substantiated. We demon- strate here, however, that the rejection of Dobell's chro- 304 L. MARGULIS ET AL midial concept by proto- and other zoologists (e.g., ref. 8) was probably premature and prejudicial. Protoctists are not little animals (14, 15, 16). This realization revives claims earlier in this century of the uniqueness of many protoctist structures and processes, including the forma- tion, cytoplasmic passage and vacuolar release of chro- midial propagules of amoebomastigotes. fNote added in proof A book written in Russian by B. Swarczewsky about the chromidial concept has come to our attention. The transliterated title is Krornidial'nyiia obrazovaniia n pro- tozoa. Published in Kiev by the Imperial University Press in 1912, the book is a Meinoire de la Societe des Natwulistes de Kieff. I >>/. XXII. A German translation, found on the title page, reads Die Chroinidien der Proio- zoen imd Hire Beziehung :ur Chromatindualismushy- pothese. From the drawings found in this 1 76-page book, we infer that some of the phenomena described in the present paper are also discussed in this volume. Acknowledgments. We are grateful to Floyd Craft for aid with electron microscopy, and to Rene Fester and Thomas Lang for manuscript preparation; we thank the Lounsbery Foundation, NASA Life Sciences, and re- search trust funds at the University of Massachusetts for financial support. Literature Cited 1. En/ien, M., II. I. McRhann, and L. Margulis. 1989. Ecology and life history of an amoebomastigote. Paraletramitus nigosus. from a microbial mat: new evidence for multiple fission, fiiol Bull. 177: 110-129. 2. Dobcll, C. 1913. Observations on the life-history of Cienkowski's "Ann hiuilci" . irch Prolislcnkd 31: 317-353. 3. Hogue, M. 1914. Studies of the life history of an amoeba of the Umax group. Arch Protistenkd. 35: I 54- 163. -1. Wherry, \V. B. 1913. Studies on the biology of an amoeba of the limax group. Vahlkampfiasp. No. I. Arch. Protistenkd- 31: 77-94. 5. Stolz, J. F. 1990. Distribution of phototrophs in the flat lami- nated microbial mat at Laguna Figueroa, Baja California. Mexico. BioSystems 23: 345-357. 6. Read, L. R., L. Margulis, J. Stolz, R. Obar, and T. K. Sawyer. 1983. A new strain of Paratetramitus jugosus from Laguna Fi- gueroa. Baja California. Mexico. Biol. Bull 165: 241-264. 7. Wilson, E. B. 1925. The Cell in Development and Heredity. 3rd edition. The Macmillan Company, New York. 8. Dogiel, V. A. 1965. General Protozoology. 2nd edition. (Revised by J. I. Poljanskij and E. M. Chejsin.) The Clarendon Press, Ox- ford, England. 9. Lee, J. J. 1990. Granuloreticulosa. Pp. 524-548 in Handbook of Proloclista; The Structure. Cultivation, Habitats and Life Cycles of the Eukarvolic Microorganisms and Their Descendants Exclusive of Animals. Plants and Fungi, L. Margulis, J. O. Corliss. M. Mel- konian, and D. J. Chapman, eds. Jones and Bartlett Publishers, Boston 10. Cleveland, L. R., and E. P. Saunders. 1930. Encystation, multi- ple fission without encystment, excystation, metacystic develop- ment, and variation in a pure line and nine strains of Eniamocha hislolvlica. Arch Prnlistcnkd. 70: 224-266. 11. Corliss, J. O. 1979. The Ciliated Prolo:oa. Characterization, Classification and Guide to the Literature. Pergamon, Press, Lon- don. 12. Raikov, I. B. 1982. The Protozoan Nucleus: Morphology and Evolution Springer Verlag, Vienna and New York. 13. Polne-Fuller, M. 1987. A multinucleated marine amoeba which digests seaweeds. ./ Protozool. 