3 x j ■•r 1 ~ L. 6 o I In il ZOOLOGICA SCIENTIFIC CONTRIBUTIONS OF THE NEW YORK ZOOLOGICAL SOCIETY VOLUME 54 • ISSUE 3 • FALL, 1969 PUBLISHED BY THE SOCIETY The ZOOLOGICAL PARK, New York Contents PAGE 6. Intact Killifish ( Fundalus heteroclitus ) as a Tool for Medically Oriented Study of Marine Neurotoxins. By John J. A. McLaughlin and Russell J. Down. Plates I-II; Text-figure 1 85 7. Studies on the Biology of Barnacles: Parasites of Balanus ebumeus and B. balanoides from New York Harbor and a Review of the Parasites and Diseases of Other Cirripedia. By Lucie Arvy and Ross F. Nigrelli. Plate I 95 Manuscripts must conform with Style Manual for Biological Journals (American Institute of Biological Sciences). All material must be typewritten, double-spaced. Erasable bond paper or mimeograph bond paper should not be used. Please submit an original and one copy of the manuscript. Zoologica is published quarterly by the New York Zoological Society at the New York Zoological Park. Bronx Park. Bronx. N. Y. 10460. and manuscripts, subscriptions, orders for back issues and changes of address should be sent to that address. Subscription rates: $6.00 per year; single numbers. $1.50. unless otherwise stated in the Society's catalog of publications. Second-class postage paid at Bronx. N. Y. Published January 9, 1970 © 1969 New York Zoological Society. All rights reserved. 6 Intact Killifish (Fundulus heteoroclitus) as a Tool for Medically Oriented Study of Marine Neurotoxins1 John J. A. McLaughlin2’3 and Russell J. Down3 (Plates I-II; Text-figure 1 ) Using Fundulus heteroclitus (killifish), an abundant teleost fish in which aggregation- dispersion of melanophore pigment is rapid and predominantly nerve regulated, it was observed that systemic administration of tetrodotoxin or saxitoxin (purified shellfish poison or derived from Gonyaulax ccitenella) caused sectoral darkening, interpreted as following the previously reported pattern of peripheral pigment-motor unervation, indicating peripheral, central nervous system effects. The test can be used as a sensitive and specific assay for these toxins. Concurrent qualitative and qualitative analysis as well as screening for neuroactive toxins is possible using intraperitoneal or intragas-bladder injection of fish weighing 6-7 gm.(adv.). A consideration of our results and those of others indicates that Fundulus herteroclitus lacks the long postulated melanin dispersing nerve. Introduction This study arose from work by Burke et al. (1960) and Ruggieri et al. (1962) with axenic cultures of Gonyaulax species and other dinoflagellate toxins, and from our discovery of a specific pigmentary response in Fundulus upon systemic administration of these toxins or tetrodotoxin. Using this response, we developed a sensitive and specific assay system for tetrodotoxin and saxitoxin. To do this, it was first necessary to further define Fundulus ( 1 ) by observing the effect of physical factors, and (2) through administration of drugs having known mechanisms of action. These were corre- lated with previously known facts regarding Fundulus and with subsequently published find- ings regarding the mechanism of action of saxi- toxin and tetrodotoxin. Relevant previously known facts are as fol- lows: 1. Fundulus heteroclitus belong to that group of teleosts whose color changes are primarily under nervous control (Fingerman, 1963) as exerted through their melanin-aggregating 1 Aided by Grant NB1198 from the U.S. Public Health Service. 2 Department of Biological Sciences, Fordham Uni- versity, New York, New York 10458. 3 Haskins Laboratories, 305 East 45 Street, New York, New York 10017. (blanching or paling) sympathetic nerves (Wy- man, 1924a) (Text-fig. 1). 2. There is no direct evidence that any opposing melanin-dispersing pigmentomotor nerves exist in Fundulus (Pye, 1964a). 3. A pituitary melanin-dispersing hormone (M.D.H.) must play a part in opposing sym- pathetic nerve effects, for although melano- phores of intact Fundulus are nonresponsive to M.D.H. (Pickford & Kosto, 1957), denervated tail melanophones cannot expand to a black background in the absence of M.D.H. (Abramo- witz, 1940; Pickford & Kosto, 1957). 4. Fundulus pituitary extracts contain, and their melanophores respond to, a melanin aggre- gating hormone (M.A.H.) (Pickford & Atz, 1957). Pineal substance aggregates melano- phores of embryonic and larval Fundulus but not adult Fundulus (Wyman, 1924b) (Lerner’s melanin aggregating hormone, melatonin, has since been isolated from the pineal gland [Ler- ner et al., 1958] ) . 5. Fundulus melanophores are neural crest derivatives and react directly to many neuro- tropic agents, as other investigators including Scheline (1963), Wyman (1924a), and Abbott (1968) have shown by work on isolated scales; however, many of these reactions are probably mediated by release of transmitter substance FEB 5 1970 86 Zoologica: New York Zoological Society [54: 3 from presynaptic membrane, as Fujii & Novales (1968) found for tetrodotoxin. 6. It has been stated by von Frisch (1911) and quoted by Brown (1957), Pye (1964a), Wyman (1924a), and others that the melanin- aggregating nerves of Phoxinus phoxinus L, a Fundulus-Wke teleost, emerge from the spinal canal at the 15th vertebra, bifurcating anteriad and posteriad (Text-fig. 1). Tetrodotoxin (Narahashi et al., 1964) and saxitoxin (Kao & Nishiyama, 1965) were subse- quently reported to interrupt neuronal function by blocking the rapid passive flux of sodium into nerve cells without affecting the sodium pump or the inward or outward shifts of potassium (Kao, 1966). No other agents are known to work precisely this way. In contrast, commonly employed pharmacological agents, such as local anesthetics and barbiturates, block both sodium and potassium ion passive flux (Frank & Sanders, 1963; Frank & Pinski, 1966; Nara- hashi et ah, 1964; Narahashi et ah, 1967). For reasons discussed in depth elsewhere (Down & McLaughlin, 1969), we believe the unique re- sponse of Fundulus to saxitoxin and tetrodotoxin reflects this difference, as well as the peculiarities of Fundulus melanophore innervation. Materials and Methods Toxin sources. Dinoflagellate toxins were ob- tained by growing Gonyaulax catenella and G. tamarensis in axenic culture. Cells were har- vested, extracted, and the poison purified using techniques previously developed by Burke et ah (1960). Samples of purified shellfish poison were obtained from Edward J. Schantz, U.S.A. Chemical Corps Biological Laboratories, Ft. Detrick, Maryland. This shellfish toxin had been prepared as previously reported (Shantz, 1960). Although the dinoflagellate toxin has not been shown to be chemically identical to the shellfish toxin, for brevity these toxins will be called “saxitoxin” (Schuett & Rapoport, 1962), as no pharmacological distinctions have been observed previously or in this work. Crystalline tetrodo- toxin prepared by Sankyo Co. was obtained from Dr. C. Y. Kao of the Downstate Medical Center, Brooklyn, New York, on May 12, 1964, and used within that month. Potency