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CVE ( 4 Qe Ca Ce G4 ac @ (C 4 a © ic Ge ¢ COLOR Ci@ eae q ¢¢ (q@C € G a C1& ¢ OEE Cee cae @ UAE CC OE GE « CAE, Cag CC: 1% COM qi Gaie @¢ «¢ mC OC Ge &7 .€ as A GH OO, <“(& Game qd « “(ORK Cm ea. ae C11 ES <<¢ & ov sist . & CAGES Rie GT a CMEEE MCGEE Hi ce (OG CE Ce gaC q( qa CU GRRE GG COS @ 4 C a%, (& r (Gi C 4G CC C(€ 4 (iC ¢ ( (CGR @ « ; Cc’ ; qi << 4. aa max «& A & (CEG aC aE eC COE CME @ & Ce (1G €@:C : (qi | x ti , a é <2 O Comgia 40 C (CME @ CO 4 @ COLNE EC emt EM CEE © (GME OT 45:5 {Oe ao vr le & ¢@ Qa c@ ave Se are mor oe ed “eqs = € cat tp CK « » ae Ut a «¢ ‘om ott © Gea @€ . 6¢ .. ¢ SBiccG ae a 4 *y Cor (a ¢ ¢€ oc iq @€ aq | COE (CC QC a COMA CCH CC 4 iia . ae CE UGE ZC aS CCC & (CM amr, eC (OLE CE EE CHE (COO CO COC em oe TOV 0a & y~ ve CK @& 4 r —_a\ & yg RY a C & C&T ¢ C4 Ce (Ge so 9) RAY. ad P| | ae, Johto ec Pg | Ae , sd | Sa | AN DIS ~~ ww Swueele ; “ UNI Ae Leyes . 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Nu fee! ~~ ~ -_ = Sas eee 4 ed ~ dh ~ 3 > Vir aed Ned wi Secs Wt Ber Sis2: Be A we: = had ww ~ 4 Jie, agen = NS =i Neng XN aC ei WN, we Sle Na ¥ "ites ah MPs Jt \ é a AT eon? . ae! oeng Ts i DEPARTMENT OF MARINE BIOLOGY OF THE CARNEGIE INSTITUTION OF WASHINGTON ALFRED G. MAYOR, Director PAPERS FROM THE DEPARTMENT OF MARINE BIOLOGY OF THE CARNEGIE INSTITUTION OF WASHINGTON VOLUME XVIII T= | “a q\iheenina latin Ss ney (& Os i MAR y 7 MB fees ee’ 1925 mead | Sloe! Husau®: PUBLISHED BY THE CARNEGIE INSTITUTION OF WASHINGTON WasHinaton, Novemssr, 1922 eat mg Ar mit gi a Mi ¢ Bh AG Y is Baek OF s St, , DEPARTMENT OF MARINE BIOLOGY THE CARNEGIE INSTITUTION OF WASHINGTON ; Pa ALFRED G. MAYOR, Director PAPERS, FROM THE DEPARTMENT OF MARINE BIOLOGY OF THE CARNEGIE INSTITUTION OF WASHINGTON VOLUME XVIII \ | oes PUBLISHED BY THE CARNEGIE INSTITUTION OF WASHINGTON Wasuincton, NovemMser, 1922 CARNEGIE INSTITUTION OF WASHINGTON PusuicaTion No. 312 TECHNICAL PRESS WASHINGTON, D. C. CONTENTS. PAGE I. Studies on the Hybridization of LEchinoids, Cidaris tribuloides. By Davin HevBONNEINT., 9 PIALOS 2S, fIPUITOR: a. ces. cce cnc ee we ene tWie iwwew cas 3-20 Part I. Embryology and Hybridization of Cidaris.............0..0eeeeeees 3 Nature and Systematic Position of the Material......................8. 8 Early Development of Cidaris tribuloides.........0.cccceee ee eee re eeees 5 Rate of Development of Cidaris compared with that of other Echinoids.. 6 Comparison with other Investigations of Cidaris............2..00+0000s 9 Formation of Mesenchyme in Echinoderms.................52+0020e00% 11 Observations ‘on Hybridization of Cidaris... 2.0.2... be ccc eee sce sere eens 13 Influence of Spermatozoon on Production of Paternal Characters........ 15 The Spermatozoon and Pertilization:. 2055 2i. 36. 6s.. ces eentrcecessere 16 Cross-Activation versus Cross-Fertilization................0eeseeeeeee 17 @lasahieationyo le EL yio Ee 8 ar.) ccho aye sigieis eeioleiste ssi vietcievele,sinen'@ #10! sles cle «ie, ecee 18 Part II. Cytology of the Eggs of Cidaris tribuloides and of Cross-Activated Carp aot taco fore orale cia oie acs oiein sa W's) wie. scas site sian) oie # s plefeiesetarcts 21-42 Simmeture smite CG SUATLa ue ne eeten ys w cleia\e straw ieisvalevshe dye vaya Rei iere Gleple + 21 Study of Chromosomes in Species-Fertilized Cidaris Eggs.............-. 23 Chromosomes in Cidaris Eggs Cross-Activated by Lytechinus Sperm...... 27 Chromosomes in Cidaris Eggs Cross-Activated by Tripneustes Sperm.... 29 PAMIMnAtION Ol. CHTOMOSOMES = clase ole rsieivione (ele oie die Blciereke ee vie. S cca elelete stois, sims 32 Alteration in Physical Characteristies of the Cytoplasm of the Egg by ection. Of POreignt SPEEIMNi veo. oe.’ . wis cele ue Mirae so alt 68 choferw.o ee 34 Nuclear Enzymes and Cytoplasmic Substrate..............0 20 ec eee ees 35 Aine Biniuelearity? FLY POUBCSIAS osc.-0)2/o bine-ae we s-owmellels 130 Gennes PEGdICG SAVIO. ON eiciks tes WAEEI c uc ec alnaM aacaan aneme eee 130 VATICEB CTR He ee IR foyds oe ere ee iat Sci Secteur halen ae 131 cy) Fg 000 a tetg] ata SSS an a ce ra ee te Ae 134 HUVEC BAGG SAVIRD Ve ere. ss, 5-5 Ay vies. wha sheen sale es 136 flava-punctata, new species.................. ee ee 136 SUVIENSIS, NEW SPECIES s,.0< 1) arose. Nas pide slew alee bone sone 138 GU DICGOLS: MOWrSPeEClesa 4.65 0. cols Gia. sero epee OMS 4 139 COCCINER NGEUDGs foe slice sien, hectare ischemia 142 ALICUIALA: NEW SPECIES... co: fe sch, 2< 25 sate oon wage aes 143 armillatay New SPECIOS. mc. ger a). eae 4d os» aie Masa ee ete 144 crassi-tentaculata, new species...................... 146 biformi-cirrata, new species..................0.000. 148 Gracili-ciFratal, NEW SPECIES)... = 2 sii si. ave cares oO eee 149 SOHN WAT DNV GA) SAMARIA oS sea So peeerired.e deo doe soos aeaie Sse eee wil ah Ses 150 Californicny NIGOTEs 4h aya) ees wales ale one 150 mackintoshi' Crossland’. - ¢ 3..c 5.0 25). cas cus wes 151 SIMPIEX, Hew SPECIES. e.. os erg ncts be sees sche Stokes we 151 Genus Paramarphysa Whlers ve. ..2- eas o's t dances es nd wo Emaar 153 EGRES. NEW SPECIES: sisi. cia oles Beas eats oot Sate 153 Genus Onuphis Audouin et Milne Edwards ....................... 154 holobranchiata v. Marenzeller..................... 154 LONUN: VSIGICE DANELDY Sete a senna caus Sidas carey thaw gi zoho ary ie ee oie 154 FUSCA, NEWSSPECIOS 7.5, 6 cbace 2's als Bio bya alloud a Pavautte wars eee 154 PAV NEW SPECIES so f55 ohh ode ok ances eta iis Helo 155 CeMNS NICIIOMMRER DET i ai8. Stasis «a ae ce dicln me ac aeieiron Caicos 156 fusca-fasciata, new species..................00e eee 156 Sig ransaty Esa RIMORCIN ED Yo. css) ua: duc.o Ma vaiotsiw soe. Gnele wavdinero renee 157 Genus; Lumbrinerois'de: Blainville: . 2 2))2.. 8.55.05 oes hacen ewes 157 spherocephala Schmarda..................... 158 brevicirraSchmarga’: , & aves Fonica. aate ee cies 158 VI Contents. PAGE VIII. Leodicide from Fiji and Samoa—Continued. Genus Arabella Grube 4 si). 3i ial aiera! «n'a a igustvis inte oben toate widens oteemis SEA ete 160 GUDICS WOW BPECIES. ./.5:.. «foe pee eat te eer sae ene 160 Genus (Driloncreis Planenedes. Ns od keane ke es eee nee 161 WUNIDPICUA; TleW APECIES 65:57 ie sok ees sce ew eee 161 2 paucidentata, new species.............e0e2eccees 162 Genus Oenone SAvinn ys oo, Slice es dase Ore te etna e tie aeelnd 163 lpi BR VIBNY ha is sc erice Pelee clare etice ote shale stores 163 Subfamily Dorvillemisy Meee occ: Co oe eieia atone > aes arene 166 Genus Dorvilloa Parity. cher de eas soa ache as cites aes Rae 166 australiensis: MelImtosh <7, 00 6922 casas bases ca cule oe 166 Bibliography sinc, « daneenet ces aoe eee ae eeie ale eae aoe 169 IX. Polychetous Annelids Collected at Friday Harbor, State of Washington, in February and March 1920. By A. L. TREADWELL. 37 figures........ 171-181 Hoch rerl\aroy id UUs | Cn an nN ee RNR AR sat Se See BR 173 Autely tus varias (Treadwell). 2% ou scmer cee beens caine ere eanen 173 amily; Phytlodocidee 2 ee roves hos oe ces Caer Se Oe tee lemon 174 Bteone maculata, new Species; . sh 6 cs ee ccna vw aiid ines ew ecu ober 174 tubereulata;*new species! fs. os ck oh atioes sees canoe 175 Family Leodicide ics. asin tee ce eet eee eS eng cei ee 175 Lumbriconereiszonata Johnson. -57 22%. so oc hes esse ede een ake 175 cervicalis, new: species.(./..c2%. a8 ee fous ciate ae 176 Onuphis stigmatis, new species.............. eMac ie hors eaine 176 Family Spionidse: eho say ausce eaiees cas ot coat oe ene eee 178 Polydora californica Treadwell...) = 3. 5 <.:ca'. oesaide es sane oeies 178 Pamitly, Crrmatymise = 6 foals a cia s Rak sce ete ATR ae nee 179 Cura tulusrobustus Johnson.) .\0 aia essay nee ace ree 179 Family ipbelnidas: 2s iit atea acc ois aise sic cetere ey eget hese ae elie eee eae 179 Ammotrypane brevis Moore.................. hes Gitefal Saleen (ere 179 STUDIES ON THE HYBRIDIZATION OF ECHINOIDS, CIDARIS TRIBULOIDES. By DAVID H. TENNENT, Bryn Mawr College. Three plates, twenty-eight figures. CONTENTS. PAGE Part I. The embryology and hybridization of Cidaris. Nature and systematic position of the material.............. 0.0.0: cecuerececeeee 3 Barly development of Cedaristrsbuloides 5c 56 Sen svote Se a cle Coe wee ee ae 5 Rate of development of Cidaris compared with that of other Echinoids............. 6 Somparison with other investigations of Cidaris................ 00. c cece eee ee 9 Formation of mesenchyme in: Echinoderms. . 00). 0.0... os oe de as Hon enms be te ee 11 Observations on hy bridizationvol iC idarts! hss tac stee cc bn et ee ue eae 13 Influence of spermatozoon on production of paternal characters ................... 15 ‘he: spermatozoon and fertuization (20 eens 3.0). eck Oe hate te eles cate Serer 16 Crass-activation ‘versuscross-fertilization. 5)... 6b. sa sd inde aloe ren bela 17 Classification Of Ny bmds) isso .c. se tute Ge nae eciele ne ars RS eA eae ee 18 Part IT. Cytology of eggs of Cidaris tribuloides and of cross-activated Cidaris eggs. Structure of the Cedaris eye 000s Petcare OE, cic oe ashe eld anes ae eee 21 Study of chromosomes in species-fertilized Cidaris eggs... 2... 00... ce eee 23 Chromosomes in Cidaris eggs cross-activated by Lytechinus sperm.................. 27 Chromosomes in Cidaris eggs cross-activated by Tripneustes sperm................. 29 Miimination: of ;ChFOMOSOMES . < s..he ax >. Settles al dle eR iti ee ee ee eee 32 Alteration in physical characteristics of the cytoplasm of the egg by action of foreign SPELOSSS. siarecccne wae siossyonschcabedstode pekeltapenewe chelate ei reer uane ledeh eke euo efoto hale eens tet atte here eter 34 Nuclear enzymes’and cytoplasmic substrate 6!) scwcd «eames oe ote = eee ee 35 Phe PMUcleAMty NypOUHNESIS) o's 505. smite aeas ih eae mo ecient ee eee ee 36 MPIC otra ape ae hohe coe 5 SRS din Wd Sos 5, Sue ae SO EAE Se ee ae 37 PSLEECELLLCL RNS Ae Cod Ble eS Oe ee aaa BORSA CAH VUE oo A cee Bar a SS 40 MPOMETARIONS 5 ee nee tac iaic ded os Mul eG betke «Basses side ceiene ReneRe e e 41 LOPES VATE © SRM Ueno ayn Lat ee MEA REL LO PMMA Sey 2k eee SAN oN 2 42 STUDIES ON THE HYBRIDIZATION OF ECHINOIDS, By Davip H. TENNENT. PART I. EMBRYOLOGY AND HYBRIDIZATION OF CIDARIS. In 1912, at Montego Bay, Jamaica, I obtained material and began the study of straight-fertilized eggs of Cidaris tribuloides Lamarck, of Cidaris eggs fertilized with the sperms of Lytechinus (Toxopneustes) variegatus, of Cidaris eggs fertilized with the sperms of Tripneustes (Hipponoé) esculenta, and of Cidaris eggs caused to develop par- thenogenetically. A brief account of some of the facts determined appeared in Publication No. 182 of the Carnegie Institution of Wash- ington. The present paper includes my completed observations. NATURE AND SYSTEMATIC POSITION OF THE MATERIAL. The nature and systematic position of the forms used demand more than passing notice. Cvidaris represents the lower extreme of a series extending from little specialized to highly specialized Echinoids; Lytechinus and Tripneustes represent the upper extreme. Jackson (1912) has shown that the Cidaroida are primitive, extending from the Lower Carboniferous to Recent times. He says: “The most primitive type of Echini, I believe emphatically, is Bothriocidaris [p. 208]. . . . The order Cidaroida is placed as derived directly from the Both- riocidaroida without known intermediate forms. The Cidarids, as regards the struc- ture of the young and adult, are the least removed from Bothriocidaris of any known echinoid, living or fossil” [p. 211]. Lytechinus and Tripneustes are members of the order Centrech- inoida (Triassic to Recent), of the suborder Camarodonta, and of the family Echinide (Cretaceous to Recent). Again quoting from Jackson (p. 210): “The sub-order Camarodonta may be considered the most specialized of modern regular Echini on the basis of the lantern, and also in various genera by the sculptured test, the degree of specialization of the ambulacrum, peristome, perignathic girdle, or the elliptical form through a sidewise axis.” In discussing the lantern, Jackson says (p. 187) that ‘‘ Trzpneustes represents the most complex structure known in the Centrechinoida.”’ H. L. Clark (1912, p. 365), in his consideration of the Echino- metridz, says: “There can be little question that this family includes the most highly specialized of the regular recent Echini, for the elongation of one axis, when combined with highly developed ambulacra, indicates an unusual complexity of structure. And yet in the 3 4 Hybridization of Echinoids. characters of the abactinal system and the globuliferous pedicellarie, the most special- ized Echinide, such as Tripneustes, are apparently more advanced than any of the Echinometride, and it is therefore merely a matter of opinion whether T'ripneustes or Heterocentrotus is considered the ‘highest’ of the regular Echini.” These ideas, based upon a morphological and paleontological con- sideration of Echinoids, have an added significance and interest when we find that Cidaris is also primitive in its development. Cvdaris shows some processes of development that have not been described, so far as I know, for any other Echinoid, and in one phase of its early development resembles the Crinoids and Holothurians more closely than it does the Echinoids. The material has also given an oppor- tunity for the study of the results following the insemination of the egg of a species having a primitive type of development with sperms of species having a modern type of development. Lytechinus and Tripneustes are about as widely removed from Cidaris as it is possible for them to be and remain in the same class. Lytechinus and Tripneustes are of the order Centrechinoida; Cidaris is of the order Cidaroida. Crosses between these forms are therefore interordinal, and in themselves are of a good deal of interest. Conklin (1915, p. 176) has stated the generally accepted belief regarding the respective potency of egg and spermatozoon in heredity: “ At the time of fertilization the hereditary potencies of the two germ-cells are not equal, all the early stages of development, including the polarity, symmetry, type of cleavage, and the pattern, or relative positions and proportions of future organs, being foreshadowed in the cytoplasm of the egg-cell, while only the differentiations of later development are influenced by the sperm. In short, the egg cytoplasm fixes the general type of development and the sperm and egg nuclei supply only the details.” There has been a considerable amount of interest concerning the time at which the influence of the sperm first becomes evident. The material at hand enables us to push back our determination of the time of appearance of paternal influence succeeding fertilization to a point beyond that which has previously been reached and estab- lished, but it is also convincing in its proof of the lack of plasticity in the egg, of the inability of the egg to follow a system of develop- ment which is not its own. The egg cytoplasm is the material which is to be differentiated, but it does not seem to be able to harmonize its inherent system of development with a foreign system. Most of the successful crosses between Echinoderms have been within one suborder of the order Centrechinoida. The cleavage pattern and the early development of the forms considered have afforded no landmarks for the guidance of the observer. In this Cidaris and Cidaris-Lytechinus-Tripneustes material there is a defi- nite, visible specificity of development, and it is possible to see the exact period at which the disharmony between two systems of devel- opment begins. Hybridization of Echinoids. 5 This study has been based on an examination of both living and fixed material. The development of the straight-fertilized eggs was followed through 6 days. Plutei were kept alive for 2 weeks, but did not advance strikingly beyond the stage of development shown by the 6-day larve. The cultures were kept in finger-bowls and the larve were given a complete change of water daily, after the third day, by transferring the contents of the bowls to centrifuge tubes and centrifugalizing gently, thus throwing the larve to the ends of the tubes, then withdrawing the water from above the larve and replacing with fresh sea-water. The plutei treated in this manner kept in better condition than those not centrifugalized, but their rate of growth was slow. The general form of the larve changed very slightly, but there was some increase in the size of the skeleton. I was at first of the opinion that the sole cause of the slow rate of growth was an inadequate supply of food, but the observations of Prouho and Mortensen on developing Cidaris larve have caused me to change my opinion to the extent of believing that, even though the quantity of food available was somewhat below normal, this was not the only cause of what seems a slow rate of growth when compared with that of other Echinoid larve. Development in Cidaris is apparently slow; these larve may have been growing at nearly the normal rate. Because of limited time, no attempt was made to rear the larve to metamorphosis by the use of cultures of diatoms. Material was fixed in sublimate-acetic (98 c. c. saturated aqueous solution of cor- rosive sublimate plus 2 ¢. c. glacial acetic acid), for 15 minutes. For some purposes, portions of this material have been stained in toto and mounted entire; for other purposes portions of the material were embedded in paraffine in the usual way, cut into sections of 5 or 7 microns, and stained by Heidenhain’s iron hematoxylin method. Larve kept for the study of skeletal rods were killed in fresh water and then transferred to alkaline alcohol. EarLty DEVELOPMENT OF CIDARIS TRIBULOIDES. The living eggs of Cidaris tribuloides are exceedingly transparent. They may be fertilized readily in the laboratory with species sperm or with the sperm of other sea-urchins abundant in the region, among these being Lytechinus variegatus and Tripneustes esculenta, and the union of the male and female nuclei may be followed without difficulty. For straight fertilization the eggs were carefully washed and inseminated within a few minutes after their removal from the gonad. The crosses are easily made. The eggs may be activated with the foreign sperm at once after their removal from the ovary, without artificial aid, although as a check against the possibility of error due 6 Hybridization of Echinoids. to chance fertilization with Cidaris sperm, they were kept for two hours before making the inseminations. A portion of the eggs was always kept as an unfertilized control. These statements regarding the ease of cross-fertilizing apply to conditions existing from March 5 through March 18, 1912, the only period in which I have had the opportunity of working on these forms. In its normal development Cidaris proved of interest: (1) because of its slowness of development when compared with Lytechinus and Tripneustes; (2) in the difference in site of its mesenchyme formation; (3) in the place of appearance of the larval skeleton; (4) in the form of the larva. RaTE OF DEVELOPMENT OF CIDARIS COMPARED WITH THAT OF OTHER EcHINOIDs. Following fertilization, the various phases of the nucleus during division may be followed readily. The beginning of the anaphase of the first division is reached about 50 minutes after insemination. The cleavage is like that of the eggs of other echinoids, the formation of the micromeres being as in Lytechinus and Tripneustes. The blastula stage is reached in 16 to 18 hours, the eggs from a given female developing uniformly. The variation in rate of development of different lots of eggs is not altogether determined by temperature, since mixed lots of eggs, although nearly all may develop normally, do not develop at a uniform rate. Gastrulation begins in 20 to 23 hours; mesenchyme formation begins in 23 to 26 hours, the mesen- chyme cells arising from the inner end of the archenteron; chromat- ophores appear in about 44 hours; the enteroccele arises as a single pouch in 44 to 50 hours; in 55 hours two enterocceles may be seen, formed by the division of the single vesicle; in 72 to 73 hours the first skeletal spicules may be noted. Cidaris. Hours. Lytechinus. Hours. Blastule# (swimming)! 16 to 18 | Blastule (swimming) 5.5 Gastrule (beginning)| 20 to 23 | Mesenchyme........ Mesenchyme........ 23 to 26 | Gastrule (beginning. ) 9 Chromatophores..... 44 Chromatophores..... 15 to 16 Skeleton (beginning).| 72 to 73 | Skeleton (beginning).| 15 to 16 IPMMteUBP aes ences 120 Pluteusicee aeecene: 24 Even at this time, the beginning of the fourth day, the body has not begun to assume the form of an echinopluteus, and it is not until the fifth day that the arms begin to push out. These facts are of interest when compared with those of the development of Lytechinus. Here the blastule reach a swimming stage in 51% hours after the insemination of the egg; mesenchyme cells begin to push into the ————— errr ee er er Hybridization of Echinoids. ri blastoccele from the flattened and thickened posterior pole of the blastula in 8 hours; the process of gastrulation begins in 9 hours; chromatophores appear in 15 to 16 hours; skeletal spicules appear in 15 to 16 hours; and the young pluteus stage is reach in 24 hours after insemination; it may be reached in as short a time as 20 hours. These facts are shown in parallel columns (p.6). The hours mentioned indicate hours after insemination. The difference in sequence of stages as well as the difference in rate of development is evident. As has been stated above, there is nothing unusual in the develop- ment of Cidaris, aside from extreme slowness, until the stage when mesenchyme formation might be expected. The blastule (fig. la; plate 3, fig. r) have a wall of rather uniform thickness. The cells at the posterior end seem very slightly larger than those of other A oO i) OL oF \O\, ie) Cc] Xo 4 See &/ ~ GOOF _sOy se i) Dae Oy oxo i) ae yy aoe Fic. la, Optical section Cidaris blastula 16 hours old. Drawn from fixed and stained material. X 285. 1b, Optical section Cidaris beginning gastrulation; 20 hours. Drawn from fixed and stained material. X 285. 