34: 159-165. 14. Corliss, J. 0. 1990. Toward a nomenclatural protist perspective. Pp. xxv-xxx in Handbook of Proloclista: The Structure. Cultiva- tion. Habitats and Life Cycles of the Eukaryotic Microorganisms and Their Descendants Exclusive of Animals. Plants and Eungi. L. Margulis, J. O. Corliss, M. Melkonian, and D. J. Chapman, eds. Jones and Bartlett Publishers. Boston. 15. Margulis, L. 1990. Introduction. Pp. xi-xxiii In Handbook of Protoctista: The Structure. Cultivation. Habitats and Life Cycles of the Eukaryotic Microorganisms and Their Descendants Exclusive ut Animals, Plants and Fungi. L. Margulis, J. O. Corliss, M. Mel- konian. M. and D. J. Chapman, eds. Jones and Bartlett Publishers, Boston. 16. Margulis, L., and D. Sagan. 1985. Order amidst animalcules: the Protoctista kingdom and its undulipodiated cells. BioSystems 18: 141-147. INDEX 5-HT, 260 A, adenosine receptor modulation ot'adenylyl cyclase of a deep-living teleost fish, Anlimora rostrata, 65 A, adenosine receptors, 65 rniai, The. 231 Efferent innervation to Limiilitx eyes in v/vn phosphorylates a 1 22 kD protein, 267 Egg jelly formation, 295 Eicosanoid, 1 Eicosatricnoic acid, I ELDRIDGE, DANA, see Keith Wight. 205 Electromyographic record of classical conditioning of eye withdrawal in the crab, 187 Embryogenesis, 231 Endodermal differentiation, 222 ENZIEN, MICHAEL, see Lynn Margulis, 300 Enzyme. 144, 160 Epidermis, 217 Epimorphic regeneration. 2 1 Eriocheir xincnxix, 94 EVANS, D. H., see D. A. Price, 279 Exocytosis, 137 Extracellular crypts. 295 Feeding, 205,217 FEINMAN, RICHARD D.. RAFAEL H. LLINAS, CHARLES I. ABRAMSON, AND ROBIN R. FORMAN, Electromyographic record of classicial conditioning of eye withdrawal in the crab, 187 FEEDER. DARRVL L., see Donald L. Lovett. 144. 160 Fertilization, 44, 210 Fertilization efficiency, 85 FLOOD, PER R., DON DEIBEL. AND CLAUDE C. MORRIS. Visualization of the transparent, gelatinous house of the pelagic tunicate Oi- kopleura vanlwdh'ni using Scpui ink, 1 18 FMRFamide, 260 Food aversion learning by the hermit crab Pagurus granosimanus, 205 Food vacuoles, 300 FORMAN, ROBIN R., see Richard D. Feinman, 187 FORWARD, RICHARD B., JR.. Behavioral responses of crustacean larvae to rates of temperature change. 195 FRANCIS, LISBETH. see Keith Wight, 205 FREEMAN. JOHN A., Regulation of tissue growth in crustacean larvae by feeding regime, 2 1 7 Functional morphology, 126 I'whiuhts hclcriicHius, 279 GALLI, S. M., see D. A. Price, 279 GARLAND, LISA, see Joseph P. Bidwell, 23 1 GARY G. MARTIN, see Jo Ellen Hose, 33 Genital ducts. 94 GERARD, ALISON SUE. see Jo Ellen Hose, 33 G, protein pertussis toxin ribosylation, 65 GNAIGER, ERICH, see Roger A. Byrne, 25 1 Gnalhophausia ingcns, 286 GOLDMAN, ROBERT, see George Dessev, 210 Granulocytes, 55 Green crab, 187 GRIFFIN, FRED J.. see Wallis H. Clark Jr., 295 Growth, 2 I 7 H Hatching envelope, 295 HAUENSCHILD, CARL, see Bernd Schierwater. 1 1 1 Heart rate, 25 1 Helisoma, radula and degrowth, 25 Hemocyanin. 46, 286 Hemocyte, 33, 55 Hemolymph, 286 Hepatopancreas, 144 HESSINGER, DAVID A., see Glyne LI. Thorington. 74 Histochemistry, 160 HOEGH-GULDBERG, O., see D. C. Sutton, 1 75 Horseshoe crab Ta<.-liyi>lcits trulcntalus has two kinds of hemocytes: granulocytes and plasmatocytes. The, 55 HOSE, Jo ELLEN, GARY G. MARTIN, AND ALLISON SUE GERARD, A decapod hemocyte classification scheme integrating morphology, cytochemistry, and function, 33 Host-factor. 