standardization of toxins. One ml volumes of diluted saxitoxin were injected intra- peritoneally (I.P.) into 20.7 — 27.1 gram white mice, and the concentration for an average 15-minute death time (1 Mouse unit, or M.U.) determined. Solutions were periodically re- standardized with mice to eliminate error from loss of potency occurring over the seven month period of use. Because fish used were smaller than mice, volumes of 0.1 ml were almost always used for fish injection. Tetrodotoxin was stand- ardized using the same technique (0.2 ug was found equal to 1 M.U.). Saxitoxin; sodiunt concentration. Two sam- ples of saxitoxin, one acid extracted, the other further purified and concentrated by passage through an ion-exchange column, were assayed for sodium and potassium content by the kidney- and-electrolyte laboratory of the Seton Hall Col- lege of Medicine and Dentistry. Except for 43 meq/L sodium in the acid extracted sample, these were present in negligible concentrations. Preparation of solutions. Solutions were in water (a few in saline) and prepared with HC1 or NaOH only if necessary to effect solution, then adjusted to pH 7. Fresh solutions were pre- pared daily, usually within two hours of use. No aseptic precautions were taken in preparing or injecting solutions. Test animal. Killifish were trapped from Oc- tober 1963 through May 1964 in estuaries and tidal pools of Cape May County, New Jersey, where water temperatures as low as — 1.5C0 were recorded. Occasionally, when this source of sup- ply was unavailable, fish were obtained from the Osborn Laboratories of Marine Sciences, New York Aquarium, Brooklyn, New York. Trans- ported in polyethylene bags, fish showed hy- poxia-induced darkening only when shipments were delayed, and such fish were used only for experiments on hypoxia damage. Fundulus were maintained in aerated sea water aquaria, kept on a neutral-shaded background in an air condi- tioned room (24-28°C) . Unless otherwise stated, light-adapted fish over a light background were used. All experiments were performed at normal room illumination. Fish were generally used within 48 hours of collection. They were placed on a light or dark background for one-half hour before injection, and after treatment were placed in culture dishes over light (white porcelain pans) or dark (wet carbon paper) backgrounds. Injection of test animals (13 mm long, 27 gauge needles). With practice, capture and re- straint of fish in a bare, moistened hand took no longer than 10 seconds. For preliminary or quantitative work, the injection method of choice proved to be intra-gas-bladder (i.g.b.). Subcu- taneous and intramuscular injections gave vari- able results, probably reflecting chance injection near nerves or blood vessels. Intraperitoneal (i.p.) injections were easier but lacked the ad- vantage of i.g.b. injections of entry confirmation by gas aspiration. Aspiration attempted before i.p. injection ruled out gas bladder or blood ves- sel entrance. To avoid damaging major vessels 1969] McLaughlin & Down: Intact Killifish (Fundulus heteroclitus) 87 during i.g.b. injection, the needle was not per- mitted to contact the vertebral bodies. Occa- sionally, i.g.b. injection caused vascular damage visible through the body wall, and fish so effected were discarded. A minimum of three fish were injected for each concentration of each solution tested. Timing of melanophore responses, melano- phore recoveries, and death times. Times of ear- liest visible melanophore changes were recorded. Death times represent the irreversible cessation of visible opercular movements. Weighing Fundulus. All fish were drained of excess water and weighed immediately. Photography. Kodak Panatomic 35-mm film was exposed by an electronic flash unit. Results Only the responses of dispersion and aggrega- tion of melanin are dealt with. Experiments were too brief for observable morphological effects, i.e. pigment mass change. Temperature sensitivity of fish. Local temper- ature change-induced melanophore effects have been described in Phoxinus (Pye, 1964c). We found that transfer of Fundulus from seawater at — 1.5°C to room temperature or vice versa had no immediate or delayed visible effect except transient equilibrium loss, without visible mel- anophore reaction. Hypoxia. Fifty fish held in a water-filled poly- ethylene bag until hypoxic became dark, and most died. Most survivors regained ability to respond to a white background, but three re- mained dark. To test melanophore integrity in permanently dark, damaged animals, L-artere- nol (norepinephrine) 0.01 mg/g was injected i.p. two days after the hypoxic episode. Within two minutes total blanching occurred, as with normal dark-adapted animals, indicating me- lanophore integrity of hypoxia-damaged fish. Size and species differences. We determined the response of various size-ranged animals. Fundulus were graded into three size-groups: small (1.3 to 2.2 g.); medium (5.5 to 6.5 g. ) ; and large (9.1 to 18.8 g. ). Mice ranged from 24 to 27 g. Saxitoxin was administered i.p. to mice and i.g.b. to fish. Separate tests showed no dif- ference between time or intensity of effects upon i.g.b. versus i.p. injection. Results are shown in chart (Table I). Dosages given to small and medium sized fish and to mice gave death times Table I time/dose relationships, saxitoxin sample containing 1.0 MOUSE UNIT/ 0.6 ml ( INTRA AIR- SAC INJECTION ) 88 Zoologica: New York Zoological Society [54: 3 which fell along a single curve. Large fish gave consistent results only with more concentrated solutions, probably because their smaller area of internal absorbing surface per gram of animal and per ml of injected solution vis-a-vis smaller fish resulted in slower toxin absorption. Thus, when animal size is approximately the same, toxin sensitivity of mice is greater than that of Fundulus. These results dictated our use of Fun- dulus weighing 7-g or less, to allow use of saxi- toxin in the same dilution as for standardization by mouse test. Results were consistent and no correction factor for size was needed. Peculiarties of Fundulus vasculature. We as- sumed tourniquets might serve to sort out nerve- vs. blood-mediated melanophore responses. Rubber band tourniquets were tried and found ineffective. Peculiarities of Fundulus melanophore inner- vation. A. Cholinergic (dispersing) pigmentomotor nerves. Melanin aggregating nerves of Fundulus are known to be adrenergic (Pye, 1964a; Wyman, 1924a). Melanin dispersing nerves when postu- lated have been assigned cholinergic activity (Pye, 1964c; Fujii and Novales, 1968). We at- tempted to unmask melanin dispersing nerve action by blocking the sympathetic nervous sys- tem with a sympatholytic agent before injecting an acetylcholinesterase inhibitor to increase the concentration of acetylcholine. Priscoline (tolazoline) , an alpha adrenergic blocking agent, upon i.p. injection of 0.02 mg caused a generalized light to dark gray reaction in light adapted animals (4 g) in ~ 10 minutes, with return to normal (light) color at ~ one hour. Eserine (physostigmine) , an acetylcholi- nesterase inhibitor injected in a 2 mg dose in other dark adapted animals caused paling in ~ 10 minutes. This was soon followed by respira- tory arrest (cessation of movements of gill oper- culae) usually preceded by tail spasms and/or convulsions. Individual drug effects thus established, we gave light adapted Fundulus ranging from 1.8-6. 