1c, Optical section Cidaris gastrula, 22 hours; drawn from fixed and stained material. X 285. parts of the wall. A section of an 18-hour blastula (plate 3, fig. F) shows that these larger cells are about to move or be forced inward. The first indication of an archenteron appears in embryos of about 20 hours (fig. 1b). In these, at the posterior end, there may be seen a small, hollow plug of cells, extending into the blastoccele. The cells forming this plug are quite characteristically rounded, standing out distinctly from each other like the seeds in a blackberry. The cavity of the archenteron, though shallow, is cylindrical and has a distinet blastopore. The archenteron continues to grow forward into the blastoccele, and 3 hours later has about doubled in length (figs. Ic, 2a; plate 3, fig.a). The inner end is strikingly irregular in form, because of the protrusion of cells from its surface. No mesenchyme cells have as yet migrated from the wall of the archenteron, but sections (plate 3, fig. H) reveal the fact that they are in process of withdrawal at this time. The apparently loose contact of the cells of the arch- 8 Hybridization of Echinoids. enteron with one another is striking. There seems to be little surface tension in the wall at this time, each cell being rounded and having rather a minimal contact with its neighbors. Many of the cells are in phases of mitotic division. Slightly later, cells begin to move away from the outer surface of the archenteron and the conditions represented in plate 3, figurestandJ,may beseen. The cells protrude long protoplasmic processes, which come into contact with one another and with the processes pushed out by adjacent cells; these then fuse and give rise to the characteristic networks which may be seen in echinoid gastrule (fig. 3a). At about 50 hours the migration of mesenchyme has come to an end and the enteroccele arises as a single pouch from the inner end of the archenteron. On the left side this opens to the exterior through a dorsal water-tube (fig. 2c). The single enteroccele, about 5 hours later, divides into two, a right and a left, the left retaining connection SN ‘is 1 a Bye mS a | P SV 7 \ a Se () a, ate PE Fic. 2a, Optical section living Cidaris gastrula; 23 hours. x 180. 2b, Optical section living Cidaris gastrula; 30 hours. X 140. 2c, Optical section living Cidaris gastrula; 55 hours; enteroceele and pore canal present. X 140. if a ——— tN if Z ‘ with the dorsal water-tube. By this time the wall of the archenteron has become thin, the cells composing it no longer standing out dis- tinctly from each other. About this time the spheroidal form of the embryo changes, due apparently in part to the more rapid growth of the dorsal region, which causes the blastopore to open on the ventral surface, and in part to the modification in form of the future oral area. The anal area remains rounded, while the oral area becomes first flat and then concave. A ciliated band appears along the posterior edge of the oral area. This is the posterior ciliated band. It becomes well established before there is any indication of an anterior ciliated band. The anterior band is present at 66 hours (fig. 4a). A stomodeal invagination appears about the seventieth hour and pushes back until it comes into contact with the future esophagus, the inner end of the archenteron having been directed toward the ventral side. Hybridization of Echinoids. 9 In some sections of embryos of an age of 73 hours the mouth opening does not seem to be established; in others the opening is present. It is therefore safe to say that the mouth opens about the beginning of the fourth day. The first appearance of skeletal spicules is about this time, these spicules arising in groups of mesenchyme cells at about the level of the outer ends of the posterior ciliated bands. As these rods increase in length, the sides of the embryo grow out to form what are probably the post-oral arms (figs. 4,b and c), the fenestrated rods in their growth keeping pace with the growth of the arms. The cultures were kept under observation until the end of our stay at Montego Bay (two weeks). During this time no essential change in the form of the larva took place. The skeletal rods increased in Fre. 3a, Optical section living Cidaris gastrula; somewhat flattened by pressure of coverslip; 41 hours; enteroccele forming; mesen- chyme cells abundant in blastoceele. X 140. 3b, Section of blastula of Lytechinus; 6 hours; from fixed material. 285. x 3c, Section of blastula of Lytechinus with mesenchyme cells proliferating; 7 hours; from fixed material. XX 285. 3d, Optical section living gastrula of Lytechinus; 20 hours. X 285. length and the inner ends of these rods assumed a more definite association as a framework at the sides of and ventral to the stomach (fig. 5b). In the most advanced specimens that have been studied the fenestrated rods show as many as 14 openings, and there has been a pronounced increase in the length of the antero-lateral rods. The body-rods extend posteriorly on each side of the stomach, while the ventral transverse rods have grown toward the median ventral line, although there has as yet been no contact or union between the ends of the rods of opposite sides. COMPARISON WITH OTHER INVESTIGATIONS OF CIDARIS. Cidaris tribuloides, as may be seen from the foregoing description, is quite unlike the more familiar echinoids in the time and place of 10 Hybridization of Echinoids. formation of primary mesenchyme and in the form of the pluteus. The only detailed description I have found of the early development of a Cidarid is that of Prouho (1887) on Dorocidaris papillata. The rate of development of this form is very slow, slower than that which I have recorded for Cidaris tribuloides. The stage of the gastrula with a primary enteroccele is reached in 6 days; the enteroccele has divided to form two pouches in 8 days; while in 10 days skeletal rods with four openings are present. Prouho kept larve of this species alive for 8 months and obtained plutei with 3 pairs of larval arms and of the characteristic echinopluteus form. Fig. 4a, Free-hand sketch of living Cidaris larva in ventral view; 66 hours. 4b, Camera sketch of living Cidaris larva in ventral view; 73 hours. X 185. 4c, Camera sketch of living Cidaris larva in ventral view; 6 days. X 135. Prouho’s description of the process of mesenchyme formation is not complete enough to enable me to decide whether the process in these two Cidarids is identical. He describes the mesenchyme as budding into the blastoccele from the posterior wall of the blastula, and gives a figure illustrating the process. This figure would serve as a satisfactory illustration of beginning gastrulation, as I have seen it, in Cidaris tribuloides. Without question the first cells that are carried into the blastoccele in tribuloides, as invagination begins, are the future mesenchyme cells, but these cells do not leave their position in the wall of the archenteron until this has grown well toward the center of the blastoccele. The appearance is somewhat that of mass budding. It might be called such if the cells formed a Hybridization of Echinovds. tf solid mass, but they form instead the inner end of the hollow archen- teron. The compact mass of cells shown by Prouho in his figure causes me to believe that the manner of formation of mesenchyme in Dorocidaris papillata will be found, on reexamination, to be of the type that I have described for Cidaris tribuloides. Mortensen (1921) has described observations that he made in 1915 on Eucidaris thouarst. His figure of a larva 6 days old dem- onstrates that the thowarsi larva is very similar to the tribuloides larva. Mortensen (1921, p. 78 et seg. and appendix) has suggested that the very unusual larva that he has described as Echinopluteus transversus is really the Cidaris larva. The additional evidence presented in this paper supports, so far as it goes, Mortensen’s suggestion. My oldest specimens have been reared for 2 weeks. None of these larvee has reached the stage of development shown by Mortensen’s plutei obtained from plankton samples from the West Indies and from the Indian Ocean. In my oldest or most advanced larve (fig. 5b) the fenestrated rods have increased noticeably in length and the antero- lateral rods have extended well forward into the anterior end of the larva (fig. 5a). No contact between the ventral transverse rods of opposite sides or of body-rods has yet occurred. ForRMATION OF MESENCHYME IN ECHINODERMS. The manner of formation of mesenchyme that I have described is similar to that occurring in Antedon as described by Seeliger, and which has been regarded as typical of Crinoids. Mortensen (1920), in his study of the development of the Crinoid Tropiometra carinata, has discovered that in this form, prior to the invagination of the wall of the blastula in the formation of the endoderm, there is a migra- tion of cells into the cavity of the blastosphere, probably from dif- ferent places in the wall of the blastosphere; ‘‘ these cells lie loosely in the cavity and look like mesenchyme cells, which, however, they are not” (p. 8). When the cavity of the blastula is nearly full of these cells the typical invagination begins and the loose cells become closely applied to the upper end of the invagination. This process is in striking contrast to the condition in Antedon, in which Seeliger found no free cells in the blastoccele until after the invagination of the endo- derm. Mortensen (1920), in his study of the development of one of the viviparous Crinoids, [sometra vivipara, found a cleavage of the kind typical of Arthropods; there is a division and migration of nuclei before any cell-walls appear; ectoderm and endoderm are differentiated in place, no invagination occurring. There are no cells in the space between ectoderm and endoderm, but some yolk grains may be seen lying in this cavity. Guthrie and Hibbard (1919) have given a summary of the facts known regarding the time of formation of mesenchyme in the various 12 Hybridization of Echinoids. classes of Echinoderms. To the list given in the paper cited should be added Mortensen’s contributions on Crinoids and that of Oshima (1918, 1921) on the Holothurian Cucuwmaria echinata, in which the mesenchyme cells are described as migrating into the blastoccele before the beginning of gastrulation. Fia. 5a, Skeletal rods from fixed and mounted Cidaris larva. 6 days. X 350. 5b, Cidaris larva, 2 weeks old, fixed and mounted. Camera sketch. X 110. 5c, Camera sketch of living Cidaris ? x Lytechinus@ gastrula. 24 hours. X 220. 5d, Camera sketch of living Cidaris 9 X Lytechinuso gastrula. 24 hours. X 220. 5e, Camera sketch of living Cidaris 2 x Lytechinus gastrula. 40 hours. X 220. Mortensen’s (1920) studies have shown that there is diversity in the manner of formation of mesenchyme in the Crinoids, a fact which our knowledge of the development of three species of Antedon had failed to reveal. These observations do not affect the conclusions that I have drawn regarding the primitive nature of the development of Cidaris. Cidaris tribuloides, in the estimation of those qualified to speak on the systematic position of echinoids, is primitive. This fact suggests Hybridization of Echinoids. 15 the probability, although it does not absolutely establish the proof, of the conclusion that the development of Cidaris is primitive. OBSERVATIONS ON THE HYBRIDIZATION OF CIDARIS. At the time when these observations on the development of Cidaris were made, March 1912, two other sea-urchins in the region were breeding. These were Lytechinus (Toxopneustes) variegatus and Tripneustes (Hipponoé) esculenta. The fertilization of the egg of Cidaris by either Lytechinus or Tripneustes sperm was easily accom- plished. No preliminary treatment of the eggs was necessary. It was possible to fertilize the eggs with either Lytechinus or Tripneustes sperm immediately after their removal from the ovary, but, as a precaution against failure to detect chance fertilization by Cidaris sperm, they were kept for 2 hours before inseminating with the foreign sperm. More than 90 per cent of the eggs so treated fertilized; the typical fertilization membrane appeared shortly after the addition of the sperm. The reciprocal crosses were not readily made, and as the time available was short and the problem with which my investigation was concerned was not closely connected with that of specificity in fertilization, no attempt was made to develop a successful technique for these crosses. In the cross-fertilized Cidaris eggs, no difference from the charac- teristic normal Cidaris development was noted before the beginning gastrula stage. The fertilization-cleavage interval was not lessened. Cleavage was regular; the micromeres were formed as in other echinoid eggs, and the blastule had the appearance of normal Cidaris blastule. In both crosses the mesenchyme cells arose from the sides and around the base of the archenteron, close to the point of union of the archenteron with the wall of the gastrula (plate 3, figs. Ato®8). In point of time the appearance of the mesenchyme seemed slightly hastened, although not sufficiently to warrant a general conclusion to that effect, as it was within the range of variation between different lots of straight fertilized eggs. As to the place of mesenchyme formation, there is no chance for individual variation in the larve or in different lots of eggs to form the basis of an error in conclusion. In Cidaris gastrule the archenteron is a straight, slender tube, at whose inner end the migrating mesen- chyme cells may be seen readily (plate 3, fig. 5). At the time of the beginning of the formation of mesenchyme the gastrula is exceedingly transparent and the observer may convince himself that there are no formed elements at any place in the blastoccele. Figures 5, c, d, and e, are of optical sections of Cidaris? xX Lytechinus J living gastrule. In these the primary mesenchyme ceils may be seen clustered at the base of the archenteron and in some cases dispersed 14 Hybridization of Echinoids. throughout the blastoeceele. In plate 3, figures A to E, sections of the posterior portion of embryos in process of gastrulation areshown. In plate 3, figure A, cells are in process of migration from the wall; in plate 3, figure B, mesenchyme cells are at the inner end of the archenteron and at its base. Figures c, p, and & of plate 3 are from the same slide and are all of embryos 24 hours old. They have been selected because they show considerable individual variation. In figure c of plate 3 no cells have left the wall of the archenteron; in figure p of plate 3, mesenchyme cells may be seen at the base of the archenteron; and in figure 5 of plate 3, mesenchyme cells are to be seen at the base of the archenteron and in process of withdrawal from its wall. It is of interest to compare these sections of the hybrids, age for age, with the sections of the normal larve. The corresponding ages are figure A of plate 3 with F and G, all 18-hour embryos; figure B with H, both 23-hour embryos; and figures c, p, and & with 1, all 24-hour embryos. Succeeding stages show considerable variation. In some the growth of the archenteron ceases and the blastoccele becomes filled with a mass of opaque cells (fig.5e). In others gastrulation continues slightly beyond the stage indicated in figure & of plate 3. In a few cases a small triradiate spicule was found in the mass of mesenchyme cells at the right and left of the base of the archenteron. I was unable to keep the hybrid material alive beyond the gastrula stage. No plutei whatever were obtained. It is at this stage of beginning gastrulation that many attempted hybridizations between species of Echinoids fail. The material from which sections represented in figures B to BE of plate 3 were made enables us to see clearly that the failure in development is accom- panied by an extreme degeneration of nuclei. In figure B the cells above the inner end of the archenteron seem to be in a process of disgorgement of chromatin; in the same figure a mesenchyme cell on the lower left side is shown which has completed such a process. In figure c two of the cells in the wall at the lower right are degener- ating, while in figures p and © numerous stages in karyolysis are shown; the nucleus in some instances seems to have disintegrated, in others to have become simply a deeply stained mass of chromatin which may even be extruded from the cell. In some sections these distinct masses of deeply stained material may be found in consider- able abundance among the scattered cells. When we review this series of changes in the development of the hybrids and compare this development with the development of Cidaris and with the corresponding blastula and gastrula stages of Lytechinus (figs. 3, b, c, and d) and of Tripneustes, it may be seen readily that the processes of development in the hybrid are inter- mediate in character, lying between those of Cidaris and those of Hybridization of Echinoids. 15 Lytechinus and Tripneustes. In Cidaris the primary mesenchyme cells are given off from the inner end of the archenteron (figs. H, 1, and J of plate 3); in Lytechinus and Tripneustes these cells arise from the posterior wall of the blastula (figs. 3, b andc), while in the hybrids they arise from the wall of the archenteron at the time this structure commences to grow into the blastoccele (figs. A, B, D of plate 3; figs. 5, c and d). The most striking result of fertilization with foreign sperm has been this change in the time and place of mesenchyme formation. INFLUENCE OF SPERMATOZOON ON PRODUCTION OF PATERNAL CHARACTERS. This result gives proof of an earlier visible evidence of the influence of the spermatozoon in the production of paternal characters than has previously been gained. ‘The reason for this lies wholly in the nature of the material. By chance, material belonging to two visibly different systems of development was obtained. In the discussion between Boveri and Driesch, in 1903-04, on this subject, evidence on the form of the larva, the skeleton, the number of chromatophores, the pigment content of the chromatophores, the arrangement of the chromatophores, the number of primary mesen- chyme cells, and, under certain conditions, the size of the larvee were considered. With the exception of the primary mesenchyme cells, these are, as events happen in early development, characters which are relatively late in the time of their appearance, and yet the visible differences in the material were not of sufficient value to enable Boveri and Driesch to reach a conclusion in common. The reason for this failure to reach such an agreement seems to lie in the fact that the forms used are closely enough related to have the same system of development, and any differences that could appear are compar- atively minor ones. If we consider the rédle of the spermatozoon in development, we find that the function of the spermatozoon may be double. It may give the initial impulse to development and carry the determiners for the development of paternal characters into the egg; or it may give this initial impulse without exerting influence in later differen- tiation. The mature egg contains material whose differentiation will result in a new individual. Treatment with egg secretions, with a great variety of reagents, or the mere penetration of the surface of an egg by a spermatozoon, or by a sharply pointed instrument, may cause the egg to develop parthenogenetically into a thelykaryotiec individual. The entrance of a spermatozoon, followed by the fusion of the sperm nucleus with the egg nucleus, may cause the egg to develop into an individual showing biparental characters. Usually we have thought of fertilization as comprising the whole series of changes occurring 16 Hybridization of Echinoids. during the time extending from the instant of contact of the sperma- tozoon with the surface of the egg to the completion of fusion of the germ nuclei, although we have tacitly admitted the logic of the position that fertilization is not completed until the conjugation of homologous maternal and paternal chromosomes. In_ practice, however, the word fertilization is often used as though it applied merely to the initial impulse to development. Strictly speaking, the development of a uniparental individual begins with the separation from the primordial germ-plasm of the material which is to form the ovum from which the body of that individual is to be differentiated. All of the so-called stages in oogenesis are stages in the development of the individual. In biparental individuals there must be added the processes of formation of the spermatozoon which conjugates with the egg. Development may be slowed down, possibly even suspended, pending the activation of the egg as brought about by the earlier stages of union of the two germ-cells. Development then goes on as a continuous series of reactions, up to a certain point, difficult to ascertain but nevertheless actual, when the progressive differentiations in both body and mind, if one were to consider higher animals, come to an end. Develop- ment may be regarded as completed when progressive differentiation ceases and regressive changes set in. THE SPERMATOZOON AND FERTILIZATION. For purposes of convenience in study and analysis, development is often considered as a series of definitely limited, consecutive stages rather than as a continuous process. Indeed, the course of development seems to fall rather naturally into periods, the beginning of each of these standing out as a critical point in the life of the organism. One of these periods begins with the activation of the egg to its development as a multicellular organism. Lillie (1919, p. 129) says concerning fertilization: ‘It is a series of reactions which can not be regarded as complete until full capacity for development and inheritance is attained by the zygote.” In his discussion of the physiology of the spermatozoon, Lillie distinguishes between cortical block to the fertilization reaction and internal block to later stages in fertilization. From my study of hybrid material I should like to go a little further in the characterization of stages, defining cortical block, as Lillie has done, as the block to the cortical reaction, but limiting the use of the term internal block to the period during which conditions which prevent the union of sperm and egg nuclei are effective, and adding developmental block as a designation of the block that may become evident after a success- ful activation of the egg and the union of the two germ nuclei. ‘This block to development may operate at an earlier or a later period, Hybridization of Echinoids. £7 and we might distinguish between early developmental block and late developmental block. In the material described in this paper, and in much material of a similar nature, normal development stops at the beginning of gastrulation, even though there has been no evident failure in any of the processes of development up to that time. It has been found possible to overcome cortical block by various preparatory treatments of the egg, and some success has been won in the search for methods of eliminating internal block, but little has been accomplished in the way of devising corrective methods for block of the later period. Godlewski’s (1911) method of treatment of Spherechinus eggs after insemination with Chetopterus sperm is an excellent example of success in the elimination of internal block. Godlewski found it possible to activate the eggs with the foreign sperm. ‘The fertilization membrane was formed, but no further development followed. After determining that development did not follow when unfertilized eggs were subjected to a short treatment with hypertonic sea-water, he found that if the eggs were first inseminated with the foreign sperm and then subjected to a treatment of 22 to 25 minutes with hyper- tonic sea-water, the sperm and egg nuclei united and the eggs con- tinued to the pluteus stage. Following this union of the nuclei, the paternal chromatin was eliminated and the later development was in effect parthenogenetic. A similar method of treatment was not successful with Spherechinus eggs inseminated with Dentalium sperm. Cross-ACTIVATION VERSUS CROSS-FERTILIZATION. Among investigators in the field of experimental hybridization, there has long been dissatisfaction with the term ‘‘cross-fertilization,”’ for the reason that it does not always describe the nature of the result obtained. The term may or may not be correctly applied. The phrase ‘“‘attempt at cross-fertilization’? does not remove the difficulty. If one speaks of successful cross-fertilization, the difficulty is even greater. In one paper (1911) I arbitrarily designated as ‘‘successful”’ those crosses that gave swimming gastrule. That was for the purpose of ruling out all cases which resulted in the develop- ment of irregular, formless masses of cells. Yet ‘‘successful fertiliza- tion’’ can not be a relative term. ‘The only test of success in fertili- zation is in the production of fertile offspring. This might be called the eugenic test. If we regard fertilization as the cortical reaction of the egg to contact with the spermatozoon, cross-fertilization would follow many inseminations, and the term ‘‘cross-fertilization,”’ as used com- monly in the biological literature of the present time, would not be incorrect. 