175 Host-zooxanthella interactions in tour temperate marine invertebrate symbioses: assessment of effect of host extracts on symbionts, 1 75 HSIEH. HWEY-LIAN. AND JOSEPH L. SIMON, The sperm transfer system in Kinbergonuphis simoni (Polychaeta: Onuphidae), 85 Humoral immunity, 137 Hydrostatic pressure. 65 Hypoxia, 46, 286 Immune defense, 55 Immunological influence, 21 JAKOBSEN, PER Pt.ouc;, AND PETER SUHR-JESSEN. The horseshoe crab Tacliyplciix tridcnliitus has two kinds of hemocytes: granulocytes and plasmatocytes, 55 Jelly layer formation in Penaeoidean shrimp eggs. 295 JONES. GLENN, see Joseph P. Bidwell. 23 1 k KIER, WILLIAM M., AND ANDREW M. SMITH, The morphology and mechanics of octopus suckers, 1 26 KUZIRIAN, ALAN, see Joseph P. Bidwell, 231 Lactate, 286 Larvae, 2 1 7 Learning, 187.205 LEE, T. D., see D. A. Price. 279 LEE, TAI-HLING, AND FUMIO YAMAZAKI, Structure and function of a special tissue in the female genital ducts of the Chinese freshwater crab Eriocheir M/ICHS/.V, 94 Life cycle. I 1 1 Life history strategy, 1 I I Limiting l~37. 267" l.imulti.t blood cell secretes «2-macroglobulin when activated, 1 37 LLINAS, RAFAEL H., see Richard D. Feinman. 1 87 LOMBARD, MARY F.. see Raymond E. Sicard, 2 1 INDEX TO VOLUME 178 307 LOVETT, DONALD L., AND DARRYL L. FELDER. Ontogenetic change in digestive enzyme activity of larval and postlarval white shrimp Penaeus setiferus (Crustacea, Decapoda, Penaeidae), 144 LOVETT, DONALD L., AND DARRYL L. FELDER, Ontogenetic changes in enzyme distribution and midgut function in developmental stages of Penaeus set items (Crustacea, Decapoda, Penaeidae). 160 LYNN. JOHN W., see Wallis H. Clark Jr., 295 M MANGUM, CHARLOTTE P., see Peter L. deFur. 46 MARGULIS, LYNN, MICHAEL ENZIEN, AND HEATHER I. MCKHANN. Revival of Dobell's "chromidia" hypothesis: chromatin bodies in the amoebomastigote Paratetramitus jugosus, 300 MARTIN, VICKI J., Development of nerve cells in hydrozoan planulae: 111. Some interstitial cells traverse the ganglionic pathway in the endoderm, 10 McKHANN, HEATHER L, see Lynn Margulis, 300 MCMAHON. ROBERT F., see Roger A. Byrne, 25 1 Midgut. 160 Molluscan radula and degrowlh. 25 Molluscs. 260 Morphologic and genetic verification that Monterey Botryllus and Woods Hole Botryllus are the same species. 239 Morphology and mechanics of octopus suckers. The, 1 26 MORRIS, CLAUDE C., see Per R. Flood, 1 1 8 Multiple fission, 300 MURRAY. THOMAS F., see Joseph F. Siebenaller, 65 Muscular-hydrostats, 126 Mysid, 286" N NADEAU, LLOYD, see Joseph P. Bidwell. 23 1 Nematocysts, 74 Nerves, 10 Notophtalmus viridescens, 21 Nuclear budding, 300 Nudibranch, 175 PILLAI, MLIRALIDHARAN C., see Wallis H. Clark Jr.. 295 Planulae. 10 Plasmatocytes, 55 Polychaeta. 1, 101 Potentiation of hypoosmotic cellular volume regulation in thequahog. Mereenaria mercenaria, by 5-hydroxytryptamine. FMRFamide. and phorbol esters, 260 PRICE, D. A.. K. E. DOBLE, T. D. LEE, S. M. GALLI, B. M. DUNN, B. PARTEN, AND D. H. EVANS, The sequencing, synthesis, and biological actions of an ANP-like peptide isolated from the brain of the killifish I-'uiululiis helernclilus, 279 Propagules, 300 Protease inhibitor. 1 37 Protein phosphorylation, 267 Protoctista, 300 Putative immunological influence upon amphibian forelimb regenera- tion. II. Effects of x-irradiation on regeneration and allograft rejec- tion, 2 1 QUIGLEY, JAMES P., see Peter B. Armstrong. 137 R Radula secretion, abnormal, 25 REESE, JOHN E.. see Peter L. deFur. 46 Regulation of tissue growth in crustacean larvae by feeding regime. 