8 g priscoline 0.02 mg i.p. and again the partial uniform darkening occurred in ~ 10 minutes. Twenty minutes after i.p. priscoline in- jection, eserine 2.0 i.g.b. was given each animal. Within 10 minutes respiratory arrest occurred without deepening of the priscoline-induced par- tial darkening. In a similar experiment light- adapted animals on a light background given doses of priscoline lOx larger (0.2 mg) survived, but melanin dispersal lasted up to five hours. When fish had again become partially light, each was given eserine 2.0 mg and the results were the same, i.e., they progressed to respiratory arrest in ~ 10 minutes without intervening change in degree of melanin dispersion. B. Adrenergic ( aggregating ) pigmentomotor nerves. It has been reported by von Frisch (1911) and widely quoted (Brown, 1957; Pye, 1964a; Wy- man, 1924a) that the melanin-aggregating nerves of Phoxinus phoxinus L., a Fundulus- like teleost, emerge from the spinal canal at the fif- teenth vertebra, bilaterally, and bifurcate ante- riad and posteriad (Text-fig. 1). We found the certain minimum lethal dose (M.L.D.) of saxitoxin, given i.p. or i.g.b. to Fundulus weighing not over 6 g, to be 0.15 M.U./g (~ 0.03 ug/g.). Half this amount (or more) evokes darkening by quadrants. Usually within one minute, 1-4 sectors (usually right or left anterior) begin to darken (PI. I, fig. 1). The darkening increases and appears in other sectors (PI. I, fig. 2), proceeding at an independent rate within each sector until all but the midsection is fully dark ( PI. II, fig. I ) . With lethal doses, total body darkening and death ensue (PI. II, fig. 2). Tetrodotoxin (M.L.D. = 0.075 MU/g = ~ 0.015 ug/g) also causes this sectoral darkening. We believe this illustrates a melanin-aggregating nerve distribution in Fundulus similar to that demonstrated in Phoxinus. However, it would be surprising to find that these sympathetic pigmentomotor nerves always emerge from the spinal cord and pass into the sympathetic chain at precisely the fifteenth verte- bra. We observed that dark sectors in hundreds of fish, elicited by i.p. or i.g.b. injection of saxi- toxin, usually resulted in the pale midzone being centered at or near the fifteenth vertebral level bilaterally. Rarely, the sharply demarcated pos- terior boundary of one anterior sector and the contralateral sector in a given fish varied in location by as much as four myotomes. Also in some fish, the pale midzone centered several myotomes anteriad, and in others several myo- tomes posteriad to the fifteenth vertebra. This indicates variability in the level of emergence of the pigment motor nerve fibers. Discussion Temperature sensitivity. Even though sudden temperature change had no visible effect upon the shade of intact Fudulus, we kept fish at room temperature for 12 hours or longer before using them. This precaution proved worthwhile, for it has since been found (Guttman & Barnhill, 1968) that the excitability of tetrodotoxin- treated axons is more temperature-dependent than that of normal axons. Hypoxia. That total-animal hypoxia resulted 1969] McLaughlin & Down: Intact Killifish (Fundulus heteroclitus) 89 Text-fig. 1. Diagram of the course of the pigment-motor nerve fibers in the minnow. Pigment-motor centers and fibers in heavy black. Nervous system stippled. (Modified after von Frisch, ’ll; pi. 4, fig. 6.) C, pigment-motor center in medulla; P, pigment-motor center in spinal cord; T, trigeminal nerve; N, spinal nerve; M, melanophore; V, vertebral column; S, sympathetic system with pigment-motor fibers; B, spinal nerve to tail. in complete generalized melanin dispersal con- trasts with statements by Wyman (1924a), who cut vessels and nerves, and Spaeth, who used iso- lated Fundulus scales, as related by Wyman (1924a), indicating the effect of anoxia upon Fundulus melanophores is aggregation. Al- though the “anemia contraction” of melanin as observed by Wyman (1924a) could be due to loss of endocrine control, alone or reinforced by loss of oxygen supply, the same reasoning can- not be applied to results with isolated scales as these remain dark in Ringer’s solution until oxygen is withdrawn. However, this direct pal- ing effect of anoxia upon melanophores can be explained if melanin dispersal is associated with maintenance of melanophore polarization, and if oxygen is required for this energy-consuming task. Then, if melanophores of hypoxic, intact Fundulus still obtain enough oxygen from sur- rounding water even after depression of the more hypoxia-sensitive nervous system, release from pigment-motor nerve control would allow them to maintain polarity and the accompany- ing melanin dispersion. Animals remaining per- manently dark following an hypoxic episode probably incurred nervous system damage and thus, permanent melanophore release from pig- ment-motor nerve control— a potentially valu- able preparation for other toxin or melanophore research. Size and species differences. The ratio of in- ternal absorbing surface to animal body mass is important when saxitoxin, tetrodotoxin, and other rapidly absorbable compounds are in- jected i.p. or i.g.b. Small animals have greater area of visceral plus parietal peritoneal surface per gram than large animals. We observed that saxitoxin m.l.d. per gram for mice (20-plus g) and Fundulus in its most abundant sizes (1.5 to 6 g) is nearly the same: the interspecies differ- ence in sensitivity cancels out the difference in sensitivity due to size when animals of these sizes are employed. This is fortuitous, and may or may not hold true for any other given toxin. Peculiarities of Fundulus vasculature. It is probable that our results using tourniquets were negative due to collateral circulation via vessels within vertebral canals. These results supplement fruitless attempts of others (Wyman, 1924a) to sort out endocrine vs. nerve mediated melano- phore effects by ligating or otherwise modifying Fundulus circulation. Peculiarities of Fundulus melanophore inner- vation. A. Cholinergic ( dispersing ) pigmentomotor nerves. While direct evidence for melanin-dispersing nerves in Fundlus is wanting, many have argued for their existence on the basis of indirect evi- dence (Fingerman, 1963; von Geili, 1942; Mills, 1932a, b; Parker, 1948; Waring, 1963). Like Scott (1965), we consider the interpretation of the results upon which this indirect evidence is based to be open to question. Electron micro- scopy has shown but one type of end-plate on Fundulus melanophores (Bikle, 1966). Others (Pye, 1964b; Healey, 1954; Healey & Ross, 1966; Abbott, 1968) found no evidence of mela- nin dispersing nerves in Phoxinus or Fundulus. Wyman (1924a) concluded that there is no in- dication of a double innervation to Fundulus melanophores. Our results support this view. Pye (1964c) showed that “regetine appears to suppress all activity in chromatic (paling) nerve tracts while leaving the melanophores free to respond to the humoral influence of large doses of pituitary extracts.” He also showed that rogentine does not influence in any way the nor- mal melanin aggregating action of adrenalin di- rectly upon melanophores. Thus, in our attempts to unmask melanin dispersing nerve activity, we propose that the alpha adrenergic blocking agent, priscoline, similar to regetine, causes 90 Zoologica: New York Zoological Society [54: 3 melanin dispersal by blocking the melanin ag- gregating nerves with little if any direct effect on melanophores. If then, fish are made partially dark by priscoline-induced alpha adrenergic nerve blockade, eserine-induced buildup of acetylcholine should cause further melanin dis- persal by stimulation only of unblocked cholin- ergic melanin dispersing nerves, if any exist. But no such effect, nor any other indication of mela- nin dispersing nerve action, was seen in our priscoline-eserine experiments. We have not yet performed trials substituting beta adrenergic blocking or anti-adrenergic compounds for pris- coline. B. Cholinergic (aggregating) pigmentomotor nerves. Anatomic features of the pigment-motor ner- vous system were mentioned under Results (see Text-fig. 1). We believe that darkening by sector, whether induced by saxitoxin or tetrodo- toxin, etc., is explainable only on the basis of the anatomic features of the peripheral melanin ag- gregating nervous system of Fundulus. No other known system within the animal (retinal ele- ments, afferent sensory nerves, pigment-motor centers in medulla or spinal cord, etc.) offers an adequate explanation. The elicitation by known neurotoxins supports this interpretation. The midsection nearest the point of nerve emergence from the spinal cord is last to darken, indicating differential nerve sensitivity reminiscent of the ascending paralysis which saxitoxin and tetrodo- toxin induce in mammals, including man. Re- sults indicating the probable ionic mechanism for peripheral pigmentomotor system > central nervous system sensitivity to saxitoxin and tetro- dotoxin and further confirming specificity of sectoral darkening is given in detail elsewhere (Down & McLaughlin, 1969). Summary Using Fundulus heteroclitus, a hardy and abundant teleost bait fish in which aggregation- dispersion of melanophore pigment is rapid and predominantly nerve regulated, we found that systemic administration of tetrodotoxin, or saxitoxin cause sectoral darkening, interpreted as following the previously undocumented (in Fundulus) pattern of peripheral pigment-motor innervation, indicating peripheral > central nervous system effect, and constituting a sensi- tive and specific assay system for the toxins. Concurrent quantitative and qualitative analysis of as well as screening for neuroactive toxins is possible using i.p. or i.g.b. Fundulus injection. A combination of our findings and old and new published information leads us strongly to doubt the existence of long-postulated melanin dispersing nerves in Fundulus heteroclitus. Literature Cited Abbott, F. S. 1968. The effects of certain drugs and biogenic substances on the melanophores of Fun- dulus heteroclitus L. Canad. J. Zook, 46 1149. Abramowitz, A. A. 1940. A new method for the biological assay of intermedin. J. pharmacol. exp. ther. 69, 156. Bikle, D. 1966. Microtubules and pigment migration in the melanophores of Fundulus heteroclitus L. Protoplasma, 61:322-345. Brown, M. E. 1957. The Physiology of Fishes. Academic Press, N.Y., 277 pp. Burke, J. M„ J. Marchisotto, J. J. A. McLaughlin, and L. Provasoli 1960. Analysis of the toxin produced by Gony- aulax catenella in axenic culture. Ann. N.Y. Acad. Sci., 90(3 ): 837-842. Down, R. J., and J. J. A. McLaughlin 1969. Submitted to Toxicon. Fingerman, M. 1963. The control of chromatophores. New York. Pergamon Press, 178 pp. Frank, G. B., and Pinsky, C. 1966. Tetrodotoxin — induced central nervous system depression. Brit. J. Pharmacol.: 26/2, 435-443. Frank, G. B., and H. D. Sanders 1963. A proposed common mechanism of action for general and local anesthetics in the central nervous system. Brit. J. Pharma- col.: 21, 1-9. Frisch, K. von 1911. Beitrage zur physiologie der pigment- zellen in der fischhaut. Arch. ges. physiol., Bd. 138, s. 319-387. Fuji, R., and R. R. Novales 1968. Tetrodotoxin effects on fish and frog melanophores. Science: 160:1123-1124. Gelei, G. von 1942. Zur frage der doppelinnervation der chro- matophoren. Z. vergl. physiol., 29:532- 540. Guttman, R., and R. Barnhill 1968. Effect of low sodium, tetrodotoxin, and temperature variation upon excitation. J. Gen. Physiol., 51:621-634. Healey, E. G. 1954. The colour change of the minnow (Phoxi- nus laevis Ag.). J. Exp. Biol., 31:473-491. 1969] McLaughlin & Down: Intact Killifish (Fundulus heteroclitus) 91 Healey, E. G., and D. M. Ross 1966. Effects of drugs on background colour response of the minnow. Comp. Biochem. Physiol., 19:545. Kao, C. Y. 1966. Tetrodotoxin, saxitoxin and their signifi- cance in the study of excitation phenom- ena. Pharmacol. Rev., 18/2:997-1049. Kao, C. Y., and A. Nishiyama 1965. Actions of saxitoxin on peripheral neuro- muscular systems. J. Physiol., 180:50-66. Lerner, A. B., J. D. Case, Y. Takahashi, T. H. Lee, and W. Mori 1958. Isolation of melatonin, the pineal gland factor that lightens melanocytes. J. Am. Chem. Soc., 80, 2582. Mills, S. M. 1932a. Double innervation of melanophores. Proc. Nat. Acad. Sci., 18:538-540. 1932b. Neuro-humoral control of fish melano- phores. Proc. Nat. Acad. Sci., 18:540-543. Narahashi, T., N. C. Anderson, and J. W. Moore 1967. Comparison of tetrodotoxin and procaine in internally perfused squid giant axons. J. Gen. Physiol., 50/5:1413-1428. Narahashi, T., H. W. Moore, and W. R. Scott 1964. Tetrodotoxin blockage of sodium conduct- ance increase in lobster giant axons. Jour. Gen. Physiol., 47:965-974. Parker, G. H. 1948. Animal color changes and their neuro- humors. Cambridge Univ. Press, 377 pp. PlCKFORD, G. E., AND J. W. ATZ 1957. The physiology of the pituitary gland of fishes. N. Y. Zool. Soc., 613 pp. PlCKFORD, G. E., AND B. KOSTO 1957. Hormonal induction of melanogenesis in hypophysectomized killifish ( Fundulus heteroclitus) . Endocrinology, 61:177. Pye, J. D. 1964a. Nervous control of chromatophores in teleost fishes. I. Electrical stimulation in the minnow ( Phoxinus phoxinus (L.)). J. Exp. Biol., 41:525-534. 1964b. Nervous control of chromatophores in teleost fishes. II. The influence of certain drugs in the minnow {Phoxinus phoxinus (L) ). J. Exp. Biol., 41:535-541. 