18 Hybridization of Echinoids. If we define fertilization as the process of conjugation of egg and sperm nuclei, and stop there without further qualification, cases in which there is only a temporary fusion of egg and sperm nuclei, fol- lowed by the rejection of the paternal chromatin, might be accepted as examples of fertilization. If, however, we follow this definition to its logical conclusion and insist further that fertilization ‘‘can not be regarded as complete until full capacity for development and inheritance is attained by the zygote,”’ or if we define fertilization as ‘‘the permanent fusion of two germ-cells, one of paternal and one of maternal origin” (Wilson, 1902), and give to this the necessary physiological qualification, there need be little difficulty in restricting the use of the term cross-fertilization to its proper signification. The real difficulty does not lie in the use of the word “‘cross,’”’ but in the use of the word ‘‘fertilization.”” Suppose that in an attempted cross we get nothing more than activation. The eggs have then been ‘“‘eross”’ activated. The sperm nucleus having entered the egg, internal block may inter- vene to prevent its union with the egg nucleus, yet the egg may develop thelykaryotically. Or suppose that no internal block to the union of the nuclei exists, yet in succeeding stages some deviation from a successful course of development occurs, such as the elimination of chromosomes in the first or any succeeding cleavage, or a complete developmental block at the time of beginning gastrulation; in none of the cases has the egg been fertilized. It has been cross-activated, and in the cases mentioned three different results have followed this activation. On the other hand, cross-activation may be followed by a series of reactions whose sum total is perfect fertilization, judged by the standard of attainment of full capacity for development and inheritance. For these different results the terms suggested by Giinther Hertwig (1918), with a slight addition that I shall suggest, will be found useful. CLASSIFICATION OF Hysrips. G. Hertwig classifies hybrids as true hybrids or orthonothi and false hybrids or pseudonothi. The true hybrids contain the full complement of maternal and paternal chromatin, while the false hybrids contain the maternal chromatin only, their development being therefore parthenogenetic. The true hybrids may be divided further into fertile individuals, sterile individuals, and misformed, pathological, non-viable individuals. The fertile and sterile indi- viduals correspond to Poll’s (1920 and earlier papers) tokonothi and steironothi. For the misformed individuals Hertwig suggests the name dysnothi. The false hybrids are of two types, those with haploid nuclei and those with diploid nuclei, those of the second type differing from the ee Hybridization of Echinoids. 19 first in the fact that their chromosomes have doubled in numbers by reason of a monaster division. This classification does not provide a place for individuals that have been obtained by cross-activation, and that have lost some, but not all, of their paternal chromosomes by a process of elimination. It is evident that such forms are not true hybrids, for they do not contain all of the maternal and paternal chromosomes; neither can they be regarded as false hybrids, since they contain some paternal chromosomes. For these individuals I suggest the name partial hybrids. It is conceivable that partial hybrids might be fertile, sterile, or misformed and non-viable. Evidence on these points is incomplete. Our classification might then be: mM 1. True hybrids, all chromosomes retained: a, fertile individuals; b, sterile individuals; c, misformed, non-viable individuals. 2. Partial hybrids, partial elimination of paternal chromosomes: a, fertile individuals; b, sterile individuals; c, misformed, non-viable individuals. 3. False hybrids, maternal chromatin only retained; parthenogenetic: a, haploid nuclei; b, diploid nuclei. Some of the misformed, non-viable, true, and partial hybrids, although they do not produce offspring, are potentially fertile, just as are the young, fertile, true hybrids that die before the age of maturity. In both cases failure to produce offspring may be re- garded as accidental. The group of misformed, non-viable indi- viduals in each class will contain sterile forms: The whole matter may be stated in another way. It is only by the application of a performance test and by cytological examination that we can determine whether true cross-fertilization has taken place. The performance test lies in the production of fertile offspring. Cytological examination alone will enable us to determine whether any of the chromosomes have been eliminated. If only the maternal chromatin is retained, the individuals obtained from the cross- activated eggs will be false hybrids (pseudonothi). If both sets of chromosomes are retained, we shall have true hybrids (orthonothi), but if these hybrids should be sterile, sterile because of an inability to produce ripe germ-cells, these hybrids can not be regarded as having been formed from ‘‘fertilized”’ eggs. The egg was activated, no internal block prevented the union of the germ nuclei, developmental block did not occur until the time of expected maturity, but full capacity for development and inheritance was not attained. Successful cross-activation lies in demonstrable cortical reactions in the egg. Granting that these reactions have taken place, and that internal block or early developmental blocks have been overcome, the cross-activated egg may give rise to sterile, or to misformed, non-viable, true hybrids. Poll (1920) points out the fact that 20) Hybridization of Echinoids. tokonothi may, by accident, be sterile, their sterility, however, not being due to a lack of ability to produce mature germ-cells. Successful cross-activation may be followed by the fusion of the germ nuclei, but because of a partial developmental block some paternal chromosomes may be eliminated. Partial hybrids will be the result. Judged by the standard of full capacity for inheritance and development, and tested by cytological examination, even fertile partial hybrids fail to meet the fertilization test. Finally, successful cross-activation may be followed by internal block to the fusion of the germ nuclei, and the paternal chromatin may be eliminated, but developmental block may fail to intervene to prevent development. The result of this activation will be the production of false hybrids. These hybrids, again, although they may be fertile, can not be said to have developed from fertilized eggs, since they have not the full capacity for development and inheri- tance nor have all of the chromosomes been retained. PART II. THE CYTOLOGY OF THE EGGS OF CIDARIS TRIBULOIDES AND OF CROSS-ACTIVATED CIDARIS EGGS. STRUCTURE OF THE CIDARIS Ecc. The living eggs of Cidaris are about 0.07 mm. in diameter. They are very transparent and the various phases of the nucleus during division may be followed readily. Because of the transparency of the living egg, I was much surprised to find that in the stained sections the cytoplasm was filled with deeply staining spherules (plate 1, a to E; plate 2,a ands). As this seemed at first a somewhat unusual feature in a sea-urchin egg, sea-urchin eggs often being described as alecithal, a somewhat prolonged study of these spherules has been made. My conclusions concerning them have been reached in part indirectly, through the examination of other sea-urchin eggs which were available for study by the aid of various micro-chemical methods. My own study has been based on the eggs of Arbacia punctulata, Echinometra mathei, Peronella lesuert, and Salmacis alexandri, while that of one of my students, Dr. Hope Hibbard, has been based on HEchinarachnius parma. The spherules in Cidaris measure from 0.2 to 2 microns in diameter. With iron hematoxylin they stain intensely black. During the resting stage of the nucleus the spherules are scattered uniformly through the cytoplasm from nuclear wall to the surface of the egg, the surface layer being filled with deeply staining microsomes. With the transition from the ‘‘resting stage” to the active phases of cell life as exhibited in mitosis—i. e., with the passage from the gel to the sol phase in the protoplasm—the spherules are carried out from the region of the nucleus, so that when the amphiaster is established it lies in a region of clear cytoplasm, one entirely free from the presence of the spherules described (plate 1, fig. a). Some eggs are characterized by a smaller number of large spherules, other eggs by a larger number of smaller spherules. After the elimination of the possible explanation that the difference in size might be due to different degrees of extraction of the stain, an attempt was made to show a correlation between size of spherules and phase of division, a preliminary examination having shown that most of the eggs in the anaphase of the first division had large spherules. It was soon found that such a generalization would be without adequate basis in fact, since numerous exceptions to the supposed condition were found (plate 2, figs. p to L), and it may be stated positively that there is no correlation between the primary size of these deutoplasmic spherules and phase of division. As the 21 22 Hybridization of Echinoids. cleavage of the egg proceeds, there is a gradual diminution in size of the spherules, until in the gastrula all trace of them has been lost. My conclusion regarding these spherules is that they are droplets of fat. In all of the sea-urchin eggs that I have been able to study by adequate methods I have found that in the clear protoplasmic matrix there are included, in addition to the so-called active inclu- sions, two types of inert bodies, fat droplets and yolk plates. The method of fixation used in preparing these Cidaris eggs for study is not adequate for the demonstration of the yolk plates, nor is it adequate for the demonstration of the fatty nature of the spherules described. These spherules, however, are in the nature of droplets, not platelets, and their distribution is similar to that of undoubted fat droplets in other sea-urchin eggs. Before making these supple- mentary studies I was inclined to the noncommittal description of these bodies as deutoplasmic spherules, but that, I now believe, would be in the nature of a rather unnecessary circumlocution. The Cidaris egg is, as I have pointed out, unusual among echinoid eggs. A study of its protoplasm by modern cytological methods should prove of more than usual interest. My material was pre- pared for the study of chromosomes and has been satisfactory for that purpose. It is wholly inadequate for the study of cytoplasmic inclusions. The fat droplets and yolk are shifted about readily in the proto- plasm while this is in a sol phase. I have already described the movement of the droplets away from the nucleus during the early stages of division. A movement in the opposite direction begins as the egg passes from the anaphases to the telophases of division. While the chromosomes are developing into the usual chromosomal vesicles (plate 1, figs. c and b; plate 2, figs. F and aq; fig. 25), there is a centripetal movement of the fat droplets, so that with the re- formation of the nucleus these again lie distributed uniformly between the wall of the nucleus and the cell wall. In some eggs, prior to the appearance of any constriction of the surface of the egg in division, there is a noticeable pressing together of spindle fibers along the line of future cleavage in the cytoplasm, and a very definite impression of internal cleavage is given (fig. B of plate 1). The microsomes visible in the surface of the egg, as shown in figures A and B, plate 1, are of a nature differing from that of the fat droplets. They destain much more readily, and it will be noted that they are not shown in any of the remaining sections of the entire eggs. It will also be noted that these are the only sections of straight-fertilized Cidaris eggs shown, and that the other sections are of cross-activated eggs. I have considered very carefully the question of their abundance in one class of eggs and their scarcity in Hybridization of Echinoids. 23 another, and feel that the facts confirm the statement I have made. As to the nature of these granules, two possible explanations suggest themselves: Either they may be bodies containing material which enters into the cortical reaction alone in fertilization, in which case it would seem that there must have been a more complete reaction in the cross-activation than in direct activation, or (and this seems the more probable explanation) they contain the sperm-agglutinating substance described by Lillie, and a large part of this has been given up by the eggs during their two hours staling in sea-water before insemination. It should be a matter of little difficulty, if suitable material were available, to test the latter explanation by determining whether there is a progressive diminution in number of superficial granules in unfertilized eggs allowed to stand for some hours in sea-water. A positive proof of this point would not preclude the idea that their substance is also concerned in the reaction of the egg to the spermatozoon. StuDY OF CHROMOSOMES IN SPECIES-FERTILIZED CipAris Eaas. The study of chromosomes both in the straight-fertilized and in the cross-activated eggs has been prolonged and has been based upon an abundance of material. Some evidence which has been of value has been obtained by the study of eggs in which development was initiated by Loeb’s butyric-acid method. The chromosomes in the straight-fertilized Cidaris eggs are crowded closely together; they are small and difficult to count. My study of the morphology of the chromosome groups has been mainly of sections showing polar views or of lateral views of the division figure in anaphase. In both types of section it is usually very difficult to distinguish between a fragment of a chromosome and an entire chromosome. In sections 5 microns in thickness, which pass through the spindle in the direction of its long axis, chromosomes will be found usually in three successive sections. Should the plane of the section be slightly oblique to the long axis of the spindle, there may still be chromosomes in three successive sections of the egg, but there will be only two sections of each anaphase plate. When the sections have been of this type I have combined the three sections in two figures, as in figures 7a and 7b, the latter containing the chromo- somes from the first and third sections of the series. In such figures as this, therefore, sister chromosomes do not stand opposite each other in the anaphase plates as shown. The danger of overcounting the number of chromosomes in the longitudinal sections of the division figures because of the sectioning of individual chromosomes is probably offset by the fact that some of the chromosomes lie directly under one another and on this account may be overlooked, even in the most careful focusing. 24 Hybridization of Echinoids. From all the evidence obtainable I have concluded that the number of chromosomes present in these eggs is 37 and 38. This difference is due to the fact that half of the eggs contain a single V-shaped chro- mosome, this shape being due to the atelomitic attachment of spindle fibers, and the remaining half a pair of these V-shaped elements. DX) RUSS ic 5 ny Vy dppf AN AMWAT A at 6 ra MN) Fic. 6, a,b, andc. Three successive sections of a first-cleavage am- phiaster, 35-41. Two heterochromosomes. Figs. 6 to 11 are of anaphase plates from Cidaris X Cidaris eggs. The figures were drawn with the aid of a Zeiss compensating ocular 12 and 2 mm. oil-immer- sion objective. The camera sketches were enlarged 2 diameters and then compared with the sections and finished. These enlarged drawings have been reduced one-half in reproduction, so that the magnification of the chromosomes in the figures described is about 2,400 diameters. I) CUA uit ( yn hie Fic. 7,aand b. Plane of section was slightly oblique to long axis of spindle. Two sections have been combined in b. On theslide, the upper part of b appears in one section, then a as drawn, then the lower part of b. Two heterochromosomes, 33-34. In most cases the arms of the V are brought so closely together, during their movement toward the poles of the spindles, that the chromo- some has the appearance of a rod of twice the thickness of the remain- ing chromosomes. In one anaphase (fig. 1la) there is a single long chromosome; no V is present. Hybridization of Echinoids. Zo During division the yolk and oil droplets seem forcibly pushed out of the region of the amphiaster, which lies in a region of clear cyto- plasm. As the cell passes into the telophases of division the yolk and oil droplets are carried inward and again lie distributed uniformly through the cell. \w \ Miah \\ JMn\y 1" \Wiy yl Fig. 8,a, b, and c. Three successive sections of a first-cleavage amphiaster. One heterotypic chromosome, 32-82. y! | \ TTL AN, \V 1 WAN) What Cc Fic. 9, a,b, and c. Three successive sections of a first-cleavage amphiaster. Two heterochromosomes. One chromosome aeided and lagging at the center. In the anaphase plates 32-32. The chromosomes divide and move apart with great regularity; lagging chromosomes are seen only rarely (figs. 9a and 9b). Figures 6, 7, and 9 are of anaphase plates showing two of the double-armed rods in each plate of chromosomes; in figures 8 and 10 but one of these chromosomes of double width could be found. In figure 1la only one section of the spindle is shown; in this egg no chromosomes of double width could be found; there is, instead, a chromosome of 26 Hybridization of Echinoids. unusual length, which may be regarded as the heterochromosome, the probable explanation being that the attachment of the spindle fiber was telomitic rather than atelomitic. In figures 11, b, c, d, and e, are shown polar views of anaphase plates; b, c, d are of the first division; e is one of the daughter plates of the second division. The chromosomes labeled h are those which I have YA yin 10 RY ly) 7 Meh eit Waxy \wa\ Fic. 10,a,b, and c. Three successive sections of a first-cleavage am- phiaster. One heterochromosome, 36-88. h ({ SP ee iiaagre « ( \ \ = he mae oe a ~~ 4\\% acer “ h 2 b Eh val e eh Wh hy te : Deve ‘ a se:° ° : oe xs erst eof ete See ie % PRO on See A) &e%h a ‘*h ’ c e Fia. 11a, A single section from an amphiaster in which the chromo- somes were much elongated. The extreme length reached by one of the chromosomes in each daughter plate may be seen readily. 11b, Polar view anaphase, first division 37 chromosomes. 11c, Polar view anaphase, first division 38 chromosomes. 11d, Polar view anaphase, first division 37 chromosomes. 1le, Polar view anaphase, second division 38 chromosomes. interpreted as the heterochromosomes, while those which are sepa- rated slightly from each other I have regarded as autosomes. Refer- ence to the figures of the lateral views of anaphase plates shows that in every plate there are several chromosomes which lie rather closely applied to each other. The reason for the presence of several slightly elongated chromosomes in each of these polar views will be seen readily when one visualizes the polar views of such sections as 8a. It will be seen at once that these, and similarly oriented chromosomes Hybridization of Echinoids. 