2 1 7 RENNINGER, GEORGE H., see Samuel C. Edwards, 267 Reproductive system, 94 Respiratory pigment, 46 Respiratory responses of the blue crab Callinectes sapidus to long-term hypoxia, 46 Revival of Dobell's "chromidia" hypothesis: chromatin bodies in the amoebomastigote Paratetramitus jugosus, 300 RICKLES, FREDERICK R., see Peter B. Armstrong, 137 Role of arachidomc acid and eicosatrienoic acids in the activation of spermatozoa in Arenicola marina L. (Annelida: Polychaeta). The, I RUSSELL-HUNTER, W. D., see David A. Smith. 25 O Octopus, 126 Oikoplcura vaiihoeffcni, 1 18 Oligochaetes, 1 I I Ontogenetic change in digestive enzyme activity of larval and postlar- val white shrimp Penaeux setij'erus (Crustacea, Decapoda, Penaei- dae). 144 Ontogenetic changes in enzyme distribution and midgut function in developmental stages of Penaeus setij'erus (Crustacea, Decapoda, Penaeidae), 160 Onuphid polychaete, 85 OSANAI, KENZI, see Masanori Sato, 101 Oxygen consumption, 25 1 Oxygen minimum layer, 286 Oxygen-binding, 286 PACEY. A. A., see M. G. Bentley. 1 Paratetramitus jugosus, 300 PARTEN, B., see D. A. Price, 279 Pavlovian conditioning, 187 Penaeoid eggs, 295 Penaeus, 144, 160 Peptide, 279 Phorbol esters, 260 Photoperiod determined life-cycle in an oligochaete worm. A, 1 1 1 Photoreceptors, 267 SAITO, YASUNORI, see Heather C. Boyd, 239 SANDERS, N. K... AND J. J. CHILDRESS. Adaptations to the deep-sea oxygen minimum layer: oxygen binding by the hemocyanin of the bathypelagic mysid. Gnathophausia ingenx Dohrn, 286 SATO, MASSANORI, AND KENZI OSANAI, Sperm attachment and aero- some reaction on the egg surface of the polychaete, Tylorrliynchus heterochaetus, 101 SCHIERWATER. BERND, AND CARL HAUENSCHILD, A photoperiod de- termined life-cycle in an oligochaete worm, 1 1 1 Sea anemones, 74 Seminal receptacles, 85 Sepia, 1 1 8 Sequencing, synthesis, and biological actions of an ANP-like peptide isolated from the brain of the killifish Fundulus heteroclitus, The. 279 Sexual and asexual reproduction. 1 1 I Shrimp, 144, 160.217 SICARD, RAYMOND E., AND MARY F. LOMBARD, Putative immuno- logical influence upon amphibian forelimb regeneration. II. Effects of x-irradiation on regeneration and allograft rejection. 2 1 SIEBENALLER, JOSEPH F., AND THOMAS F. MURRAY, A, adenosine receptor modulation of adenylyl cyclase of a deep-living teleost fish, Antinwra roslrala, 65 SIMON, JOSEPH L., see Hwey-Lian Hsieh, 85 SMITH, ANDREW M., see William M. Kier, 126 SMITH, DAVID A., AND W. D. RUSSELL-HUNTER. Correlation of ab- normal radular secretion with tissue degrowth during stress peri- ods in IlclisKina fr/vo/vw (Pulmonata, Basommatophora). 25 308 INDEX TO VOLUME 178 Soft coral, 175 Specification of cell late. 222 Sperm activation. I Sperm attachment and acrosome reaction on the egg surface of the polychaete, Tylorrhynchus heterochaetus, 101 Sperm transfer system in Kinbergonuphis simoni (Polychasta: Onuphi- dae). The. 85 Spermatheca. 94 Spermatophores. 85 Spirocysts. 