1964c. Nervous control of chromatophores in teleost fishes. III. Local temperature re- sponses in the minnow (Phoxinus phoxi- nus ( L) ) . J. Exp. Biol. 41:543-551. Ruggieri, G. D., R. F. Nigrelli, and J. J. A. McLaughlin 1962. The effects of dinoflagellate toxins on development of Arbacia punctulata. Amer. Zool. 2 (4) : 366. SCHANTZ, E. 1960. Biochemical studies of paralytic shellfish poisons. Ann. N.Y. Acad. Sci., 90(3) :843- 855. SCHELINE, R. R. 1963. Adrenergic mechanisms in fish; chroma- tophore pigment concentration in the cucoo wrasse, Lahrus ossifagus L. Comp. Biochem. Physiol., 9:215-227. SCHUETT, W., AND H. RAPOPORT 1962. Saxitoxin, the paralytic shellfish poison. Degradation to a pyrolopyrimidine. J. Amer. Chem. Soc., 84:2266. Scott, G. T. 1965. Physiology and pharmacology of color change in the sand flounder Scoptnalmus aquarsus. Limnol. Oceanog. 10 Supp. R. 230. Waring, H. 1963. Color Change Mechanisms of Cold Blooded Vertebrates. New York, Aca- demic Press, 266 pp. Wyman, L. C. 1924a. Blood and nerve as controlling agents in the movements of melanophores. Jour. Exp. Zool., 39:73-132. Wyman, L. C. 1924b. Administration to larval, embryonic and adult Fundulus heteroclitus of dessicated pineal gland. J. Exp. Zool., 40:161. 92 Zoologica: New York Zoological Society [54: 3 EXPLANATION OF THE PLATES Effects of saxitoxin (M.L.D.) Plate I Fig. 1. Darkening in right anterior sector begin- ning. Fig. 2. Both anterior sectors totally or near to- tally dark. Plate II Fig. 1. All four sectors dark, midsection still light, fish listing. Fig. 2. Postmortem, showing body-wide darken- ing and muscular atony (held by one gill cover, head down). MCLAUGHLIN & DOWN PLATE I FIG 1 FIG. 2 INTACT KILLIFISH (FUNDULUS H ETEROCLITUS ) AS A TOOL FOR MEDICALLY ORIENTED STUDY OF MARINE NEUROTOXINS MCLAUGHLIN & DOWN PLATE II FIG 1 FIG. 2 INTACT KILLIFISH (FUNDULUS H ETEROCLITUS ) AS A TOOL FOR MEDICALLY ORIENTED STUDY OF MARINE NEUROTOXINS 7 Studies on the Biology of Barnacles: Parasites of Balanus eburneus and B. balanoides from New York Harbor and a Review of the Parasites and Diseases of Other Cirripedia1 Lucie Arvy2 and Ross F. Nigrelli3 (Plate I) Three species of organisms previously reported as predators or as parasites of barnacles and two commensal peritrichs have been found in populations of Balanus eburneus and Balanus balanoides occurring on the rock jetties from Sea Gate and immediately adjacent to the Osborn Laboratories of Marine Sciences at Seaside Park, Coney Island, New York City, respectively. The species found were Cephaloidophora communis (Protozoa: Sporozoa: Gregarinida: Eugregarinina: Cephalinoidea: Cephaloidophoridae) from the intestine of Balanus eburneus; Epistylis horizontalis and Epistylis nigrellii (Protozoa: Ciliophora: Ciliata: Peritrichidae) from the branchial lamellae of Balanus balanoides and B. eburneus, respectively; Stylochus ellipticus (Platyhelminthes: Turbellaria: Polycladia: Acotylea: Stylochidae) from the internal wall of the opercular valves of Balanus eburneus attached to Mytilus edulis; and metacercariae, possibly of Maritrema arenaria (Trematoda: Digenea: Microphallidae) , on the external gut wall and other tissues of Balanus balanoides. The presence of these organisms in local barnacles represents a new geographical record. These and their effects on the host are briefly described together with a review of the literature on other parasites and diseases of barnacles. Introduction There is very little information in the lit- erature on the diseases and parasites of Cirripedia, and apparently only the mem- bers of the Balanidae have been investigated with any degree of thoroughness. The most com- mon group of parasites found in these sessile crustaceans are the Gregarinida (Protozoa: Sporozoa), fungal parasites, larval digenetic trematodes (metacercariae), and a single species (Hemioniscida balani) of parasitic isopod have also been reported. Some of these parasites may be more common than indicated in the litera- ture. Studies in our laboratory have shown that one species of gregarine ( Cephaloidophora com- munis) and the metacercaria of a microphallid trematode, together with a predatory turbellarian ( Stylochus ellipticut ) and a commensal peritrich ciliate ( Epistylis horizontalis), occur in bar- 1 Supported by grant from The Rockefeller Founda- tion, RF 64078. 2 Visiting Research Associate, Osborn Laboratories of Marine Sciences; Lab. Histoenzymology, Faculty of Medicine, 45 rue des Saints-Peres, Paris VIeme, France. 3 Osborn Laboratories of Marine Sciences, New York Aquarium, Brooklyn, New York 11224. nacles (Balanus eburneus and B. balanoides) in the New York area. These are reported below together with a review of the literature. Gregarinida Gregarines have been repeatedly observed in barnacles since they were first described by Kolliker in 1847; the most recent report deals with electron microscope studies on Cephaloido- phora communis in the intestine of Balanus tin- tinnabulum (Reger, 1966). C. communis was first described in Balanus improvisus, B. ebur- neus, and B. amphitrite by Mawrodiadi in 1908, and has since been reported by several authors in other Balanidae. This species, together with Pyxinioides bolitoides in Balanus nubilis from the Pacific coast (see Table I), was recently dis- covered by the present investigators in Balanus eburneus collected on the jetties adjacent to the Osborn Laboratories of Marine Sciences. Since development occurs when the sporozoites pene- trate the cells of the gut epithelium, some patho- logical effects (e.g. necrosis and desquamation) must occur in spite of the seemingly innocuity of the bioassociated sporadins (PI. I, fig. 1). Barnes (1955) suggested that infections with gregarines have some profound effect on the 95 96 Zoologica: New York Zoological Society [54: 3 Table I Protozoa: Gregarinida Host Parasite Author Balanus pusillus Pollicipes polymerus Balanus improvisus Balanus perforatus Balanus perforatus Pollicipes cornucopia Balanus improvisus Balanus eburneus Balanus amphitrite Chthamalus stellatus Chthamalus stellatus Balanus eburneus Balanus amphitrite Balanus eburneus Balanus improvisus Balanus tintinnabulum Balanus amphitrite Balanus crenatus Balanus gland ula Balanus cariosus Balanus nubilis Balanus nubilis Balanus nubilis Balanus balanus pugetensis Balanus rostratus heteropus Balanus balanus pugetensis Mitella polymerus Balanus eburneus Balanus balanoides Balanus amphitrite Chthamalus stellatus Chthamalus stellatus Balanus tintinnabulum Balanus eburneus Balanus nubilis Balanus balanoides Balanus eburneus Gregarina balani26 Gregarina valettei 7 Unnamed gregarine Nematoides fusiformis* * 3 Nematoides fusiformis Nematoides fusiformis Cephaloidophora communis 7 Cephaloidophora communis Unnamed gregarine Frenzelina chthamali 4 Cephaloidophora communis Unnamed gregarine Pyxinioides balani Pyxinioides balani Cephaloidophora communis Cephaloidophora communis Cephaloidophora communis Cephaloidophora communis Cephaloidophora communis Cephaloidophora communis Pyxinoides bolitoides 5 Gregarina spissa Cephaloidophora magna Cephaloidophora multiplex Cephaloidophora multiplex Pyxinoides pugetensis 5 Gregarina valettei Cephaloidophora communis Gregarina balani Cephaloidophora communis Bifilida rara Pyxinioides chthamali Cephaloidophora communis