2¢ in other plates, would appear in the polar view as short rods because of their oblique position. With this interpretation the number of chromosomes in these plates is either 37 or 38. The final proof of these specific numbers of chromosomes in Cidaris was gained by a study of the eggs activated artificially. Unfor- tunately, in the material that I have studied I have been unable to find any amphiasters. The figures that show well are all of mon- aster plates. The most favorable of these is shown in figure 28. The chromosomes have all divided longitudinally on the monaster. Nineteen such divided chromosomes were present in one section, the remaining section of the egg containing no additional chromo- somes. In this plate Iam unable to designate the heterochromosomes with any degree of certainty; it may be one of either of the two rounded elements in the lower right-hand corner of the plate. The U- or V-shaped chromosomes may, however, be seen in most plates, as in figures 26 and 27a. Figure 26 was drawn from two sections. With the exception of two chromosomes, all were in one section. The total number of chromosomes here also was 19. In all sections in which the count could be made with certainty, 19 was the number of chromosomes present. Such figures as 27a and 276 are inconclusive as to numerical count, because of the evident fragmentation of the chromosomes in sectioning. CHROMOSOMES IN Ciparis Eccs Cross-ACTIVATED BY LYTECHINUS SPERM. Cidaris 9? X Lytechinus o. Figures 12 to 17. A comparison of figures 6 to 11 with figures 12 to 17 shows a general similarity in the character of the anaphase plates, but it also reveals some rather striking differences. As a rule, division seems to be rather regular, but even in the most regular figures some evi- dence of lagging in the separation of the chromosomes is evident (figs. 12 to 15). So far as the morphology of the chromosome groups is concerned, I have found that the eggs may be divided into two groups: one group in which each anaphase plate contains two of the V-shaped or broader chromosomes, and one group in which each anaphase plate contains three of these heterochromosomes. In the preceding section evidence is presented that the Cidaris egg contains one of these heterochromosomes. It is therefore evident that the Lytechinus sperms are dimorphic, carrying either one or two V-shaped heterochromosomes. Figures 12, a and b, show two chro- mosomes of double width; figures 13, a and b, show three such hetero- chromosomes in each plate; figures 14, a and 6b, show three; figures 15, a and b, show three such chromosomes in the lower half of the figure and three in the upper half. 28 Hybridization of Echinoids. The evidence here confirms my conclusions reached in 1912, namely, that in Lytechinus (Toxopneustes) the fertilized eggs contained either 3 V’s or 4 V’s, and that this variation in number was due to the fact that the sperms were dimorphic with respect to these chromosomes, carrying either 1 or 2 V’s into the egg. Figs. 12 to 15 are of anaphase plates from Cidaris eggs activated with Lytechinus sperm. They were drawn and enlarged in the manner described for figures 6 to 11. They have been reduced one-half in reproduction. The magnification shown is of about 2,400 diameters. 4 fe Pe a ' h | / if itgy 0 Hh yy a 13 4, A | pi ‘ | / dy NG) lh Ml) i.e Vic. 12, a and b, Cidaris 9 x Lytechinuso&. Two heterochromosomes, 33-35. Fic. 18, a and b, Cidaris? X Lytechinuso’. Three heterochromosomes. Much lagging; 28?-26?. The fact that two heterotypic chromosomes in figure 15) he among the lagging chromosomes, and the knowledge that two of these elements were brought into the egg by the Lylechinus sperm, make it seem probable that the lagging chromosomes are those of Lytechinus. The same type of evidence is given by the sections of the egg of which figure 17 is one. In the half of the spindle shown in figure 17 division is very regular and a V-shaped chromosome may be seen clearly in each plate. In the next section of the egg (not shown in the illustrations), two pairs of lagging V’s lying among a very irregular and much twisted mass of autosomes may be seen, the evidence again lending support to the conclusion that the lagging chromosomes are those of Lytechinus. The number of chromosomes in these plates varies greatly. In figures 12, a and b, the number is 33-35; in figure 13, 28?—26?; in figure 14, 27?-35?; in figure 15, 29-28. The expected number is 38-38 or 37-37. Figures 16, a, b, and c, are of a tetrapolar spindle in anaphase. While spindles of this type are usually associated with double fertili- zation, it is evident in this example that the chromosomes of but one Hybridization of Echinoids. 29 sperm nucleus are involved in division, and that of a sperm carrying but one heterochromosome. The expected number of chromosomes in this case would thus be 19+ 18 = 37; these have all split in division, which would give a total of 74. The number shown in the figure is 80, of which some are evidently fragments. One of the V’s has been cut in sectioning. I am unable to designate its parts. CHROMOSOMES IN C1pARIS Eacs Cross-ACTIVATED BY TRIPNEUSTES SPERM. Cidaris 2 X Tripneustes o. Figures 18 to 25. In general, the division figures of this cross are more regular than those of the Cidaris-Lytechinus cross. There are some lagging chromosomes, but these do not form so striking a feature as in the preceding cross. Some of the sections show the heterochromosomes especially well. In figure 18, for example, 3 V-shaped and 1 hook- like chromosome are present; in figure 19, 3 V-shaped chromosomes ify iL ih vl | | ned \ | J / 4 / 15 yap ANIM vy) ip ; ATT Fic. 14, a and 6, Cidaris 9 X& Lytech- inus o’. Three heterochromo- Fia. 15, aand b, Cidaris 9 X Lytechinuso’. Thev- somes. Lagging chromo- shaped form of the heterotypic chromo- somes at center, 27?-35? somes shows clearly 29-28. were present in the egg, but no hook-shaped chromosome was present. These two illustrations represent the two classes of eggs that were present in these cultures. In figures 20, b and c, 3 V’s; in figures 21, a and b, 3 V’s and a hook; in figures 22, a and 6, 3 V’s; in figures 23, a and b, 3 V’s and a hook. The sections from which figures 24, a and b, were drawn are of more than usual interest. Most of the chromosomes appearing in b are longitudinal sections of the chro- mosomes shown in a, as indicated both by position and width of the chromosomes. The third section of this spindle (not shown in the illustrations) contains a much involved mass of chromosomes, which 30 Hybridization of Echinoids. obviously could not have divided normally. The edge of this mass of chromosomes lies between the anaphase plates in figure 24b. Here again the morphology of the groups of chromosomes confirms the expectation based on our knowledge of the chromosomes in Tripneustes (Hipponoé). From the studies of Tennent (1911a) and Pinney (1911) we know that the dimorphism in straight-fertilized eggs of Tripneustes is due to dimorphism of the groups of chromosomes in Tripneustes sperms, the sperms of this species introducing either 2 V’s or 2 V’s and a hook into the egg. i (\ —= Fia. 16, a, b, and c, Cidaris 9 X Lylechinus @. ‘Three successive sections of a tetrapolar spindle 80 chromosomes, inclusive of fragments. X 2,400. The expectation as to number of chromosomes in the cross-activated eggs is 19-+16=35, or 19+17=36. The realization as to the number of chromosomes in the sections drawn is: Figures 20, a, b, and c, 38-35; 21, a and b, 25-34; 22, a and b, 29-28; 23, a and b, 22-26. It is evident that success in division of the chromosomes in the cross-activated egg isrelative. Figures 20, a, b, and c, show anormal division. It is evident that at least two of the rods in the upper anaphase plate are fragments of chromosomes; probably a third is also a fragment. With the rejection of three, the count would be 35-35, the expected number following fertilization by the sperm carrying 2 V’s only. In figure 21, the plane of section is slightly oblique to the long axis of the spindle. I do not feel confident that I have been able to see all of the chromosomes which are massed Hybridization of Echinoids. 3l in the upper plate in figure 21a. In figure 22, 6 or 7 chromosomes have been eliminated; in figure 23, 9 or 10; while in figure 24 indica- tions are that nearly all of the paternal chromosomes were in process of elimination. The amount of chromatin in the Zwischenkorper seems to vary (compare plate 1c with plate 2B), which may mean that a greater { Wan uy ie. a , | lt | ih Mid 4 Ty Fia. 17, Cidaris 9 X Lytechinusc. \/-shaped heterochromosome readily seen. X 2,400. Figures 18 to 24 are of anaphase plates from Cidaris eggs inseminated with T'ripneustes sperm. They were prepared in the manner described for the figures 6 to 11 and reproduced at a magnification of about 2,400 diameters. Fia. 18, Cidaris? xX Tripneustes#. A section selected because it contains all of the heterochromosomes, three V’s and one hook, in each anaphase plate. Fic. 19. Cidaris? xX Tripneustes#. Section showing three V’s in each plate; no hook. ARM “ ie A ly HOY GAN a t \ (\/ i { Wl) a a \ : | i {i ,] Fra. 20, a, b, and c, Cidaris? X Tripneustes a Three V’s, 38-35. amount of eliminated chromatin is included in some bodies than in others. Plate 28 was drawn from a preparation in which the process of destaining had been carried further than in that from which plate lc was drawn. In addition it will be seen that figure B of plate 2 represents a stage of completed division. Figure a of plate 2 shows a2 Hybridization of Echinoids. lagging chromosomes which will be at the line of division of the cel! and which will lie at the position of the Zwischenkorper. alt iy) ANA Sy NUD yan wy WM al Fia. 21, a and b, Cidaris? X Tripneusteso. Two V’s and hook, 25-34. Fia. 22, a and b, Cidaris? X Tripneusteso. Three V’s, 29-28. AG yyy J “Ny Al ML Ming) 710" a a i vs 7 b Me 23 a 24 vA Fia. 23, a and b, Cidaris 9? X Tripneusteso. Two V’s and hook, 22-26. Fic. 24, a and b, Cidaris? X Tripneustes. Early anaphase; the position and width of the chromosomes in b give clear evidence that these are merely sections of those shown ina. Two V’s. ELIMINATION OF CHROMOSOMES. In 1912 I summarized the facts regarding retention or elimination of chromosomes in cross-activated Echinoid eggs as follows: (1) Elimination of no chromosomes and dominance of one species over the other with respect to the character of the skeleton. (2) Elimination of part of the chromosomes and dominance of one species over the other with respect to the character of the skeleton. (3) Elimination of no chromosomes and skeleton of intermediate character. (4) Elimination of part of the chromosomes and skeleton of intermediate character. (5) Elimination of part of both maternal and paternal chromosomes and inhibition of development. Hybridization of Echinoids. 30 In group (2) I included such cases as result from the activations of the Echinoid egg by the sperm of annelids and mollusks (Godlewski, 1911; Kupelwieser, 1906) and the fertilization of eggs which had been given a certain impulse to parthenogenetic development by means of chemicals, which give, as these authors have pointed out, thely- karyotic larve. I stated further that, practically, larvee derived from such crosses inherit from the egg parent alone just as strictly as if the eggs had been caused to develop from the first by artificial chemical fertilization. They are false hybrids. = 25 Sheed A 4 F] a ie » Op bi hi sve" aay it ay * 2A ge sie 28 2 am Fia. 25, Cidaris9? X Tripneusteso’. Median section of three, other two not shown, showing chromosomes becoming transformed to chromosomal vesicles before contraction of chromosome has taken place. X 2,400. Fig. 26, Parthenogenetic Cidaris egg. Prophase of monaster division. X 2,400. Fig. 27, a and b, Parthenogenetic Cidaris egg. Two successive sections of monaster plate. Many elements shown in b are sections of those shown ina. X 2,400. Fia. 28, Parthenogenetic Cidaris egg. Monaster division. All chromosomes have divided; 18 pairs. XX 2,400. These Cidaris-Lytechinus and Cidaris-Tripneustes crosses consid- ered in this paper constitute another group. (6) Elimination of part of the chromosomes, presumably paternal and failure in development. These may be regarded as partial hybrids. Probably several additional crosses that have been made, but which have not received adequate cytological examination, will ultimately be found to belong in this group. In these crosses, development seems to proceed well as long as the generalized type of development, common to each, continues; devel- opmental block occurs when the period of specialized development begins. ‘The egg seems unable to accommodate itself fully to the impulse for a divergent type. 34 Hybridization of Echinoids. ALTERATION IN PuysicaAL CHARACTERISTICS OF THE CYTOPLASM OF THE Eae By ACTION OF FOREIGN SPERMS. In my interpretation of the phenomena exhibited in the develop- ment of Cidaris and its hybrids I have been influenced, to a consider- able extent, by the consideration of facts concerning the transformation of deutoplasmic materials to cytoplasm. The Cidaris egg, despite its transparent character, contains a relatively large amount of yolk and fat. This material gradually disappears as development con- tinues. I have also been guided by results which I have obtained from another inter-ordinal cross, Arbacia X Moira (Tennent, 1920). It is unnecessary to review in this place the facts presented in that paper, and it seems sufficient to say that certain granules and rods, present in sections of cross-activated eggs and absent in sections of straight-fertilized eggs, were explained as coarse precipitates formed as a result of the emission of a foreign enzyme from the nucleus. Even though we were to regard these bodies as artifacts produced by the fixing fluid used (acetic-sublimate)—for we know that certain metallic salts may cause the precipitation of proteids—the fact remains that physical conditions in these eggs must have been differ- ent than in the straight-fertilized eggs which were fixed at corre- sponding stages and in the same fixing fluid, and which show no bodies of this kind. In continuation of the investigation mentioned above, one of my students, Miss Hibbard, has made a study of Tripneustes eggs fertil- ized with Lytechinus sperms, comparing these with the straight- fertilized and parthenogenetic eggs, and has found that in the cross- activated eggs which are developing normally granules appear and later disappear as the nucleus passes into the later phases of division. Here, again, the only explanation seems to lie in the action of the enzyme introduced by the foreign spermatozoon. Miss Hibbard has also determined that some of the cross-activated eggs, which segment less normally, show other and characteristic changes in their cytoplasm. These changes are clearly pathological. The most striking result of the removal of cortical block followed by cross-activation that has come within my experience is that following the insemination of the Lytechinus egg, after preliminary treatment with NaOH or CaCl, with the sperms of Holothuria floridana (Tennent, 1911, p. 140). In this instance the sperm, upon entering, in most cases tears the egg to pieces. A deep notch or pathway made by the sperm may be seen and then the egg suddenly disintegrates. In other cases the entrance of the spermatozoon is followed by a slower, but none the less complete, cytolysis of the egg. Gray (1913) studied the eggs of Echinus acutus fertilized by the sperm of Echinus esculentus and species-fertilized eggs of Hchinus ee Hybridization of Echinoids. 515) aculus treated by hypertonic solutions. The results were similar. There was an elimination of a certain number of chromosomes from the nucleus in both the cross-activated eggs and in the straight-fer- tilized eggs which had been treated with hypertonic solutions. The eggs of Echinus esculentus were not affected by similar treatment. Gray suggests, tentatively, after consideration of the results of McClendon (1913) and R. 8. Lillie (1909, 1911), on increased perme- ability of the egg membrane after the entrance of the spermatozoon, that the degree of change in permeability after fertilization is a fune- tion of the sperm and that the cytological behavior of reciprocal crosses is explicable on this hypothesis. Gray points out that changes in permeability of the cytoplasm as induced by the sperm can not be a sufficient explanation of all abnormalities observed in cross- fertilized eggs. Doncaster and Gray (1913), in considering the probable fact that the vesicles in the eggs of the cross acutus X esculentus are derived from the acutus chromosomes, suggest that the explanation may lie in an alteration of the permeability or osmotic condition of the egg, consequent upon the development within it of a foreiga spermatozoon, i. e., the physical condition of the cytoplasm is altered by the develop- ment within it of a foreign sperm nucleus. NucLearn ENzyYMES AND CYTOPLASMIC SUBSTRATE. The study of a sufficient number of species, both closely related and widely separated, has now given, I believe, an adequate basis for generalization. The egg contains the mechanism necessary for development. It may develop parthenogenetically, giving a maternal larva and adult. In that development there is a harmonious interaction between the nuclear and cytoplasmic material. The results of cross-activation in which there is a complete rejection of paternal nuclear material and production of false hybrids, as in the Echinoid-Mollusk, or Echi- noid-Annelid cross, are closely related to examples of artificial par- thenogenesis. Above this level there are crosses showing maternal or paternal influence in lesser or greater degree. We might pass in review a great number of cases of partial hybridization and reach finally undoubted examples of true hybridization. If we restate these facts on the basis of chromatin content, we find that in false hybrids there is a complete elimination of paternal chromosomes, in partial hybrids a partial elimination of chromosomes, and in true hybrids no elimination of chromosomes. The fact that the results obtained from a given cross differ from those obtained in the reciprocal cross indicates that the differences are not due to lack of harmony between the substances of the germ 36 Hybridization of Echinoids. nuclei. It is clear that cytoplasmic differences must be taken into account. The life of the cell lies in the interaction between nucleus and cytoplasm. ‘The relative importance of each is that of enzyme to substrate. There can not be cytoplasmic control, nor can there be nuclear control, purely as such, for the processes of life lie in the interaction of both. ‘There is nuclear control in that by the intro- duction of enzymes we obtain synthetic products; there is cytoplasmic control in that with a given substrate we provide material from which products are synthesized. From the evidence given by Arbacia-Moira material (Tennent, 1920), it seems clear that the reactions in the cytoplasm caused by foreign sperms are unlike the reactions caused by species sperms. That fact fixes the attention on the nature of the activities of the nucleus. Tue BinucLearity HyporuHesis. Various binuclearity hypotheses, founded in part on Richard Hertwig’s chromidial hypothesis, have influenced interpretations of nuclear phenomena. The nucleus shows two phases, an active and a resting. These two phases, kinetic and interkinetic, have been made to lend themselves to an analogy with a true binucleate con- dition and to an assumption of dichromaticity. Of these two kinds of chromatin, one is supposed to be propagatory (idiochromatin), in evidence at the time of cell division; the other trophic (tropho- chromatin, somatochromatin), formed by the idiochromatin, but resident in the cytoplasm. The somatic phase of the nucleus covers a period during which it may be assumed that nuclear enzymes have passed from the nucleus into the cytoplasm, and the cytoplasm has become the seat of synthetic activities. The nucleus at this time is in a ‘‘resting’’ condition; it seems comparatively empty, is acido- phile, and basophilic bodies may be found in the cytoplasm. Under the influence of the chromidial hypothesis, supposed particles of chromatin in the cytoplasm have been interpreted as chromidia, or as trophochromidia. The basophilic bodies found in cross-activated Arbacia eggs might have been interpreted similarly had there been any evidence that they were emitted from the nucleus. The possi- bility of the emission of large particles is foreign to our conception of the nature of the nuclear membrane. Hybridization of Echidoids. ot DISCUSSION. Masing (1910), selecting for study one of the nuclear constit- uents, nucleic acid, found that while during development in Arbacia pustulosa up to the blastula stage there is an increase of about a thousand fold in nuclear mass, there is no perceptible increase in the nucleic-acid content of the egg. He reached the conclusions that the nucleic acid of the cleavage nuclei arises from a preformed stock in the egg plasma and that nucleic acid and the chromatin of the his- tologists must be different things. (Quoted from Godlewski, 1911.) During the period studied by Masing there has been no increase in the mass of organism. Godlewski (1908) showed that the total volume of plasma in the blastula is about one-third less than in the unsegmented egg. The nuclear material has increased in volume at the expense of the cytoplasm. Godlewski’s investigations show a change in ratio between cytoplasm and nucleus from 550:1 in the unfertilized ege to 6:1 in the blastula. In the light of the results of Conklin (1912) on the growth of protoplasm during cleavage, and in the light of my own results from a study of the eggs of Arbacia punctulata, as yet unpublished, and of those of Hibbard on the eggs of Hchinarachnius parma, in press, these figures need some revision. This revision will not affect Godlewski’s general conclusions. The unpublished results mentioned above show that in the eggs of Arbacia punctulata and Echinarachnius parma there is a considerable mass of deutoplasmie material, which is in process of transformation to cytoplasm during the cleavage stages. In the echinoderm egg, despite its frequent transparent character, there is a stock of yolk and fat. Cell volume in these eggs is not equivalent to cytoplasmic volume. The growth in nuclear mass is due, in part at least, to the assimilation of deutoplasm. Marcus (1906) and Erdmann (1908) have shown that the size of the chromosomes diminishes during cleavage. Erdmann has concluded that the chromosomes of the pluteus