74 Spisula,2lO Strontium, 231 Structure and function of a special tissue in the female genital ducts of the Chinese freshwater crab Knuclicir .viicnxix, 94 Suckers, octopus, 1 26 SUHR-JESSEN. PETER, see Per Ploug Jakobsen, 55 Surf clam oocytes, 210 SUTTON, D. C., AND O. HoEGH-Gui DBERG, Host-zooxanthella inter- actions in four temperate marine invertebrate symbioses: assess- ment of effect of host extracts on symbionts, 175 Symbiosis, 175 Vacuolar release, 300 Valve gaping, 25 1 Valve-like tissue, 94 Vasorelaxation, 279 Visualization of the transparent, gelatinous house of the pelagic tuni- cate Oikuplciiru vanhocffcni using Sepia ink, 1 18 Vitelline envelopes, 210 Volume regulation, 260 w WEISSMAN, IRVING L., see Heather C. Boyd, 239 WHITTAKER, J. R., Determination of alkaline phosphatase expression in endodermal cell lineages of an ascidian embryo, 222 WIEBE, ERIC M., see Samuel C. Edwards, 267 WIGHT, KEITH, LISBETH FRANCIS, AND DANA ELDRIDGE, Food aver- sion learning by the hermit crab Pagurus granosimanus, 205 Tachypleux, 55 Taxonomy. 239 Temperature. 195 THORINGTON, GLYNE LI., AND DAVID A. HESSINGER, Control of cnida discharge: III. Spirocysts are regulated by three classes of chemoreceptors, 74 Tunicate, 1 18 Tunicate taxonomy. 239 Tvlorrliviichiix heterochaetus, I o 1 \-irradiation. 21 YAMAZAKI. FUMIO, see Tai-Hung Lee, 94 VUDIN, ASHLEY L. see Wallis H. Clark Jr., 295 u infrastructure. 160 Llndulipodia, 300 Zoanthid, 175 Zoozanthcllae. 175 36/45 OR I CONTENTS BEHAVIOR Feinman, Richard D., Rafael H. Llinas, Charles I. Abramson, and Robin R. Forman Electromyographic record of classical conditioning of eye withdrawal in the crab 187 Forward, Richard B.,Jr. Behavioral responses of crustacean larvae to rates of temperature change 195 Wight, Keith, Lisbeth Francis, and Dana Eldridge Food aversion learning by the hermit crab Pagurus granosimanus 205 DEVELOPMENT AND REPRODUCTION Dessev, George, and Robert Goldman Effect of calcium on the stability of the vitelline enve- lope of surf clam oocytes 210 Freeman, John A. Regulation of tissue growth in crustacean larvae by feeding regime 217 Whittaker,J. R. Determination of alkaline phosphatase expression in endodermal cell lineages of an ascidian embryo .... 222 PHYSIOLOGY Byrne, Roger A., Erich Gnaiger, Robert F. McMa- hon, and Thomas H. Dietz Behavioral and metabolic responses to emersion and subsequent reimmersion in the freshwater bivalve, Corbicula ///ii/uinu 251 Deaton, Lewis E. Potentiation of hypoosmotic cellular volume regula- tion in the quahog, Mercenaria IIIOICIKIIIII, by 5-hy- droxytryptamine, FMRFamide, and phorbol esters 260 Edwards, Samuel C., Anne W. Andrews, George H. Renninger, Eric M. Wiebe, and Barbara-Anne Bat- telle Efferent innervation to Liniuliis eyes in viva phos- phorylates a 1 22 kD protein 267 Price, D. A., K. E. Doble, T. D. Lee, S. M. Galli, B. M. Dunn, B. Parten, and D. H. Evans The sequencing, synthesis, and biological actions of an ANP-like peptide isolated from the brain of the killifish Finirliilin In-tfrix-litiia 279 Sanders, N. K., and J. J. Childress Adaptations to the deep-sea oxygen minimum layer: oxygen binding by the hemocyanin of the bathype- lagic mysid, Gnathophausia ingi'»\ Dohrn 286 GENERAL BIOLOGY Bidwell, Joseph P., Alan Kuzirian, Glenn Jones, Lloyd Nadeau, and Lisa Garland The effect of strontium on embryonic calcification ofApl\siu californiai Boyd, Heather C., Irving L. Weissman, and Yasu- nori Saito Morphologic and genetic verification that Monterey Botr\llus and Woods Hole Bolr\llu\ are the same spe- cies . 231 239 SHORT REPORTS Clark, Wallis H., Jr., Ashley I. Yudin, John W. Lynn, Fred J. Griffin, and Muralidharan C. Pillai [elly layer formation in penaeoidean shrimp eggs 295 Margulis, Lynn, Michael Enzien, and Heather I. McKhann Revival of Dobell's "Chromidia" hypothesis: chro- matin bodies in the amoebomastigote Pnniti'tniinitin jugosits 300 Index to Volume 178 . 305 MBL WHOI I.1BKAKY UH 1B2H .