Cephaloidophora communis Pyxinioides bolitoides Epistylis horizontalis Chatton1 Epistylis nigrellii Kolliker, 1847 Nussbaum, 1890 Solger, 1890 Mingazzini, 1891 Labbe, 1899 Labbe, 1899 Mawrodiadi, 1908 Mawrodiadi, 1908 Mawrodiadi, 1908 Leger and Duboscq, 1909 Leger and Duboscq, 1909 Budington, 1910 Tregouboff, 1912 Tregouboff, 1912 Tregouboff, 1912 Tregouboff, 1912 Ball, 1937 Henry, 1938 Henry, 1938 Henry, 1938 Henry, 1938 Henry, 1938 Henry, 1938 Henry, 1938 Henry, 1938 Henry, 1938 Henry, 1938 Ball, 1950 Ouspenskaia, 1960 Heckman, 1961 Tuzet and Ormieres, 1964 Tuzet and Ormieres, 1964 Reger, 1966 Arvy and Nigrelli (Present paper) Arvy and Lacombe (recorded in present paper) Arvy and Batisse, 1968 Arvy and Batisse, 1968 4 Arvy, and Batisse (in press) recently rediscovered the peritrich ciliate Epistylis horizontalis Chatton among the ovarian follicles in Balanus balanoides collected off the rock jetty adjacent to the Osborn Laboratories of Marine Sciences; they also reported a new species, E. nigrellii, from the gills of B. eburneus in the same locality. 2 The barnacle parasites included in the Gregarina must be reclassified since the members of this genus are exclusively found in insects (Tuzet and Ormieres, 1964). 3 Sporocysts of Gregarina and Nematoides in Cirri- pedia are unknown. 4 The name Frenzelina is now used for a testate sar- codina of the family Difflugiidae; F. chthamali, however, is a true gregarine that has been reclassified as Cephaloi- dophora and/or Pyxinioides (see Henry, 1938, for dis- cussion of this species). s Misspelling for Pyxinioides, the generic name cre- ated by Tregouboff (1912). sRamm (1922) believes that Gregarina balani is a synonym of Pyxinioides balani and redescribes Cepha- loidophora communis, Pyxinioides chthamali, Gregarina valettei, and Nematoides fusiformis from various spe- cies of barnacles. t Henry (1938) redescribes Cephaloidophora com- munis and Gregarina valettei in more detail; the former was also redescribed by Tregouboff (1912) and by Tuzet and Ormieres (1964). 1969] Arvy & Nigrelli: Studies on the Biology of Barnacles 97 physiology of the developing barnacle larvae. Apparently, in some unknown way, the infection causes a delay in the liberation of the nauplii; the latter, however, continue to develop within the mantle to a physiological state that shortens the transformation time into the second naupliar stage during its free existence. Trematoda As may be noted from Table II, the meta- cercariae of only three species of digenetic trematodes of the family Microphallidae have been reported in barnacles. Lebour ( 1 908- 1911) was apparently the first to describe metacer- cariae from a species of barnacles on the North- umberland coast of England, which she named Cercaria balani. More recently, Ouspenskaia (1960) reported similar type metacercarial cysts in Balanus balanoides from the Barents Sea. However the description and figures are not de- tailed enough for us to make a comparison with our species, although they were described as the metacercariae of Maritrema linguilla (L. A. Jagerskiold) and Maritrema gratiosum (W. Nicoll). The latter are sexually mature stages that occur in the intestine of gulls, terns and other marine birds. In our routine studies on the biology of the barnacles, similar type metacercariae were also found in Balanus balanoides collected in local waters. The worms are contained in thin-walled, yellowish, more often white, and refringent spherical cysts measuring 0.3 mm in diameter; the body wall of the parasite is covered with delicate spines, except for its posterior third; oral and ventral suckers are approximately of equal size; the cecae are relatively long, each branch measuring on the average 0.5 mm in length, and easily demonstrated when stained vitally with neutral red (PI. I, figs. 2-4). At- tempts at excystment by feeding isolated cysts to killifish, Fundulus heteroclitus, were unsuc- cessful. Further experiments to induce this process were dropped in view of the failures re- ported by Hadley and Castle ( 1940) when they fed cysts from infested Balanus balanoides col- lected from Woods Hole, Massachusetts, and from the coast of Maine to young Larus argen- tatus, Sterna hirundo hirundo, Trigonoides ma- cularius, white mice, white rat, kitten, and domestic fowl. On the basis of circumstantial evidence, i.e., the discovery of sexually mature adults together with metacercarial cysts in the intestine of the turnstone, Arenaria intrepes morinella, a bird that is known to feed on bar- nacles, Hadley and Castle concluded that the metacercariae and the adults were the same; they considered this to be a new species for which the name Maritrema arenaria was given. The strik- ing similarities of our form with those figured by Hadley and Castle lead us to conclude that the cysts from Balanus balanoides taken from local waters are also the metacercariae of Mari- trema arenaria. The pathological effects of metacercarial in- festations in barnacles are not too well known. Our observations show that in light infestations, the cysts are usually localized on or near the gut; when the infestations are exceptionally heavy, all parts of the body, except for the appendages and the lumen of the gut are involved. In such instances, the metacercariae are firmly em- bedded in a relatively thick connective tissue formed around the external gut wall. Barnacles showing such extreme conditions have ovaries that are reduced to filiform cords of follicular tissue and entirely devoid of oocytes. Whether or not this is a consistent pathological feature remains to be established. Table II Trematoda Host and Locality Parasite Author Balanus sp. (Northumberland coast) Cercaria balani (metacercariae) Lebour, 1908/11 B. balanoides (Woods Hole) Metacercariae of Maritrema arenaria Hadley and Castle, 1940 B. balanoides (Barents Sea) Metacercariae of Maritrema gratiosum W. Nicoll Ouspenskaia, 1960 B. balanoides (Barents Sea) Metacercariae of Maritrema linguilla L. A. Jagerskiold Ouspenskaia, 1960 B. balanoides Metacercariae of undetermined Arvy and Nigrelli (Coney Island) Microphallidae; in all probability Maritrema arenaria (present paper) 98 Zoologica: New York Zoological Society [54: 3 ISOPODA Infestations of barnacles by the protandrus hermaphrodite Hemioniscus balanus (Crusta- cea: Isopoda: Ipicaridea: Hemioniscidae) have been known since the latter part of the 19th cen- tury and have since been reported in these hosts from various parts of the world. In its develop- mental cycle, the males (cryptoniscus stage) be- come transformed into females when they take up the parasitic existence. The female is accom- panied by grotesque changes in form, eventually becoming an enlarged star-shaped egg-sac (ab- domen), with evidence of its crustacean charac- teristics indicated by the retention of certain head and thoracic appendages. In the early stages of the transformation processes, the para- sitic female sucks the body fluids of the barnacle, which are stored into two large “liver” lobes; this nutrient material