of Strongylocentrotus have only about one-fortieth the volume of those of the first cleavage spindle. She has also determined that there is a constant increase in total quantity of chromatin. Her figures show that at the stage of the blastula without mesenchyme, which Godlewski has estimated as a 1,256-cell stage, the chromosomal volume is 78 times greater than it was at the time of the first division. This would amount to an increase of about 6 per cent with the division of each cell. Godlewski (1908) estimated that the total volume of nuclear material is 47 times greater in the 1,256-cell stage than in the unfertilized egg, which would be 23.5 times the total volume in the fertilized egg. Inasmuch as he believed that practically all of this growth took place between the 1-cell stage and the 64-cell stage, and that but little increase of 38 Hybridization of Echinoids. nuclear mass occurred between the 64-cell stage and the blastula stage, this would mean an increase of about 37 per cent with the division of each cell. Godlewski speaks of the increase as being nearly in a geometric ratio, this calculation being based on the volume of nuclear mass in the unfertilized egg. It should be noted that Erdmann’s figures are of volume of chromosomes and that Godlew- ski’s are of nuclear volume. Conklin (1912) has estimated that in Crepidula the chromosomal mass grows at the rate of 8 per cent for each division up to the 32-cell stage. Baltzer (1909) has indicated the large probable error in estimates of total chromatin volume that are based on the assumption that the chromosomes in an Echinoid egg are of equal size. Mathews believes that nucleic acid is the chromatin of the his- tologists. We have seen that Masing found no evidence of increase of nucleic acid during development in Arbacia up to the blastula stage. The evidence of Conklin, Erdmann, and Godlewski is con- clusive in its demonstration of increase of total volume of chromo- somes during this period. If there has been no increase in the amount of nucleic acid, there must have been increase in its basic protein portion. Goldschmidt’s (1917) idea that the chromosomes act as adsorbents of enzymes which constitute the chemical basis of heredity finds much to support it in the facts demonstrated in this paper. We are able to show the presence of nucleic acid by our stains; we have no means of making the nuclear enzymes visible. Our only test for them is in the things that they do. We can not hope to demonstrate consecutive stages in processes of synthesis by specific stains. We can demonstrate, and the material discussed in this paper has demonstrated, the fact that foreign nuclear material produces reac- tions in the cytoplasm that do not occur in straight-fertilized eggs. Closeness of relationship is by no means indicative of the readiness with which the initial impulse to development may be received, nor a sure criterion of the extent to which it may proceed. In the Cidaris- Lytechinus and Cidaris-Tripneustes crosses under consideration there seems to be little cortical block to the entrance of the spermatozoon. There is little internal block during early development. Develop- ment proceeds regularly to the period immediately before gastrula- tion. ‘To this point it has been following the general path of develop- ment taken by most Echinoderms. At the point of deviation of special from general, abnormalities appear. Development ends in the gastrula stage, as in many crosses between Echinoderms. ‘The cells which should have gone to the completion of the archenteron and the prospective mesenchyme cells are the first to die. Sections show the nuclei to be swollen with chromatin. The nuclei finally burst, extruding irregular masses of chromatin into the cytoplasm. Hybridization of Echinoids. 39 Developmental block in this case seems to be associated with what might be described as nuclear indigestion. Fulton (1921, p. 167), in describing the result of continued stimulation of a muscle on its nucleus, says: ‘‘With continued stimulation the nucleus must liter- ally become overwhelmed with material to be oxidized; as a result, it would become fatigued, and would no longer be capable of perform- ing its normal functions.” The word “overwhelmed” describes vividly the conditions in- volving the nucleus in these hybrids. From the Arbacia-Moira material, the evidence obtained was interpreted as showing that enzymes are emitted through the nuclear membrane and that changes occur in the protoplasm following this emission. ‘The materials formed pass back to and are taken into the nucleus. ‘There is no evidence that the emitted material is chromatin. There is nothing in these views that detracts from the chromosomal theory of inheritance. It is evident that what is transmitted from generation to generation is a course of development—what Brooks in “The Foundations of Zoology” calls a “‘capacity for nurture.” The results of these cross-activations show that an orderly series of developmental reactions may be disorganized by the introduction of foreign nuclear material. In the cross-activated eggs, in the inter- action between nucleus and cytoplasm, the nucleus seems to be pro- vided with more material than it can handle. In straight-fertilization the accumulation of product would have checked the action of enzymes before that condition arose. The foreign enzymes are specifically different and their action is not checked until a greater than normal supply for this egg has accumulated. Even though the nucleus may take care of products formed, development may come to an end because of an actual exhaustion of substrate. AQ Hybridization of Echinoids. SUMMARY. Part I. 1. Cidaris tribuloides is a primitive Echinoid whose early develop- ment is less modified than that of the more modern Echinoids. 2. In its normal development it is of interest (1) because of the slowness of its development when compared with Lylechinus and Trip- neustes, (2) in the difference in site of the formation of its mesenchyme, (3) in the place of appearance of the larval skeleton, and (4) in the form of the larva. 3. The eggs of Cidaris are activated easily by the sperms of Lytechinus or Tripneustes. These give interordinal crosses. 4. The larve obtained from such cross-activations die in the gastrula stage. 5. Cross-activation with these foreign sperms causes mesenchyme cells to pass into the blastoccele before the beginning of gastrulation, while in the species-fertilized eggs immigration of mesenchyme cells does not occur until after the archenteron has been formed. 6. It is suggested that the use of the term internal block be limited to block, or inhibition of the conjugation of the germ nuclei, and that the term developmental block be used in designating later inhi- bitions of development. 7. The use of a modification of the Giinther Hertwig classification of hybrids is suggested. Parr II, 1. The eggs of Cidaris, although transparent, are rich in fat and yolk. 2. A study of species-fertilized eggs shows that half of the eggs contain 37 chromosomes and half 38. Eggs of the group containing 37 chromosomes have one V-shaped element, while those of the group containing 38 chromosomes have two V-shaped elements. 3. Parthenogenetic eggs have 19 chromosomes. 4. Cidaris sperms are dimorphic, half containing a V-shaped chromosome, half being without this element. Half of the sperma- tozoa contain 19 chromosomes, half contain 18. 5. Some of the paternal chromosomes lag in division and are eliminated during cleavage in cross-activated eggs. 6. Investigations on Lytechinus-Tripneustes and Arbacia-Moira material show alteration in the physical characteristics of the cyto- plasm of the egg by action of the foreign sperm. 7. The phenomena exhibited suggest that the effects are due to the action of foreign enzymes on the cytoplasmic substrate. 8. The nucleus in cross-activated Cidaris eggs seems to be supplied with more material than it can utilize. a es ee lel eT io Hybridization of Echineids. 4] 9. The facts suggest an emission of enzymes rather than an emis- sion of chromatin. 10. The results show that in the cross-activated Cidaris eggs an orderly series of developmental reactions is disorganized by the foreign nuclear material at the time when divergence of two systems of development occurs. 11. Through its organization and in its capacity as substrate the egg fixes the course of development. CONCLUSIONS. The consideration of the facts established during the study of the material which has formed the basis of this paper gives a deep impres- sion of the fundamental fact of organization. It gives additional proof of the existence of an underlying basis for development, which we may be able to distort, but which we are not yet able to shape at will. The normal development of the Cidaris egg is of a less-specialized type than that of the eggs of the species whose sperms were used in the cross-activations. As long as the two courses of development lie parallel, we say that development is normal. When the point of divergence between the two paths is reached, characters appear which we call aberrant. Differentiation lies in a series of reactions between nucleus and cytoplasm. In attempting to superimpose a specialized on a non-specialized type of development we fail, because of our lack of ability to harmonize two disharmonious systems of development. The consideration of this material emphasizes again the fact that the thing inherited by offspring from parent is the capacity for development. What that development will be depends on the interactions between nucleus and cytoplasm and on adjustment to environment. The cytoplasm is the material that is shaped during the series of reactions. It is because of the fact that the cytoplasm of the egg is the material basis of the body that Conklin’s statement that the egg cytoplasm ‘‘fixes the general type of development”’ is true. 42 Hybridization of Echinoids. LITERATURE. Bautzer, F, 1909. Die Chromosomen von Strongylocentrotus lividus und Echinus micro- tuberculatus. Arch. f. Zellforsch., Band 2. Cruark, H. L. 1907. The Cidaride. Bull. Mus. Comp. Zool., Harvard Coll., vol. 51, No. 7. 1912. Hawaiian and other Pacific Echini. Mem. Mus. Comp. Zool., Harvard Coll., vol. 34, No. 4. Conky, E.G. 1912. Cell size and nuclear size. Jour. Exp. Zool., vol. 12. 1915. Heredity and environment in the development of men. Princeton Univ. Press. Doncaster, L., and J. Gray. 1913. Cytological observations on the early stages of segmentation of Echinus hybrids. Quart. Jour. Mie. Sci., vol. 58. ErpMANN, Ru. 1908. Experimentelle Untersuchung der Massenverhaltnisse von Plasma, Kern und Chromosomen in dem sich entwickelnden Seeigelei. Arch. f. Zellforsch., Band 2. Futon, Jonn F., sr. 1921. Studies on neuromuscular transmission: I. Amer. Jour. Physiol., vol. 57. Goupscumipt, R. 1917. A further contribution to the theory of sex. Jour. Exp. Zool., vol. 22. Goptewskl, E., yr. 1908. Plasma und Kernsubstanz in der normalen und der durch aussere Faktoren verinderten Entwicklung der Echiniden. Arch. f. Entw. Mech., Band 26. 1911. Studien tber Entwicklungserregung. Arch. f. Entw. Mech., Band 33. Gray, J. 1913. The effects of hypertonic solutions upon the fertilised eggs of Echinus. Quart. Jour. Mic. Scei., vol. 58. Guturie, M. J., and H. Hrpparp. 1919. Cleavage and mesenchyme formation in Toxopneustes variegatus. Biol. Bull., vol. 37. Hertwic, Gintner. 1918. Ireuzungsversuche an Amphibien. Wahre und falsche Bastarde. Arch. Mikr. Anat., Band 91. JACKSON, Ropert Tracy. 1912. Phylogeny of the Echini, with a revision of the Paleozoic species. Mem. Boston Soc. Nat. His., vol. 7. Lituiz, F. R. 1919. Problems of fertilization. Univ. Chicago Press. Linu, R. 8. 1909. The general biological significance of changes in permeability of the surface layer or plasma-membrane of living cells. Biol. Bull., vol. 17. 1911. The physiology of cell division: IV. Jour. Morph., vol. 22. Marcus, H. 1906. Uber die Wirkung der Temperatur auf die Furchung bei Seeigeleiern. Arch. f. Entw. Mech., Band 22. McCuenpon, J. F. 1912. The osmotic and surface tension phenomena of living elements and their physiological significance. Biol. Bull., vol. 22. Mortensen, Tu. 1920. Studies in the development of Crinoids. Papers from Depart- ment of Marine Biology, Carnegie Inst. Wash. Pub. No. 294. 1921. Studies of the development and larval forms of Echinoderms. Published at expense of Carlsberg fund, G. E. C. Gad., Copenhagen. Osuima, H. te On the development of Cucumaria echinata. Quart. Jour. Mic. Sci., vol. 65. Pinney, Epira. 1911. A study of the chromosomes of Hipponoé esculenta and Moira atropos. Biol. Bull., vol. 21. ———. 1918. A study of the relation of the behavior of the chromatin in development and heredity in teleost hybrids. Jour. Morph., vol. 31. Pout, H. 1920. Mischlingsstudien VIII. Arch. f. Mikr. Anat., Band 94. Prouno, H. 1887. Recherches sur le Dorocidaris papillata et quelques autres Echinides de la Méditerranée. Arch. Zool., espér. génér. 2., Sér. V. Tennent, D. H. 1911. Echinoderm hybridization. Papers from Department of Marine Biology, Carnegie Inst. Wash. Pub. No. 1382. ———. 191loe. A heterochromosome of male origin in Echinoids. Biol. Bull., vol. 21. ——. 1912. Studiesin cytology. Jour. Exp. Zool., vol. 12. ——. 1914. The early influence of the spermatozoon upon the characters of Echinoid larve. Papers from Department of Marine Biology, Carnegie Inst. Wash. Pub. No. 182. ; 1920. scaeacenice on the nature of nuclear activity. Proc. Nat. Acad. Sci., vol. 6. PLatTE 1 Fig. Fia. Fia. Fic. Fia. PLATE 2. Fig. Fic. Fic. Fic. Fia. Fic. Fie ILLUSTRATIONS. . All of the figures on this plate were drawn with a Zeiss compensating ocular 8 and 2-mm. immersion objective, giving a magnification, when drawn with a camera lucida on an inclined drawing-board slightly above table level, of about 1,800 diameters. These figures have been reduced one-half in reproduction. A, Cidaris X Cidaris. Anaphase first cleavage. B, Cidaris X Cidaris. Later anaphase first cleavage. c, Cidaris 9 X Tripneustess. 'Telophase first cleavage. Zwischenkorper at line of contact between cells. D, Cidaris 2? X Tripneustes co. Late telophase second cleavage. E, Cidaris9 X Tripneustesc’. Earlier telophase first cleavage. Magnification and reduction as in plate 1. A, Cidaris 9 X Tripneusteso. Anaphase second cleavage; lagging chromosomes at center. B, Cidaris 9 X Tripneusteso. Second cleavage completed; Zwischenkorper. c, Cidaris X Cidaris. Prophase of mitosis; fat droplets small and have been moved out from region of nucleus. D, Cae x Cidaris. Late prophase of mitosis; region of asters freed from fat roplets. E, Cidaris9 X Lytechinuso’. Large fat droplets surrounding nucleus. F, CidarisX Cidaris. Late telophase; chromosomal vesicles; small fat droplets moving in. . G, Cidaris X Cidaris. Chromosomal vesicle; large fat droplets. Fics. u to L, Cidaris X Cidaris. Small area of cytoplasm from one side of the amphi- Fig Fic Fic Fic Fic aster during anaphase of mitosis, showing variation in size and relative abun- dance of droplets. . H, Clear area surrounding each droplet. . 1, Cytoplasm with a few large droplets. . J, Cytoplasm with both larger and smaller droplets. . K, Cytoplasm with numerous small droplets only. . L, Cytoplasm with numerous large droplets. PuaTE 3. In all of the detailed figures on this plate only the archenteron and a small portion of the posterior end of the gastrula are shown. The relation of the part shown to the entire gastrula may be seen by reference to figure g. All figures except figure J were drawn at a magnification of 1,900 diameters. They have been reduced one-half in reproduction. . A, Cidaris 9 X Lytechinusc. Blastula, 18 hours. One cell in blastoccele; cells in wall seem to be in process of withdrawal. . B, Cidaris2 X Lytechinus#. Gastrula, 23 hours. Seven cells in blastoccele. :. C, Cidaris9 X Lytechinuso. Gastrula, 24 hours. . D, CidarisQ xX Lytechinus@. Gastrula, 24 hours. 3. E, CidarisQ X Lytechinus@. Gastrula, 40 hours. . F, Cidaris X Cidaris. Blastula, 18 hours. . G, Cidaris X Cidaris. Gastrula, 18 hours. . H, Cidaris X Cidaris. Gastrula, 23 hours; 2 mesenchyme cells are in process of withdrawal from wall of archenteron. .1, Cidaris X Cidaris. Gastrula, 24 hours; 5 mesenchyme cells in blastoccele at plane of section. . J, Cidaris gastrula, 23 hours; X 180. Camera sketch of living gastrula; 3 mesen- chyme cells in the blastoccele. PLATE 1 TENNENT ribet eo % o * > ot Sey ie — me + TENNENT PLATE 2 PLATE 3 TENNENT HT. THE PRODUCTION OF LIGHT BY THE FISHES PHOTOBLEPHARON AND ANOMALOPS. By EK. NEWTON HARVEY, Princeton University. eee lad ~ THE PRODUCTION OF LIGHT BY THE FISHES PHOTOBLEPHARON AND ANOMALOPS. By E. Newron Harvey. GENERAL ACCOUNT OF THE FISHES. In the Banda Islands, southeast of Amboina, the Moluccas, Dutch Kast Indies, occur two luminous fish with relatively very large luminous organs. One, Photoblepharon palpebratus, called ikan (= fish) leweri (= ?) batu (= stone) by the natives, is known only from this general region, and was first described by Boddaert (1781) from a specimen obtained at Amboina. It is caught with hand-nets in shallow water, swimming singly or few together, among the stones or corals. The other, Anomalops katoptron, called ikan leweri ajer (= water) or laut (= sea) by the natives, occurs also, although rarely, at Amboina, Menado (North Celebes), Fiji, New Hebrides, and the Paumotus. It was described by Bleeker in 1858. Unlike Photoblepharon, it swims in schools of 100 or more near the surface, in somewhat deeper water. In no other place in the world can these fish be caught so easily or in such numbers as at Banda. During a trip to this region under the auspices of the Department of Marine Biology of the Carnegie Institution of Washington, in the autumn of 1920, I was able to obtain all the material needed for my investigation without encountering any more difficulty than the general indisposition of the fishermen to work beyond their usual amount, or the advent of rough weather. In dealing with the fish- ermen, my friend Sech Ahmed bin Said Baadilla acted as interpreter, and it gives me great pleasure to acknowledge his kindness during my stay at Banda Neira. I am also very greatly indebted to Dr. A. L. J. Sunier, director of the Laboratorium voor het Onderzoek der Zee, at Batavia, whose kind hospitality, letters of introduction, and interest in my undertaking made my stay in Netherlands India a pleasure and a success. Through his efforts also I was able to study luminous forms of the Java Sea on the Dutch government’s fisheries steamer Brak. I take this opportunity of expressing my thanks to the Dutch officials for their courtesy. These luminous fish of Banda possess more or less commercial value to the fishermen. Both species are caught at night by the natives and used as bait for other larger fish. The luminous organ is cut out and placed on a hook, the light, which is said to last a night, serving as a lure for other fish. From my own experience in the 45 46 The Production of Light by the laboratory the light from both species lasts between 7 and 8% hours. While the fish occur the year around, they are most easily obtained in October-November and April-May, during the change of monsoon, when the weather is calm. They differ in this respect from the lumi- nous squid (Watasenia scintillans) of Japan, which come in toward shore to spawn at only certain seasons of the year and then return to the deeper water. Although the Banda Sea about these islands is very deep (12,000 feet), these luminous fish keep near the surface, living in the relatively shallow water immediately around or between the islands of this voleanic archipelago. They are not so readily caught on moonlight nights, but this is because their light can not be so easily seen by the fishermen and not because they avoid the light of the moon or show any such periodicity in appearance as that of the palolo or other worms. Nothing is known of the life history of these fish. In October eggs can be squeezed from the females of both species. ‘These are surrounded by jelly, are transparent, and contain large oil droplets, but sink in sea-water. Eggs of Photoblepharon are about 1.7 mm. in diameter, and those of Anomalops 1 mm. in diameter, without the jelly. Anomalops eggs plus sperm did not develop under laboratory conditions, but these were not very favorable. They showed no luminescence on stimulation mechanically, or upon the addition of ammonia to the sea-water containing them. It is quite natural that the earlier observers, with preserved material at their disposal, should be ignorant of the purpose of the luminous organ. Boddaert thought the function of the organs of Photoblepharon was to shield the eyes of the fish from injury by the branches of the coral among which it lived. Lacépéde thought it a protection of some sensitive tissue against the rays of the tropical sun. Giinther first considered that the structures were light-organs, while Vordermann (1900) actually observed the living fish in 1897 and saw its light. The short paper of Vordermann and the descrip- tion recorded in the narrative of the Siboga expedition by Weber (1902) contained all our knowledge of these fishes until the appear- ance of an extensive paper by Steche (1909). This excellent mono- graph deals largely with the histology of the luminous organ and also contains a few physiological observations. Thus, Steche determined that the light shone day and night continuously with an intensity (for one organ of Photoblepharon) of 0.0024 meterkertze. Mechanical pressure or chemical stimulation is quite unable to increase the intensity of the light, which can be “turned off’? completely or not at all only by mechanical contrivances connected with the organ. Discussion of Steche’s histological observations will be given in describing the histology of the organ. The Anomalops obtained by me varied in length from 4 em. to 11 em. Photoblepharon is about the same size, but the proportions +e scone get: Fishes Photoblepharon and Anomalops. 47 of the two fish are different, Photoblepharon being stouter in pro- portion to the length. Sometimes the former grows to large size and is caught by hook and line. Mr. Baadilla, of Banda Neira, told me of one about 250 mm. long. The usual size for Anomalops is 102 mm. from head to base of tail, 832 mm. high, and 28 mm. thick at the gills. Its luminous organ will measure 12.3 by 5.7 by 1.4 mm. and weigh about 0.08 gram. In Anomalops the two luminous organs together make up about 0.58 per cent of the body-weight. ‘This is not as large a proportion as we might expect when we consider that the organ looks very large and is actually one-eighth to one-tenth the length of the fish itself. No doubt the eggs which filled the abdomen of the Anomalops examined greatly reduced the weight proportion of luminous organ to whole fish, that would be observed in a fish without eggs. In both fishes the luminous organ is a compact mass of white to cream-colored tissue, flattened oval in shape, lying in a depression just under the eye and in front of the gills. The organ looks as if made for experimentation, as it is attached only at the dorso-anterior end and can be cut out with the greatest ease, giving a piece of prac- tically pure luminous tissue. The back of the organ is covered with a layer of black pigment which serves to keep the light from shining into the tissues of the fish. In both fish there is a mechanism for obscuring the light, but curiously enough the mechanism developed is totally different in the two species, notwithstanding the fact that in structure the organ is identical in the two and in every detail except proportion the fish are very similar. In Anomalops, the organ is hinged at the antero-dorsal edge and can be turned downward until the light surface comes in contact with a fold of black-pigmented tissue forming a sort of pocket. The light is thus cut off. In Photoblepharon, a fold of black tissue has been developed on the ventral edge of the organ socket, which can be drawn up over the light surface like an eyelid, thus extinguishing the light. Why these two fish, so similar in most respects, and especially in the general structure of the luminous organ, should have developed such totally different means of extinguishing the light is a mystery. As observed by Steche, I find that the organ itself emits light continuously and steadily, in the day as well as at night. No amount of mechanical or electrical (strong interrupted induced shocks) stimulation causes any effect whatever on the intensity. In this respect it differs completely from all other animals and resembles the bacteria and fungi, which also produce a steady light independent of stimulation. One other fish, Monocentris japonica, with a paired light-organ at the tip of the lower jaw, behaves as do Anomalops and Photoblepharon. This peculiarity of steady light, independent of stimulation, should be borne in mind, as it has a very important bearing on the nature of the light of these fishes. 48 The Production of Light by the In life, the light of Anomalops is constantly being turned on and off, according to Steche, 10 seconds light and 5 seconds dark. Photo- blepharon in its natural environment shows its light continuously, but in glass Jars, either as a result of partial asphyxiation or excite- ment, its light is also intermittent. The animals appear to be swim- ming about with bright flash-lights which are turned on periodically. According to the natives, the use of the organ is as a search-light. Its use, according to Steche, is as a ‘“‘Scheinwerfer’’ or search-light to attract prey, and the flashing is to mislead its prey. When brought into the laboratory, in small glass jars, where the supply of air is limited, the fish lose control over the closing mechan- ism and the flashes become irregular. Just before death from asphyx- lation, when the fish swim upside down and slowly, the light is usually not visible, but on actual death, when movement ceases, the organ is exposed and gives forth light, both in Anomalops and Photoblepharon. ‘This simply means that the muscles involving the closing mechanism pass into the relaxed condition on death. The relaxed condition of these muscles, then, corresponds with the exposed position of the organ. HISTOLOGY OF THE LIGHT-ORGAN. Although this paper deals with the chemistry of light-production in Anomalops and Photoblepharon, an accurate description of the structure of the organ is of considerable interest in view of the rather startling conclusion to which I have come from chemical investiga- tion, namely, that the light-organ is a mass of tissue designed for the nourishment of luminous bacteria, which produce the light. The gross anatomy and histology of the organ is the same in both fishes, with the exception of the movable screen to obscure the light, pos- sessed only by Photoblepharon. Steche describes the organ as an acinose gland made up of a large number of gland-tubes parallel to each other and extending completely across the organ, from the back pigmented surface to front trans- parent surface, where the light emerges. The ascini have been elongated and arranged parallel to each other. In a parallel section of the fresh organ it is very easy to see these tubes and also the blood- vessels which run between them. A cross-section of the tubes shows that they are pressed into a polygonal shape, separated from one another by sparse connective tissue and arranged in a ring about a blood-vessel, whence they get their oxygen and food-supply. The back ends of these tubes meet a layer of cells containing small gran- ules of guanin, which are believed to act as a reflector layer, in analogy with such a layer found in the luminous organs of other fish. Steche observed that these granules dissolved in formalin and were doubly Pn ee, el ed Fishes Photoblepharon and Anomalops. 49 refractive, but made no chemical tests for guanin. Back of this reflector layer is a layer of cells containing small black pigment granules which effectively screen the tissues of the animal from its own light. In addition, the socket in which the light-organ lies, the movable screen in Photoblepharon, and the whole of the light-organ except the front, almost flat, surface, are covered with this black pigment layer. Just below the front surface of the organ a number of the tubes unite to form a common reservoir which connects with the exterior by a pore (20 to 30 microns wide) passing through cutis and epidermis. The pores are scattered over the surface of the organ and were over- looked in Steche’s first description of the tissue. ‘There is no doubt of these pores, however, as they are very clearly visible in sections of the gland made by Professor Dahlgren, of Princeton University. At the base, the light-tubes are protoplasmic, but the rest of the tube appears to be one large vacuole of secretion made up in fixed material of small droplets and granules uniformly distributed from base to front of tube. The outlines of the tubes are so difficult to see at the front that Steche gained the impression that the cell itself is directly converted into the secretion. Although no mitoses were observed, the appearance of cells with large nuclei at the base of the tube was such as to suggest a growing layer there which supplies new gland-tube cells to take the place of those wasting away by formation of the light-giving secretion. No luminous material is passed out of the gland, a point which I can confirm from my obser- vations of the living fish, so that Steche believes the light to be burned in the reservoir at the front surface of the gland. The light would therefore be extracellular but intraglandular. At the front end of the gland-tubes there is a 1- to 2-layered epi- thelium that passes at the pores into the epithelium of the outer surface of the organ. Here, also, is a tough connective tissue in which are embedded blood-vessels and nerves. The red blood- vessels are very clearly visible in the living organ outlined against the white of the gland. They arise as 9 to 13 vessels passing from both the lower and upper edges of the front surface of the gland and branch to smaller vessels meeting near the middle. A peculiar valve occurs where branch vessels leave the main artery. The blood-supply is exceedingly rich. Steche could not determine the point of ending for the nerves which follow the blood-vessels, but thinks they pass to the gland- tubes and control the secretion. I should judge it more likely that they are vasomotor in function, but I have no evidence for this point. The nerve is a large branch of the trigeminal-facial complex. There was no indication of a marked center in the brain, such as that possessed by some electric fishes to control their electric organs. 50 The Production of Light by the Steche describes the living organ as clear and transparent and bright yellow in color, but becoming cloudy and coagulated in fixing fluids or on death of the fish. JI am quite unable to agree with this description. The luminous organ of living fish is white to cream- colored and not transparent. It is fairly firm in consistency, and I have never noted any marked change on death of the fish or removal of the gland from the body of the fish. The fresh gland is firm enough to be cut with a razor into fairly thin sections, in which appear very clearly the parallel gland-tubes with blood-vessels running between them and filled with a row of oval, nucleated red blood- corpuscles. On application of pressure to the cover-glass the gland- tube contents flow downward and mingle with the fluid (sea-water) bathing them, forming a white, milky emulsion. In this emulsion can be seen a great number of small granules and rods, often arranged in spirillum-like rows. The rods are unquestionably bacteria, as they can be seen to move of their own accord, often with a corkscrew- like motion. Some of the granules are probably end views of bacteria, but others may be cell granules of one kind or another. The granules have a weak Brownian movement. ‘The spirilla-like rows may be almost as long as a red blood-corpuscle is wide. The contents of the luminous cells, then, give an emulsion of bac- teria in the sea-water. In the dark this emulsion glows brightly, but whether the light comes from the bacteria or not can not be directly proved. ‘The light is perfectly homogeneous under the microscope, showing no trace of light-giving granules so character- istic of the medusz and pennatulids. Needless to say, the light of luminous bacteria would also appear homogeneous, as the light of a single bacterium is too weak to be seen through the high power of the microscope and the bacteria are too small to be observed as individuals with the low power of the microscope. If the material of the gland-tubes is pressed out into fresh water a coagulation appears to occur and the material assumes a finely granular net structure in which it is difficult to make out individual particles. Brownian and bacterial movements cease and the emul- sion also ceases to luminesce. Bacteriolysis has taken place. Fresh smears of the luminous gland, kindly stained for me by Pro- fessor Dahlgren, show the bacteria very nicely and in great abun- dance. While Steche’s description of the structure of the light-organ is essentially correct, failure to examine the fresh organ material has led him, I believe, to a misinterpretation of its nature. The bacteria are so numerous and the chemical behavior of the emulsion of the organ (as we shall see) so similar to an emulsion of luminous bacteria that I feel that they are the real source of the light of these fishes. The organ becomes, then, not a gland for the production of a secretion Fishes Photoblepharon and Anomalops. 51 whose combustion is intraglandular but extracellular, but an organ for the nourishment of symbiotic luminous bacteria. On this view the extraordinary richness of the blood-supply is at once apparent—to supply the oxygen for respiration of the organ cell, for respiration of the bacteria, and for the production of light by the bacteria. Luminous bacteria require an unusually abundant supply of oxygen, as those who are familiar with the growth of them will readily under- stand. On this view the existence of pores in an organ which does not produce an external secretion becomes explicable—namely, a means of exit of dead bacteria. It is very likely that other fish may also be found to possess luminous organs for the growth of luminous bacteria. ‘To decide this it will be necessary to study the living animals, as no certain remains of bacteria are to be made out in fixed material. A fish which I suspect may prove to be similar to Photo- blepharon and Anomalops is Monocentris japonica of Japan. My suspicion is based on the fact that the light of this fish, two specimens of which I observed at Misaki in 1917, is produced continuously day and night and without change in intensity, just as the Banda fishes. The view that animal light is due to bacteria is not a new one. It has been advocated by Pierantoni (1918) for some years. He had grown cultures of luminous bacteria from the luminous material of squid and has even gone so far as to suggest that in forms where no bacteria can be demonstrated we are dealing with ultramicro- scopic organisms similar to ultramicroscopic pathogenic forms sup- posed to be present in filterable viruses. While it is certainly not true that the light of all forms is due to luminous bacteria, I think the chemical evidence which follows, together with the ocular evi- dence already described, is very strongly in favor of the view that the light of these fishes is due to symbiotic luminous bacteria. LACK OF OXYGEN. One of the conspicuous anatomical peculiarities of the light-organ is its rich supply of blood-vessels. ‘These can be seen in life, running from the lower (principally) and upper edges of the organ to branch over its surface into capillaries which run between and parallel to the columns of luminous material. The organ is also in a position just anterior to the gills to receive fully oxygenated blood, and in cutting the organ out blood flows freely, showing the presence of large arteries in this region. It is not surprising, then, to find that the organ is very sensitive to lack of oxygen, the light disappearing promptly in its absence. ‘This can be shown for both Anomalops and Photoblepharon by three methods. First, it may be readily observed that the light-organs of fish dying in sea-water from lack of oxygen become dimmed. [If the fish 52 The Production of Light by the are now lifted out of the water to the air, the light immediately becomes bright again. This is not due to stimulation of the fish in handling, because the same amount of stimulation while the organ is under sea-water containing less than the proper amount of oxygen does not increase the intensity of the light. It is interesting to note, in this connection, that strong, interrupted, induced electrical shocks will not cause a luminous organ, dim from insufficiency of oxygen, to glow more brightly. This shows that, for this material at least, the cell is able to absorb all the oxygen available and that electrical stim- ulation can not cause additional oxygen to enter as a result of increased permeability from stimulation. Second, if a luminous organ is removed from a fish and laid face down on a piece of glass, the light, as observed through the glass, disappears almost instantly, except about the edges of the organ in contact with air. On lifting from the glass the organ glows brightly over its whole surface. The glass keeps the air away and the light then disappears with extraordinary rapidity. Finally, if one makes an extract of the organ by grinding in sea- water with quartz sand and places the extract in a test-tube, the light quickly disappears, except at the surface of the extract in contact with air. Shaking the tube causes its contents to glow throughout. In these experiments it is the rapidity with which the oxygen is used up that is astonishing. Similar experiments can be performed with an extract of Cypridina or pennatulids, but oxygen is not con- sumed nearly so rapidly. The situation is exactly as if one had, in sea-water, an emulsion of luminous bacteria, which also use up oxygen with extraordinary activity. The difference between lumi- nous bacteria and extracts of Cypridina lies in this, that the bacteria use oxygen not only for light-production but also for respiration. Large amounts of oxygen are necessary for the latter. The extract of these fish behaves like an emulsion of luminous bacteria and fur- nishes an additional fact in favor of the view that the light is really of symbiotic bacterial origin. DESICCATION. If the light-organs of Anomalops or Photoblepharon are cut out, carefully freed of adherent sea-water, and placed in a desiccator, they dry to a hard, leathery mass which is powdered only with dif_i- culty in a mortar. On adding water, there is no light, or at most a faint light, if the tissue has been dried very rapidly by placing it very near lumps of CaCl, No doubt a vacuum desiccator would give better results, but certainly there would be no light after drying to compare with the great intensity before drying. In this respect these fish behave as luminous bacteria, which give light on moisten- =e ae E Fishes Photoblepharon and Anomalops. Oo ing only if dried very rapidly and if tested with water within a short time after they have been desiccated. DILUTION. Despite the fact that there is so little light-material in an organ of these fish which can be dried, there is an amount in the fresh glands which will give a visible light when distributed through an immense amount of sea-water. In‘one experiment with Anomalops, the light-organ was carefully cut out, ground with sand inamortar, and 10 c. ce. sea-water added; 5 c. c. of this brilliant emulsion was then diluted with equal volumes of sea-water successively until the light could no longer be seen. The organ distributed in 1,280 c. e. still gave a good light and in 5,120 ec. ¢c. to 10,240 c. c. the light was just visible. The organ measured 12.3 by 5.7 by 1.4 mm. and the organ of the opposite side weighed (fresh) about 0.08 gram. This tissue, therefore, gave light visible in, let us say, about 8 liters of sea-water, or 1 part in 100,000 sea-water. A strong emulsion of the organ is milky in appearance, due to the small, suspended particles. It resembles a suspension of luminous bacteria in sea-water, and like them the luminous material passes ordinary filter-paper readily. As no porcelain filter was obtainable, I can not say whether the particles will pass one of these or not. DURATION OF LUMINESCENCE. A concentrated sea-water extract of the luminous organ of either Anomalops or Photoblepharon allowed to stand will give only a very faint light after 7 hours and no light after 84 hours. More dilute extracts In sea-water give light for a shorter time, as do also extracts in more dilute sea-water (1 part fresh water to 2 parts sea-water and 1 part fresh water to 1 part sea-water). These experiments with diluted sea-water were tried in the hope of getting a medium more like the blood of teleosts, which usually have a salt-content considerably lower than sea-water. If the light of these fishes is of bacterial origin, the bacteria are so dependent on cultural conditions within the living organ that they will not live for more than 7 or 8 hours when removed. The organ of Anomalops or Photoblepharon kept intact in sea-water or in the dead fish will also give no light if ground in a mortar after a period of 8 hours. These experiments were performed at 29° C. temperature. Once the light has disappeared from an extract of luminous organs on standing, it can not be regenerated in any way. In this respect the extract of these fish differs from those of pennatulids and jelly- fish, which again give light upon addition of fresh water, and from 54 The Production of Light by the Cypridina and fireflies, which again give light on mixing with luci- ferin. Neither Anomalops nor Photoblepharon gives the luciferin- luciferase reaction. If fresh water is added to a sea-water extract of the organ of these fish, the light grows rapidly dimmer and dis- appears. There is no sudden increase in intensity, such as one gets on adding fresh water to extracts of pennatulids and jelly-fish. All these peculiarities are identical with those of an emulsion of luminous bacteria, and again serve to strengthen the evidence that light is due to symbiotic bacterial organisms. LUCIFERIN AND LUCIFERASE. In Cypridina, Odontosyllis, Pholas, and fireflies, the presence of luciferin and luciferase can be demonstrated. In Noctiluca, penna- tulids, jelly-fish, luminous bacteria, and Chetopterus it is not possible to demonstrate them; it is also impossible in these fish. Luciferin, if present in the organ, should be prepared by some one of the following methods: (1) Adding boiling water to the excised luminous organs and extracting them in a mortar; (2) heating the concentrated luminous sea-water extract of the organ to (a) boiling or (6) to a temperature (55° C.) which permanently extinguishes the light. Luciferase, if present in the organ, should be prepared by some one of the following methods: (1) Extracting with sea-water and allowing the extract to stand for 8 hours till the light disappears; (2) extracting with fresh water; (3) extracting with sea-water and adding such substances as saponin or sodium glycocholate, which causes the light to disappear quickly. Dark solutions which should contain luciferin and luciferase have been obtained by these various methods, but on mixing no light whatever has appeared. The luci- ferin! of these fish also gave no light with Cypridina luciferase, nor the luciferase! of these fish with Cypridina luciferin. In the impos- sibility of demonstrating a luciferin-luciferase reaction, despite an apparent abundance and persistence of luminous material, Anomalops and Photoblepharon again agree with luminous bacteria. The luci- ferin-luciferase experiments may be summed up as follows: Photoblepharon luciferase X Photoblepharon luciferin—negative “ “e Anomalops as as “ ‘“ “ x Cypridina CT “ Anomalops . > Anomalops f is x Photoblepharon iz “ ‘ x Cypridina ‘ Cypridina ch x “ ch r < Photoblepharon ““ —negative ce cc x Anomalops ce ee “ 1 Using these words for solutions which, according to the method of preparation, should have contained luciferin and luciferase. Gu Qu Fishes Photoblepharon and Anomalops. TEMPERATURE. The temperature of the water in which these fish normally live varies but little from 27° C. the year around. If a sea-water extract of the luminous organ of Anomalops is made and gradually heated in a test-tube, the light dims at 38° and disappears at 41° to 42°. If