is eventually transferred to the ripening eggs as reserve food material (see Wimpenny, 1966). Each barnacle may harbor one or more para- sitic females; Perez (1923) found as many as seven individuals in a single Balanus balanoides. It has been suggested that such a heavy infesta- tion inhibits the development of the gonads as the result of mechanical pressure, or may actu- ally cause a destruction of the ovaries. How- ever, such ovariectomized barnacles are still capable of carrying on most life functions as indicated by the continued rhythmic movements of the cirri of the parasitized animals. Fungus and Lichens Three species of fungi have been reported as infecting barnacles. Two of them, namely Didymella balani and Pharcidia marina from the tests and shell of Balanus balanoides and Chthamalus stellatus were originally classified as ascomycetes by Hariot (1887) and Bommer (1891), but have since been recognized (San- tesson, 1939) as marine lichens of the genera Arthropyrenia and Didymella (for clarification of the taxonomy, see Johnson and Sparrow, 1961). The third species, Lagenidium chthamalo- philum (Phycomycete) , is a virulent fungal agent that was found to be the cause of an epizootic in 1957 in the barnacle Chthamalus fragilis denticulata from Beaufort, North Caro- lina, with an incidence that ranged from 12.5% to 100% (Johnson, Jr., 1958). This highly Table III Isopoda: Hemioniscus balanus Species Locality Author Balanus sp. German coast, North Sea Buchholz, 1886 Balanus sp. Wimereux, French coast, English Channel Caullery and Mesnil, 1899 Balanus improvisus Gironde estuary, France Perez, 1900 Balanus perforatus Roscoff, Brittany, France Perez, 1923 Balanus balanoides Roscoff, Brittany, France Prenant, 1923 Chthamalus stellatus Roscoff, Brittany, France Prenant, 1923 Balanus balanoides Atlantic coast north to Tromsp (Norway) Crisp, 1951 Balanus amphitrite English estuaries Crisp and Molesworth, 1951 Balanus sp. South Africa Sandison, 1954 Balanus balanoides Southwest coast of England Crisp and Southward, 1954 Balanus porcatus 7 Crisp, 1954 Chthamalus dalli North American Pacific coast Cornwall, 1955 Elminius modestus ? Crisp and Davies, 1955 Balanus balanoides Atlantic French coast Crisp and Fischer-Piette, 1959 Balanus perforatus 7 Crisp and Patel, 1960 Eliminius modestus Roscoff Bourdon, 1963 Balanus balanoides Roscoff Bourdon, 1963 Balanus balanoides Halifax area (New Scotland) Crisp, 1968 Balanus balanus From Labrador to Massachusetts Crisp, 1968 Balanus glandula Friday Harbor Crisp, 1968 Chthamalus dalli Friday Harbor Crisp, 1968 Balanus balanus Irish Sea, Faroe, Shetlands Crisp, 1968 Balanus hameri Irish Sea Crisp, 1968 Remarks: Crisp (1968 ) states that barnacles such as B. improvisus, B. algicola, Chthamalus dentatus, and Elminius modestus, may also be parastilized by Hemioniscus balani. Forms such as Verruca stroemia, Balanus crenatus, B. perforatus, and B. stellatus are never infested; Perez (1900) has made a similar observation, e.g. in the Gironde estuary Balanus improvisus is heavily infested by Hemioniscus balani but Chthamalus stellatus are never parasitized; the reason of this apparent immunity remains unexplained. 1969] Arvy & Nigrelli: Studies on the Biology of Barnacles 99 Table IV Lichens and Fungus Host Locality Parasites Author Lichen Chthamalus stellatus test Didymella balani Hariot, 1887 Balanus halanoides shell Pharcidia marina Bommer, 1891 Fungus Chthamalus fragilis ova Lagenidium Johnson, 1958 var. denticulata chthamalophilum pathogenic fungus infects the ova and appar- ently is specific for Chthamalus fragilis, since Balanus amphitrite in the same waters is resist- ant to infection both under natural and experi- mental conditions. Johnson reported that the fungus develops in the ova of the barnacle at any time between gastrulation and the emergence of the nauplii; neither the released nauplii nor the somatic tis- sues are involved. The degree of infection varies with the stage of development of the egg mass. Thus, embryos with three or more appendage buds are most often infected; earlier stage em- bryos, i.e. with one or more appendage buds, are completely destroyed, leaving only clusters of egg membranes filled with fungus mycelium. Lamellae with more mature embryos apparently are more resistant, since some embryos escape invasion by the fungus and develop into normal nauplii. The infection is initiated by laterally biflagellate planonts that become transformed into spores when they settle on the egg. Within three minutes after attaching to the egg mem- brane, the spore protoplasm penetrates the membrane, increases in size into a hyphal rudi- ment, and grows along the embryo. The infection is visible as pallid grey or grey-green lamellae. The infection spreads rapidly through the entire cluster so that within two days all the embryos are invaded. There can be little doubt that Lagenidium chthamalophilum attacking the ova of the barnacle Chthamalus fragilis may have caused a reduction in population density of this species in Beaufort, North Carolina, during and after the epizootic. As pointed out by Johnson (1958), further studies are needed to establish the importance of this fungus. Studies must be undertaken on distribution and severity of in- fection; conditions favoring the development and spread of the infection; the factors respon- sible for host susceptibility; and, whether or not the epizootics are cyclic. Turbellaria Members of the genus Stylocluis, sometimes called the oyster “leech,” (Platyhelminthes: Turbellaria: Polycladia: Acotylea: Stylochidae) are predators occurring free or “encapsulated” (walled-off by chitinous secretions of the host) in oysters, barnacles, pangurid crabs, and in other invertebrates (see Hyman, 1951; Cheng, 1967). Those in the barnacles are usually found free or encapsulated on the internal wall of the opercular valves, and sometimes closely associated with the ovaries (Skerman, 1960). Stylocluis ellipticus, which according to Loosan- off (1956) may be responsible for the destruc- tion of large numbers of oysters on the flats at Milford, Connecticut, was found locally in a population of Balanus eburneus attached to Mytilus edulis. The worms were found free on the internal wall of the opercular valves or deep within the host on which they were feeding. There was no evidence that the flatworms were feeding on the Mytilus. It has been estimated that a single oyster- inhabiting turbellarian lays about 22,000 eggs in a month, which at 28° C hatch in a few days into pelagic ciliated larvae, become transformed into adults in two months, live a free existence in the littoral zone for about a year, and even- tually encysting in great numbers on all parts of the oyster spat, causing heavy mortality. It remains to be seen whether or not Stylochus Table V Turbellaria Host Predator Author Balanus sp. Stylochus neapolitanus Lang, 1884 Balanus sp. Stylochus zanbibaricus Skerman, 1960 Balanus eburneus Stylochus ellipticus Arvy and Nigrelli (present paper) 100 Zoologica: New York Zoological