cooled quickly, the light returns and the heating and cooling can be repeated several times with alternate disappearance and reap- pearance of luminescence. If heated to about 50° and cooled quickly, there is no return of the light, but there is some recovery on heating to 48° and cooling quickly. An extract of Photoblepharon behaves in just the same way as Anomalops on heating under similar conditions, except that the light dims at 40° and disappears at 48° to 44°. Heating to the neighbor- hood of 51° likewise extinguishes the light permanently. It should be noted that there is no marked increase in brightness on slight heating, so characteristic of extracts of pennatulids and jelly- fish. The sudden brightness in these forms I would interpret to be a heat cytolysis of cells containing luminous material or perhaps a granulolysis of light-producing granules. In the absence of this heat-effect these fish again resemble luminous bacteria. The temper- ature of extinction is significant, lying as it does in the neighborhood of 40°. This is the general region for extinction of the light of lumi- nous bacteria, whereas Cypridina, Cavernularia, and several other luminous forms which produce light are affected only by much higher temperatures. SPECTRUM AND INTENSITY. Steche reported the intensity of the light of Photoblepharon to be 0.0024 meterkertze. This value was arrived at by determining in Banda that he could just read his watch easily by the luminescence of the fish at a distance of 2 meters, after a 5-minute dark adaptation of his eyes. On arrival home, Steche prepared an illuminated slit giving about the same intensity and color as the light of Photo- blepharon, and found he could read his watch at a distance of 1.75 meters after a 5-minute dark adaptation. ‘The intensity of the slit was therefore 0.75 of that of the luminous organ, and in comparison with a candle the slit was found to be 0.0018 meterkertze. Hence the intensity of one light-organ of a fish was four-thirds of 0.0018 meterkertze or 0.0024 meterkertze. I have made no measurement of the intensity of the light of these fishes, but may remark in passing that it is extraordinarily bright and the living fishes present a remark- able sight as they swim through the water flashing their lanterns— so large in proportion to the body—like great electric torches. To my eyes the color of the light of both Anomalops and Photo- blepharon is greenish blue. In lamplight it looks green, as does an 56 The Production of Light by the emulsion of luminous bacteria. It is greener than Cypridina, which looks blue in lamplight and decidedly blue in the dark. Examined with a Zeiss comparison microspectroscope, the spectrum extends, in the case of both fish if lighting brightly, from about »\ = 0.45 to \ = 0.64 micron. When brightly lighting, the red end is distinctly visible; when somewhat dimmed from lack of oxygen no red can be seen, but only a strip of greenish blue. There are no dark or bright bands, but a continuous spectrum is formed. CYTOLYSIS. A luminescent extract of the photogenic gland of Cypridina made with sea-water is quite unaffected by the ordinary cytolytic agents. There is neither extinction of light nor increased brightness on mixing with fresh water, on saturation with chloroform, or on addition of small amounts of saponin or sodium glycocholate, or on slight warm- ing. By these means cells are caused to swell and become more or less completely fluid, many of the granules within the cell dissolving at the same time. A luminous extract of pennatulids (Cavernularia, Ptylosarcus) or of jelly-fish (4fquorea, Mitrocoma) becomes much brighter on appli- cation of cytolytic agents, and then the light disappears completely and can not be resuscitated in any way. Either some photogenic cells in the extract, which are still intact, cytolyze with liberation of photogenic material and production of light, or photogenic granules in the extract dissolve with production of light. The light of an emulsion of luminous bacteria, on the other hand, is immediately extinguished by the above-mentioned cytolytic means, without any preliminary increase in brightness. Light-production is dependent upon an intact bacterial cell, and when bacteriolysis has occurred the photogenic power is lost completely. Both Anomalops and Photoblepharon extracts behave toward cytolytic agents as an emulsion of luminous bacteria. The light disappears on addition of three volumes of fresh water, but not on addition of three volumes of isotonic salt or cane-sugar solution. A drop of chloroform added to a test-tube of luminous emulsion puts out the light immediately, as does also a pinch of saponin or of sodium glycocholate, the latter more readily than the former. There is no initial increase in brightness in the case either of the bacteria or of these fish. Like an emulsion of luminous bacteria, the light disappears from an extract of Anomalops photogenic organ, if crystals of cane sugar, MgSO,, or (NH,).SO, are added, but returns if the mixture is Iimme- diately diluted with sea-water, most readily and bright in the case of the cane sugar. Addition of alcohol also causes a reversible ex- tinction. As this behavior toward sugar and salts is similar to that Fishes Photoblepharon and Anomalops. o7 of luminous extracts in general, as well as suspension of luminous bacteria, it throws no light on the bacterial nature of the luminescence of these fish. The effect of cytolytic agents, however, does indicate a bacterial origin of the light. SODIUM FLUORIDE. Sodium fluoride is often used to determine if a process depends on the integrity of the cell, on some vital peculiarity, or is of enzyme nature. Sodium fluoride in 1 per cent concentration is said not to affect the activity of enzymes. It is certainly true that 1 per cent NaF does not affect the luminescence of Cypridium extract (Harvey) or of Pholas extract (Dubois). Sodium fluoride in 1 or 1.5 per cent concentration does, however, extinguish rapidly the light of an extract of Anomalops made with 3 per cent NaCl (to prevent pre- cipitation of the NaF by the Mg and Ca of sea-water). POTASSIUM CYANIDE. Although potassium cyanide affects many kinds of oxidations, it has very little effect on the luminescence of extracts of luminous animals. This is true for Cypridina, Cavernularia, fireflies, and Noctiluca. Very small amounts will kill animals and even 0.00025 per cent KCN is sufficient to cause asphyxiation and death of fresh-water fishes in 24 hours. TaBLe 1.—Effect of KCN on Luminous Bacteria. Concentration of KCN. Light after— Light disappears in— Gram mols. Per cent. 10 min. 60 min. 24 hrs. per liter. M/10 ONG Oe researc acter creravertoueycaebell fe) oy ahete cia? costello. ois enero snes 3 min. M/20 Oe rl | [Pri gi elle REL eg Cok Pat wean ea 4 min. M/40 EG ZIP || ctate revere ok ak ereriavell site eee cis tela eicavs tots! |foeeteract betes 6 min. M/80 .081 Faint. Weryetaintees|aee 80 min., about. M/160 04 Faint. Werytainten lien M/320 .02 Slightly dim. | Slightly dim. |........ M/640 01 Good. Goode Ul jcecene M/1280 .005 Good. Goode Wisse M/2560 .0025 Good. Good. Good. Sea-water. Good. Good. Good. It was found that KCN extinguishes the light of these fish more readily than that of Cypridina or Cavernularia and about as readily as that of luminous bacteria. A comparison can be made from two tables which I append, although the times observed and concentra- tions used are not exactly the same in the two cases. One gives the effect on a strain of luminous bacteria which I studied in 1915, but have not previously published the results. The second gives the 58 The Production of Light by the action of KCN on an emulsion of the luminous organ of Photo- blepharon. ‘The KCN was dissolved in sea-water, and this solution was diluted with an equal volume of sea-water each time. The con- centration of KCN necessary to inhibit luminescence of these fish is sufficiently close to that inhibiting the luminescence of bacteria to supply one more fact in favor of the view that the light of Photo- blepharon and Anomalops is of bacterial origin. TABLE 2,—Effect of KCN on Emulsion of Photoblepharon Luminous Organ. Light after— Concentration | eses eas eek eee eee Light dis- of KCN. appears in— 1 min. 10 min. 60 min. O25. pact. D600 Ba vl lPeogerenta eet att Gl SAO paCNERcHhiChe About 8 min. a20 Dim. Mery faintealsehiate ese About 20 min. a2 Fair. TES a regen whe Ei cera yes cee About 30 min. .062 Good. Maire) bul oes aietees exereneeare About 40 min. 031 Good Hairsti) frailties .015 Good. | Fair. Very faint. .008 Good. Good. Very faint. .004 Good. Good. Very faint. .002 Good. Good. Fair. Sea-water. Good. | Good. Good. CULTURE EXPERIMENTS. Despite the parallel in behavior of an extract of the luminous organ of these fish and an emulsion of luminous bacteria, final proof of the bacterial nature of the light must come with the artificial cultivation of the organisms. Although not equipped for bacteri- ological work in Banda, I have endeavored to obtain luminous cul- tures by growth of bits of the luminous gland on various media. Growth of some kind of organism has been abundant, but no light has appeared in any case. It is possible that this growth was actually made by the luminous organism, but that no light was produced under artificial conditions. Giard and Billet have described a malady of sand-fleas, an infection of the animals with luminous bacteria, which eventually led to their death. They were able to inoculate unin- fected sand-fleas, which would then become luminous, with the organ- isms, and grow them on artificial media, but light never appeared under these artificial conditions. However, the cultures inoculated into living sand-fleas would luminesce in the animal. Apparently some special nutrient material is necessary for luminescence in this particular organism, and it is not improbable that a luminous bac- terium which has developed into a symbiotic organism, such as we may suppose to be present in these fish, would require special nutrient substances. The ordinary luminous bacteria of the sea can be grown and luminesce with great regularity on almost any medium. ‘Tar- a ee, Fishes Photoblepharon and Anomalops. 59 chanoff has inoculated them in frogs and produced luminous animals. However, non-luminous strains of luminous bacteria have been de- scribed by Beijerinck. There is a possibility, then, that the growth I observed on my culture media was the form which produced light under special conditions of the luminous organ of the fish. On the other hand, although the transfer was made with sterile instruments from the interior of the organ, there is always the possibility of contamination, and nothing certain can be stated regarding the nature of the cultures obtained. The culture media used were the following: . Sterile muscle of Anomalops in sea-water. . Unsterile muscle of Anomalops in sea-water. . Sterile muscle of Photoblepharon in sea-water. . Unsterile muscle of Photoblepharon in sea-water. . Potato slab (sterilized) in sea-water. -12, Agar-peptone-amino-acid-sea-water of a reaction varying from colorless to decidedly pink to phenolphthalein. or) oe i The culture medium for 6 to 12 was made by digesting the white of one boiled egg for 24 hours with trypsin solution in 50 c. c. water, diluting to 425 c. ce. with sea-water, adding 1.5 per cent agar-agar, and sterilizing. The material was then tubed and NaOH added in suc- cessively increasing amounts to each tube to give a range of acidity on each side of neutrality. These tubes all produced a good growth of bacteria, except the deep-pink decidedly alkaline ones; 5 produced no (?) growth; 1 to 4 abundant growth of bacteria. The best growth occurred on the light-pink medium. No light appeared in any tube. Further work is therefore necessary to settle this interesting and important question of the artificial cultivation of bacteria from the luminous organs of Anomalops and Photoblepharon. SUMMARY. The light of the luminous fishes Photoblepharon and Anomalops appears to be due to luminous bacteria living in the luminous organ. Previously the organ had been considered a gland, producing a luminous secretion oxidized within the gland. Despite the general appearance of an organ of secretion, no luminous material is excreted to the sea-water by the living fish. If the organ is teased in sea-water and examined under the microscope, innumerable motile rod-shaped bacteria, sometimes forming spirilla-like chains, can be seen. Stained smears of the organ show the bacteria nicely. In chemical respects an emulsion of the organ behaves just as an emulsion of luminous bacteria and differs in one way or another from extracts of other luminous animals. These various charac- teristics are as follows: 1. The light-organ is extraordinarily well supplied with blood-vessels and the emulsion is fully as sensitive to lack of oxygen as are luminous bacteria. Light ceases very quickly in absence of oxygen. 60 The Production of Light by Certain Fishes. 2. If dried, the organ will give only a faint light when again moistened with water. This is characteristic of luminous bacteria. The luminous organs of most other forms can be dried without much loss of photogenic power. 3. Luciferin and luciferase can not be demonstrated; this is also true of luminous bacteria. 4, The light is extinguished without a preliminary flash by fresh water and other cytolytic (bacteriolytic) agents. The significance of this is discussed in the text. 5. Sodium fluoride of 1 to 0.5 per cent concentration extinguishes readily the light of an emulsion of the gland. 6. Potassium cyanide has an inhibiting effect on light production in about the same concentration as with luminous bacteria. To these observations must be added the very suggestive fact that the light of Photoblepharon and Anomalops continues night and day without ceasing and quite independently of stimulation. This is a characteristic of luminous bacteria and fungi alone among organisms, and very strongly suggests that the light is actually due to symbiotic luminous bacteria. Actual proof that the bacteria found in the organ are luminous can come only when these are grown artificially. My attempts in this direction have failed. Good growths of bacteria were obtained on pepton-agar, but they produced no light. One might expect that a symbiotic form would require rather definite food materials to produce light, and it is, perhaps, not surprising that culture experiments have failed. Certainly, the ocular and chemical evidence, if not the cultural evidence, supports the view that the light of these living fish is bacterial in origin. LITERATURE. VorDERMANN, A. G., 1900, Twee Lichtgevende Visschen van Banda: Ikan leweri Batoe (Heterophthalmus palpebratus Lacépéde) en Ikan leweri Ajar (Heterophthal- mus Blkv.?). Naturk. Tijdschrift voor Nederlandisch.-Indie, Lrx, p. 72. Weser, Max, 1902. Siboga Expeditie. Introduction et description de l’expédition, p. 108. StecHE, Orro, 1909, Die Leuchtorgane von Anomalops katoptron und Photoblepharon palpebratus, zwei Oberflachenfischen aus dem Malaiischen Archipel. Zeitsch. Wiss. zool., xctil, p. 349. PIERANTONI, UmBErTO, 1918, I microrganismi fisiologica e la luninescenza degli animali. Scientia, xxu1, pp. 102-110. Hil. HYDROGEN-ION CONCENTRATION AND ELEC- TRICAL CONDUCTIVITY OF THE SURFACE WATER OF THE ATLANTIC AND PACIFIC. By ALFRED GOLDSBOROUGH MAYOR. Three charts. 61 errr ie j i ] ’ yy Be ‘ = é » 7 in et * Fc a 7 ; 7 al belt, i a Lek, f se ; un 4 a @ A Fa. ty < ae a : i ve y } DIA a. yo ten y) reins! i } : fi d & at) HYDROGEN-ION CONCENTRATION AND ELECTRICAL CONDUCTIVITY OF THE SURFACE WATER OF THE ATLANTIC AND PACIFIC. By ALFRED GoLDsBoROUGH Mayor. The hydrogen-ion concentration of sea-water was determined by placing 0.4 c. ce. of 0.1 per cent of the red dye thymolsulphoneph- thalein in 70 per cent alcohol, in a test-tube of resistance glass, 24 mm. in caliber, then adding sea-water so as to make up 30 c. c. of solution. The more alkaline the sea-water the more blue the solution, while a yellow color appears more and more pronounced in less and less alkaline sea-waters. Thus comparing the color of the solution in the test-tube with a graded series of sealed tubes whose colors cor- respond with known hydrogen-ion concentrations of sea-water, we can readily determine the hydrogen-ion concentration of our sample. A series of such tubes, ranging from 7.95 to 8.3 PH, was standardized by Professor J. F. McClendon and kindly presented to the author in 1917, and I have since then restandardized these tubes at intervals of two years by comparison with determinations of PH made by a Leeds and Northrup potentiometer, but the color of the tubes has not changed appreciably in the interval. When not in use the tubes are, however, kept in the dark to avoid the possibility of light changing their color. The method for making up the solutions in these standard colori- metric tubes is described by McClendon, Gault, and Mulholland in publication No. 251,page 44,Carnegie Institution of Washington,1917. As is well known, Kohlrausch found that in the purest distilled water the molecules are only slightly dissociated, so that there is only about 1 gram of hydrogen ions in 10,000,000 liters of water; or the hydrogen-ion concentration is about 107. These hydrogen cations are of course balanced by 10’ concentration of OH anions in the water. In sea-water, however, the hydrogen-ion concentration is about 108, and the OH-ion concentration 10°. In order to avoid writing negative exponents, Sérensen (1909, p. 28) devised the symbol ‘‘Pu”’ to indicate the negative logarithm of the hydrogen-ion concentration. Thus 10-§ would be Pu 8 in ; ; : 1 Sérensen’s system, and a hydrogen-ion concentration of 25 108 = 0.4 X 10-* would be written PH 8.398, because 0.398 is the loga- rithm of 2.5 and 8.0 is the logarithm of 108; hence 8.0 + 0.398 = 63 64 Hydrogen-ion Concentration and Electrical Conductivity 8.398. It is, however, difficult to think in terms of negative loga- rithms, and so the tables at the end of this paper give the PH and also the hydrogen-ion concentration of the sea-water expressed arith- metically. Despite its artificiality, however, one soon finds that the Pu system gives a clearer idea of the alkalinity or acidity of a solution than does a direct expression of the hydrogen-ion concentration. Thus, in testing water, PH 7 would indicate practical neutrality; Px above 7, alkalinity; and below 7, acidity. The carbon-dioxide tension of the sea-water was calculated from the Pu and the temperature by the method devised by McClendon, Gault, and Mulholland (1917, Carnegie Inst. Wash. Pub. No. 251, p. 36). McClendon found that the Pu of sea-water falls 0.01 for 1° C. decline in temperature. Thus if the Px be 8.25 at 27° C., it may be expected to be 8.24 at 26°C. While this is true under normal conditions, if the sea-water be diluted with river-water, or if there be upwelling of cold currents carrying water rich in CO, to the sur- face, the PH may rise while the temperature declines. Thus, near the equator in the Pacific, I have observed a rise of 0.13 in the Pu while the temperature sank 0.45° C., due to the upwelling of water from the depths. The salinity of the sea is expressed in grams of total salts per 1,000 grams of sea-water, and was determined by the well-known method of using a standard AgNO; solution with K,CrO, as an indicator, and testing against a sample of standard sea-water obtained from Pro- fessor Martin Knudsen. The thermometers used read to 0.1° C. and were compared with a thermometer which had been standardized by the U. 8. Bureau of Standards. Tests made on the yacht Anton Dohrn in Florida showed that one might take samples of sea-water from the stern of the vessel while in motion without any detectable error in the Pu, the result being the same as if one dipped the sample up from the bow or stopped the ship and went out in a small boat for it. Of course the only certain way for determining a current while at sea is to anchor and use current meters, but in default of this possibility we were obliged to rely upon the difference between the position the ship expected to make and what she did make—the dif- ference being ascribed to “‘currents.”’ This is, of course, a crude and inaccurate method, for it makes no allowance for leeway due to wind or for errors in steering, but it was the only method available. The thymolsulphonephthalein colorimetric tubes gave correct readings when used for testing the PH of sea-water of salinity 0.32 or 0.33 per cent, but for higher salinities a correction of —0.01 is to be applied to the PH as read on the tube for every unit in rise of salinity. Thus, for salinity 34, if the tubes read 8.22 the correct of the Surface Water of the Atlantic and Pacific. 