Society [54: 3 ellipticus is as important a predator for young, newly set barnacles as it is for the spats. The abnormal arrangement of the plates of Balanus eburneus attacked by the Stylochus ellipticus, which we observed, may be indicative of an invasion early in its growth. Discussion Much has been written on the biology of barnacles, especially on their distribution, nu- trition and growth, factors atfecting mortality of natural populations, and particularly the role of temperature on the life cycle. These topics have been reviewed by several authors (Henry; Bookhout and Costlow, Jr.; Connell; Barnes) in a symposium on “Marine Boring and Foul- ing Organisms” held at the Friday Harbor Laboratories in 1957 (edited by Dixy Lee Ray and published in 1959 by the University of Washington Press). Very little information is included on the enemies of barnacles; it is ap- parent from the present paper that more studies are needed to establish the possible role of predators, parasites, and diseases in barnacle ecology. It is possible that one or more of these agents, under certain specific conditions, may be important for the biological control of these economically significant fouling organisms. More studies are especially needed on possible fungal and bacterial infectious agents that may play such a role. Summary Three species of organisms previously re- ported as predators or as parasites of barnacles and two commensal peritrichs, have been found in populations of Balanus eburneus and Balanus balanoides occurring on the rock jetties from Sea Gate and immediately adjacent to the Os- born Laboratories at Seaside Park, Coney Island, New York City, respectively. The species found were Cephaloidophora communis (Protozoa: Sporozoa: Gregarinida: Eugregarinina: Cepha- linoidea: Cephaloidophoridae) from the intes- tine of Balanus eburneus; Epistylis horizontalis and Epistylis nigrellii (Protozoa: Ciliophora: Ciliata: Peritrichidae) from the branchial la- mellae of Balanus balanoides and B. eburneus, re- spectively; Stylochus ellipticus (Platyhelminthes : Turbellaria: Polycladia: Acotylea: Stylochidae) from the internal wall of the opercular valves of Balanus eburneus attached to Mytilus edulis; and metacercariae, possibly of Maritrema arenaria (Trematoda: Digenea: Microphallidae), on the external gut wall and other tissues of Balanus balanoides. The presence of these organisms in local barnacles represents a new geographical record. These and their effects on the host are briefly described together with a review of the literature on other parasites and diseases of barnacles. Literature Cited Arvy, L., et A. Batisse 1968. 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Arkiv fur Bot., 29A: 1-67. Skerman, T. M. 1960. Note on Stylochus zanzibaricus Laidlaw ( Turbellaria , Polycladia ) a suspected predator of barnacles in a port of Auck- land, New Zealand. New Zealand J. Sci., 3:610-614. Solger, B. 1890. Notiz fiber eine im Darmkanal von Balanus improvisus Darw. (var. Gryphi- cus Muenter) lebende Gregarine. Mittheil. Naturwis. Ver. Neu-Vorpom Rfigen., 22:99-102. Tregouboff, G. 1912. Sur les gregarines des balanes. Arch. Zool. exp. gen., 10, notes et revue No. 3, LIII, LXI. Tuzet, O., et R. Ormieres 1964. Sur Cephaloidophora communis Mawro- diadi (1908), Pyxinioides chthamali (Leg. (Dub.) (1909) et Bifilida rara n.g. n. sp. Eugregarines parasites de Cirripedes. Leurs sporocystes. Arch. Zool. exp. gen., 104:153-161. Wimpenny, R. S. 1966. The plankton of the sea. Faber edit., Lon- don, 1966. pp. 127-128. EXPLANATION OF THE PLATE Fig. 1. Sporadin of Cephaloidophora communis Mawrodiadi (1908) from the intestine of Balanus eburneus collected on rock jetties adjacent to Sea Gate, Coney Island, Brooklyn, New York. Figs. 2-4. Metacercariae of Microphallid digenetic trematode, probably Maritrema arenaria Hadley and Castle (1940), from Balanus balanoides collected on rock jetties on Coney Island Beach; Fig. 2: unstained living specimens; Fig. 3: cyst stained with neutral red; Fig. 4: excystment. ARVV & NIGRELLI PLATE I STUDY ON THE BIOLOGY OF BARNACLES NEW YORK ZOOLOGICAL SOCIETY The Zoological Park, Bronx, N. Y. 10460 OFFICERS Laurance S. Rockefeller President Robert G. Goelet Executive Vice-President Chairman of the Executive Committee Henry Clay Frick, II Vice-President John Pierrepont T reasurer Howard Phipps, Jr. Secretary Eben W. Pyne Assistant Treasurer Edward R. Ricciuti Editor & Curator, Publications & Public Relations EDITORIAL COMMITTEE Joan Van Haasteren Associate Editor William G. Conway Donald R. Griffin Hugh B. House Robert G. Goelet Chairman F. Wayne King Peter R. Marler Ross F. Nigrelli George D. Ruggieri, S.J. William G. Conway General Director ZOOLOGICAL PARK William G. Conway . . . Director & Curator, Ornithology Hugh B. House .... Curator, Mammalogy Grace Davall . . Assistant Curator, Mammals & Birds Walter Auffenberg . . . Research Associate in Herpetology Joseph Bell . . Associate Curator, Ornithology F. Wayne King .... Curator, Herpetology William Bridges . Curator of Publications Emeritus John M. Budinger . . . Consultant, Pathology Ben Sheffy Consultant, Nutrition James G. Doherty . Assistant Curator, Mammalogy Donald F. Bruning Ornithologist Joseph A. Davis, Jr Scientific Assistant to the Director AQUARIUM Ross F. Nigrelli Director Christopher W. Coates . . . Director Emeritus Nixon Griffis .... Administrative Assistant Jay Hyman Robert A. Morris Curator U. Erich Friese Assistant Curator Louis Mowbray . Research Associate in Field Biology Consultant Veterinarian OSBORN LABORATORIES OF MARINE SCIENCES Ross F. Nigrelli . . . Director and Pathologist Martin F. Stempien, Jr. . . . Assistant to the Director & Bio-Organic Chemist George D. Ruggieri, S.J. . . . Coordinator of Research & Experimental Embryologist William Antopol . . . Research Associate in Comparative Pathology C. M. Breder, Jr. ... Research Associate in Ichthyology Jack T. Cecil Virologist Jay Hyman Harry A. Charipper . . Research Associate in Histology Kenneth Gold Marine Ecologist Myron Jacobs Neuroanatomist Klaus Kallman Fish Geneticist Vincent R. Liguori Microbiologist John J. A. McLaughlin . . Research Associate in Planktonology Research Associate in Fish Endocrinology Martin P. Schreibman Research Associate in Comparative Pathology INSTITUTE FOR RESEACH IN ANIMAL BEHAVIOR [Jointly operated by the Society and The Rockefeller University, and including the Society’s William Beebe Tropical Research Station, Trinidad, West Indies] Peter R. Marler Director & Senior Roger S. Payne Research Zoologist Research Zoologist Fernando Nottebohm . . . Research Zoologist Richard L. Penney Assistant Director George Schaller Research Zoologist & Research Zoologist Thomas T. Struhsaker . . . Research Zoologist Donald R. Griffin . . . Senior Research Zoologist C. Alan Lill Research Associate Jocelyn Crane .... Senior Research Zoologist Paul Mundinger Research Associate O. Marcus Buchanan .... Resident Director, William Beebe Tropical Research Station SMITHSONIAN INSTITUTION LIBRARIES 3 9088 01405 9232