65 reading would be 8.21; and for salinity 30, if the tubes read 8.22 the true PH would be 8.24. ‘These corrections are, however, of minor significance for Pacific sea-water, for one can not read the PH on the colorimetric tubes to within 0.02 Pu, although one can detect a difference of 0.03 Pu. Upon being taken from the sea, the water was at once tested for temperature and Pu, and a sample was preserved in a rubber-stop- pered bottle for determination of salinity; this was done as soon as possible after the end of the voyage. Some of the samples were tested by Professor L. R. Cary for oxygen, using Winkler’s method, and in all such cases the water was tested immediately upon being taken from the ocean. OBSERVATIONS. In connection with these tests of hydrogen-ion concentration, the electrical conductivity of the sea-water off Tutuila, Samoa, and at Tortugas, Florida, was determined by Kohlrausch’s method, using a Wheatstone bridge designed by Leeds and Northrup for this special purpose, and having a tunable telephone for a detector. As will be seen in table 1, the sea-water of Tortugas has a higher coefficient of electrical conductivity TABLE 1. than that of Samoa, due to the higher salinity of | erp || lee cual Atlantic water. The table gives the data of electrical conductivity of Samoan sea-water at 2, various temperatures, that of N/10 KCl being 23 3.8996 : : : 24 3.8980 unity at the corresponding temperature. This 25 3.8958 sea-water was collected one-fourth mile east of ee eae Cape Matutula, the easternmost point of Tutuila, 28 3.8871 on April 28,1917. The salinity was 34.83, cor- a nee responding to a chlorine content of 19.28 grams of chlorine in 1,000 gramsof water. The hydro- i gen-ion concentration was 0.563 < 10—* (PH 8.25) at 28.2°C. The second column shows the electrical conductivity of sea-water, that of N/10 potassium chloride at corresponding temperature being unity. At Tortugas, Florida, the conductivity of sea-water having 20.06 grams of chlorine in 1,000 grams of water, corresponding to a salinity of 36.24, was determined by the same apparatus, and with a portion of the same KCl solution used in Samoa. It was found that the conductivity of this Tortugas sea-water at 25° C. was 4.163, and at 30° C. it was 4.117 times as great as that of N/10 KCl solution at the same temperature. The average of 62 observations gives 34.87 as the salinity of the surface-water between the Hawaiian Islands and Samoa, whereas in the American tropical Atlantic the salinity is above 36. As McClendon states, temperature is the most important factor determining the Pu of the surface water of the sea; for, as the temper- 66 Hydrogen-ion Concentration and Electrical Conductivity ature rises, the carbon dioxide is driven out of the water and the alkalinity increases; whereas, if the temperature falls, the water attains an increased capacity for absorbing CO, from the atmosphere or of retaining CO, derived from animals or plants, and the H-ion in- creases in the water. Thus, in our observations of Px off theAtlantic coast of North America, the cold water off Yarmouth, Nova Scotia, which was 1.4° C., had 7.96 Pu on March 26, while the tropical water of the Sargasso Sea had 8.25 Pu at 23.55° C. on March 10, 1918. In lagoons such as that of Tortugas, Florida, and inclosed shallow areas, McClendon found there was a diurnal variation in the Pu, the water becoming more alkaline by day and relatively acid during the night. This he correctly attributed to the effect of photosynthesis by plant life, which is active in daylight but ceases during the night. Over the deep sea this effect is apparently neutralized by the stirring of the water due to waves, and perhaps by a moderate amount of inter- change between the surface water and the deeper layers. Over shallow regions, where the water may become impounded in tide-pools at low tide, the effect of photosynthesis is often very marked, the PH chang- ing greatly while the temperature may change but little. Thus, over the Aua reef-flat at Pago Pago Harbor, Samoa, where the water was impounded and stagnant at low tide of the spring tide of July 16, 1920, the conditions as compared with those of the ocean-water just seaward of the outer edge of the reef-flat were as shown in table 2. TABLE 2: | Salinity Oxygen Palo in grams of per Date. Locality. Depth. | Temp. iene total salts liter of ‘| per 1,000 water by grams of Winkler’s water. test. 1920 i 6 Cc. C. July 16, 400 ft. from shore, impound- 1541™ p.m.| ed water of reef-flat. Peon eet Ae 34.79 8.44 iJuly 16, 1530™ p.m.| Open sea, just off reef-flat. 5 fath. | 26.75) 8.25 34.7 4.67 At 27° C. the sea-water would be saturated with oxygen if it con- tained 6c. c. of oxygen per liter of water, but we see that the ocean- water outside the reef contained only 4.67 ec. c. of oxygen and was therefore below saturation, while the shallow water of the reef-flat which had been impounded in sunshine for about 2 hours at low tide had 8.44 ec. ec. of oxygen per liter, thus having gained 3.77 ce. e. of this gas per liter in this short time, and becoming supersaturated by about 2.4 c. ec. of oxygen per liter. The rise in Pu from 8.25 to 8.6 was of course due to the loss of CO, resulting from the photosynthesis of of the Surface Water of the Atlantic and Pacific. 67 the numerous alge growing over the floor of the reef-flat. In fact, the CO, tension in this impounded water was reduced to 0.8 ten- thousandth of an atmosphere, whereas in the sea-water outside the reef-flat it was 2.7 ten-thousandths. The Pu of sea-water is also lowered by fresh water pouring out from rivers or brackish bays, and one observes this effect as one passes the entrance to Delaware Bay, the Chesapeake, New York Harbor, or Long Island Sound. The water from these bays, and especially from New York Harbor, is more or less charged with decomposing animal matter derived from sewage which drifts southward along the coast. Such water is low in salinity and laden with carbon dioxide, which lowers its Pu. Thus, off Barnegat, New Jersey, at 10" 15™ a.m. on June 25, 1919, the water showed 8.06 PH at 19.5° C., and its calculated CO, tension was 4.2 ten-thousandths of an atmosphere; so it must have been discharging CO, into the air. Similarly, off Golden Gate, San Francisco Harbor, the water on May 1, 1917, was 7.85 Pu at 10.5° C., and its CO, tension was apparently 5.4 ten- thousandths of an atmosphere. In both these cases the water was discolored and evidently contained decomposing land waste. Dr. R. P. Cowles tells me that he found the Pu of the bottom water of Chesapeake Bay to range from about 7.75 to about 7.28. The dull-gray-green water that creeps down the coast of the United States from the Gulf of St. Lawrence southward is about 8.0 to 8.1 Pu, while the deep-blue water of the Gulf Stream is 8.2 to 8.24. The dull color of this shore water may to some degree be due to pelagic plant life, but it is discolored in greater measure by the drainage and waste from the thickly inhabited shore. One could readily ascertain when the ship passed out of the Gulf Stream into the shore current or vice versa by simply observing the sudden change in the Pu from about 8.22 to 8.1 or less when one entered the shore current; but this method is not likely to be of use in navigation, for the change in the color of the water itself is equally good as an indication, and, moreover, the soundings on this gradually shelving shore are so characteristic and reliable that they alone afford good information respecting the position of the ship. Along the Pacific coast of the United States the upwelling of deep water which has been demonstrated by McEwen (1910, 1918, ete.) lowers the temperature and brings water rich in CO, to the surface, thus lowering the Px, but this water with a low Pu of about 8.0 may extend for 300 miles or more offshore; so in this region we could not be certain of the close proximity of the coast merely by observing a lowering of the Px of the water. The most remarkable upwelling of cold, deep water to the surface is encountered in the mid-Pacific, at or near the heat equator, which ranges from near the geographical equator to about 5° N. latitude, 68 Hydrogen-ion Concentration and Electrical Conductivity dependent upon the season. This fact has been known since the voyage of the Challenger in 1873 (Narrative of the Challenger Expedi- tion, vol. 1, part 2, p. 758, chart No. 19). I have crossed the equator in about longitude 165° W. eight times since 1917, and at or near the equator we usually entered a region wherein the surface water was of lower temperature than to the northward or southward of this place (table 3). TABLE 3. Temp. of Direction toward which Date and time. Latitude. |surface water surface current was of sea. flowing. OC: Mar. 1, 1917, noon...... 6°35’ N. 26.1 E. strong. Mar. 2, 1917, noon...... 107 N. 24.2 NW. Mar. 3, 1917, noon...... 4 34 S. 26.4 No current. Apr. 20, 1917, noon...... 5 10 S: 26.75 Easterly. Apr. 21, 1917, noon...... Equator. 24.95 E. strong. Apr. 22, 1917, noon...... 5°42’ N. 25.9 No current. June 28, 1918, noon...... PINE 28.5 Easterly. June 28, 1918, 5430™ p.m.| Equator. epee Easterly June 29, 1918, noon...... 4°23’ S. 28.3 Easterly. Sept. 8, 1918, noon...... PISS 28.9 Westerly slight. Sept. 8, 1918, 4554™ p.m..| Equator. 29.1 Westerly slight. Sept. 9, 1918, noon...... 4°36’ N. 29.5 Westerly slight. July 17, 1919, noon...... 6735) Ni 28.3 No current. July 18, 1919, noon...... 118 N 28.6 No current. July 19, 1919, noon...... 4 06 S. 28.8 No current. Sept. 18, 1919, noon..... 4510S: 28.8 No current. Sept. 19, 1919, noon..... 031 N 28.6 W. slight. Sept. 20, 1919, noon..... 605 N 29.4 W. slight.* Mar. 29, 1920, noon..... 709 N. 7 feb Westerly. Mar. 30, 1920, noon..... LSLSN: 26.9 Westerly. Mar. 81, 1920, noon..... 4 32 S. 29.1 Southerly. July 30, 1920, noon...... 408 S. 28.4 No current. July 31, 1920, noon...... 118 N 27.5 No current. Aug. 1, 1920, noon...... 644 N 28.5 Strong easterly. * At 5 p. m. September 20 current was strong easterly until 9 p. m. It will be seen that on six of these eight voyages we crossed a region of low temperature near the equator, with warmer water both to the north and to the south of it. Moreover, on five of the eight voyages we encountered a surface current setting toward the east in this region, and thus contrary to the prevailing westerly drift of the surface water over the tropical belt of the Pacific. Now, it is well known that a surface current moving toward the west will set up a current toward the east in the deeper waters. This subject has been admirably treated by W. J. Sandstrém in his Hydrodynamics of Canadian of the Surface Water of the Atlantic and Pacific. 69 Atlantic Waters, Canadian Fisheries Expedition, under the Direction of Dr. Johan Hjort, 1914-1915. Sandstrém applies the Bjerknes theory of circulation to the waters of the Newfoundland region, which shows that Archimedean forces have more to do with the formation of ocean currents than has the wind. Thus, the Gulf Stream flows from the tropics toward Spitzbergen because the light surface layer of the ocean, which is heated by the sun, is 600 meters deep in the tropics and only 200 meters deep at Spitzbergen; hence the thicker, deeper part of this wedge of light water must constantly flow toward its thin edge at Spitzbergen in a futile attempt to overcome this difference in thickness and reduce the whole wedge to a horizontal surface layer of uniform thickness. Of course, if this were ever accomplished, the current would stop after a few oscillations, but the unequal heating ability of the sun in the tropics and in the Arctic regions keeps the current moving. We can not dwell upon this interesting subject, nor is it necessary so to do, for it could hardly be more clearly and simply explained than by Dr. Sandstrém. Suffice it to say that the prevailing westerly drift of the hot, light surface waters in the tropics must produce an easterly current in a heavier and colder layer of water which lies beneath the surface. At or near the heat equator, however, we would expect that this deep layer would at times be brought to the surface by the upwelling known to exist in this region. This would, however, not interfere with its horizontal movement until it reached the surface and moved against the prevailing wind. As is well known, the rotation of the earth about its axis gives to ocean currents in the northern hemisphere a tendency to swerve toward the right. On the equator, however, there is no such tendency, but it increases as the sine of the latitude, although in a latitude so low as 5° N. it would be practically negligible. Hence, in this region, if the surface current moves toward the west the deep layer would move toward the east, and there would be but little tendency for it to diverge toward the southeast and south. This tendency to swerve toward the right is of course not wholly lacking even in low latitudes in the northern hemisphere, and thus the great westerly drift of the surface water of the tropical Pacific eventually turns northwestward and finally northward, and flows along the coasts of China and Japan as the “Japan Current.” If the easterly surface current which is sometimes met with in about 5° north latitude in the mid-Pacific consists of deep water which has been brought to the surface by upwelling, it might, for a time at least, retain some of the characteristics of the deep water of the ocean, even after it reaches the surface. Thus we may readily account for the slightly lower temperature of the water of these easterly currents as compared with that of the 70 Hydrogen-ion Concentration and Electrical Conductivity westerly surface drift to the northward or to the southward. More- over, the Pu of this easterly-moving water ought generally to be lower than that of the westerly drift, for the cold waters of the depths are richer in CQ, than is the warm surface layer. Our observations indicate that this is probably the case; the results upon the eight voyages across the region in the mid-Pacific from 6° N. to 2° S. are shown in table 4. TABLE 4. ; s maf) = J ae Direction of surface Observed range in Average Pu for all current. Pu of surface water. observations. Westerly ss. 2. c S17 tos.20 8.21 INoseurrentin..cs «spas 8.08 to 8.22 8.19 HastenlvVecnon sae oc Sol tors: 2a 8.18 A low Pu when the current is easterly is, however, not always observed. Thus, on August 1, 1920, the ship ran out of the prevailing westerly drift at about 8 a.m. and was in astrong easterly current until about 4a. m. August 2, and yet the Pu of this easterly current was exactly the same as that of the westerly drift to the north or the south of it. Naturally, if deep water rich in CO, comes to the surface, it soon becomes warmed, and thus its excess of carbon dioxide is discharged into the air until the CO, tension of the water is about in balance with that of the air; and after this process is completed it might, due to momentum, still retain some of its easterly movement, but its PH would be the same as that of the prevailing westerly surface drift. It seems probable, therefore, that in cases where we find an easterly drift in the mid-Pacific in about 5° N. latitude, and its Pu is about 8.23, it has been for so long a time on the surface that it has attained the temperature and Px characteristic of the westerly surface drift. If these easterly drifts be derived from deep water which has up- welled to the surface, their average CO, tension ought to be higher than when the current is moving in a westerly direction, and averaging the calculated CQO, tensions of the water between about 6° N. lati- tude and 2°58. latitude, observed on our eight voyages across this region in the mid-Pacific, we find that this seems to be the case, as appears in table 5. It seems, then, that the CO, tension of the surface water in these easterly counter currents is appreciably higher than in that of the prevailing westerly surface drift. The atmospheric CO, has a tension of about 0.0003 of an atmosphere, and thus it appears that these easterly currents probably discharge CO, into the air. In this con- nection it may be of interest to observe that L. R. Cary (1917) found that the oxygen content of these easterly currents was higher than that of the westerly drift. of the Surface Water of the Atlantic and Paczfic. pul Thus the facts seem to warrant the inference that easterly counter currents, such as are found in about 5° N. latitude in the mid-Pacific and possibly also the Guinea Stream of the tropical Atlantic, are composed of water which has upwelled to the surface. Henderson and Cohn (1916, p. 621) conclude from laboratory experiments that upon the whole, in most places and at most seasons, carbon dioxide must be escaping from the sea into the air, although they also state that the balance is doubtless restored by CO, entering the water from the air in the polar regions. These authors did not consider the effect of photosynthesis by marine plants, which McClendon afterward showed to be such an important factor. Were it not for photosynthesis, it is probable that large quantities of carbon dioxide would escape from the sea in the tropics, but, instead of this, McClendon, Wells, and I find that the warm waters are nearly in balance with the atmosphere. TABLE 5. Direction toward Range in CO tension | Average CO» tension which surface current | of water in terms of in terms of 0.0001 was moving. 0.0001 atmosphere. atmosphere, asterlyaeccuen oe oe SI to: 3.5 INorcummentioc « ocuies 3 to 4.4 3.3 Wiesterlyc.tocncas.-. PATE MOY Bier Sal My observations along the Atlantic coast between Nova Scotia and Florida, from December to March, show also that the coastal current during these cold months has a CO, tension somewhat below that of the atmosphere, and this may be due to the great concentration of plant life in these cold waters. Thus, according to my observations, the averages for the shore current, the salinity of which ranges from 30 to 33, between Nova Scotia and northern Florida in winter, are: Temperature 6.7° C., salinity 31.7, Pu 8.05, and CO, tension 2.5 ten-thousandths of an atmosphere, while similar data for the Gulf Stream at the same season between the Straits of Florida and Cape Hatteras are: Temperature 22.3° C., salinity 36.35, Pu 8. 21, and CO, tension 2.8. Thus the cold shore-water seems to be in a condition to absorb CO, from the air, while the warm Gulf Stream waters are more nearly in balance with the atmosphere. In summer, when the shore current is warmed to about 22° C., its CO, tension rises to be quite in balance with the atmosphere. It is well known, from the extensive work of Blackman and Smith, that photosynthesis about doubles in effect for 10° C. rise in temper- ature, but the tropical waters are deficient in nitrogen and can thus support only a meager plant life in comparison with that of colder 72 Hydrogen-ion Concentration and Electrical Conductivity regions. Thus, McClendon found less than 0.01 mg. of nitrogen per liter as nitrates and nitrites at Tortugas, while Raben (1910) found more than ten times these amounts in the North Sea; and, as shown by McClendon, the tropical ocean, despite its high temperature, can eliminate only a small part of its free CO. by photosynthesis, due to the scarcity of pelagic plant life. Krogh (1904) calculated that if the average CO, tension of the ocean is the same as that of the air (about 0.0003 atmosphere), it must contain 27 times as much CQ, as the air. Thus, if the ocean gave off one-tenth of its CO: to the air, the carbon-dioxide tension of the sea would sink to 0.0002 atmosphere. He found that the CO, in the air of Disko Island, Greenland, ranged from 0.00025 to 0.007, being high with winds from the north and west and low when the wind blew from the south and east. The turbid sea-water at Disko Island had a COQ, tension of 0.0001 to 0.00035, while the clear water in the same region had a tension of 0.00035 to 0.0006 atmosphere, thus apparently lower than that of the surrounding air. Also, according to Krogh, the CO, tension of the surface water between Cape Farewell, southern Greenland, and the Shetland Islands was distinctly lower than that of the air. The average CO, tension of the air over the ocean seems to be about 0.000295, this being the mean of 51 observations made by Thorpe between Brazil and England. In 1917, however, using apparatus given to me by Professor McClendon, I tested the CO, tension of the air over the Pacific between Samoa and San Francisco, but there was apparently no relation between the local CO, tension of the air and that of the water under the air, this lack of coordination being due in all probability to the great mobility and rapid fluctuation in CO, tension in the air as compared with that of the water. It would apparently be necessary to obtain several thousand determinations of the CO, tension of air over the ocean, taken at all seasons and in all weathers, to determine its mean CO, tension with accuracy, but the determinations that have been made indicate that it is not far different from that of the air over the land. My average for 83 observations made during eight voyages over the tropical Pacific from 23° north to about 10° south latitude is: Temperature 27°, Pu 8.22, CO, tension 2.99 ten-thousandths of an atmosphere, and salinity 34.87 (range 33.96 to 35.71). Thus the tropical waters appear to have a CO, tension about in balance with that of the atmosphere. If we consider the subtropical and temperate surface water 400 miles and more off the California coast and extending from 35° to 23° north latitude near Honolulu, we find for the average of 46 obser- vations that its temperature appears to be 19.8° C., Pu 8.18, the CO, tension 2.51 ten-thousandths of an atmosphere, and salinity of the Surface Water of the Allantic and Pacific. 73 34.53 (range 32.94 to 35.64). Thus it seems to be absorbing CO, from the air. The surface water within 400 miles of the coast of California, due to upwelling of deep water in this region, seems as a whole to be about in balance with the atmosphere, although closer to shore it is doubtless giving out CO, into the air. Thus, my average for the eight voyages is: Temperature 13.45° C., PH 8.09, CO, tension 3 ten-thousandths of an atmosphere, and salinity 33.15. We lack sufficient data for a definite statement as to whether CO, is on the whole passing from air to sea, or vice versa, but the surmise may seem reasonable that a balance is maintained; the absorption of CO, from the air by the Polar Seas being offset by its passing slowly out of the ocean over the wide area of the tropics, at least during the warmer months of the year. ‘The temperate regions, on the other hand, stand in an intermediate relation, the water absorbing CO, during the winter and giving it out to the air during the summer. TABLE 6. Locality. Pu of sea-water. 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Bicetow, H. B. 1917. Bulletin Museum of Comparative Zoology, Harvard University, vol. 61, pp. 163-357, 2 plates. Cary, L. R. 1919. Year Book of the Carnegie Institution of Washington, No. 17, p. 168. Henverson, L. J., and E. J. Conn. 1916. Proc. Nat. Acad. Sci., vol. 2, pp. 618-622, 1 fig. Krocu, A. 1904. Meddelelser om Grénland, Heft 26, pp. 333-335; 409-434. Mayer, A.G. 1917. Proc. Nat. Acad. Sci., vol. 3, pp. 548-552. 1919. Proc. Amer. Phil. Soc., vol. 58, No. 2, pp. 150-160. McCuenpvon, J. F. 1916. Journ. Biol. Chem., vol. 24, pp. 519-526, figs. 1-5; Ibid., 1917, vol. 30, pp. 265-288. 1916. Medical Review of Reviews, vol. 22, pp. 333-365, 14 figs. 1918. Carnegie Inst. Wash. Pub. No. 252, pp. 213-258, 25 tables, 8 figs. , C. C. Gaurt, and S. MuLHoLiaNnD. 1917. Carnegie Inst. Wash. Pub. No. 251, pp. 21-69, 24 figs. McEwen, G. F. 1910. University of California Publications, Zool., vol. 6, pp. 189-204, 2 text-figs.; Ibid., vol. 15, pp. 255-356, 38 plates, figs. a-c. 1918. julien Scripps Institution for Biological Research, Nov. 8, 1918, No. , 20 pp. Pauitzscu, S. 1911. International Council for Study of Sea, Publications de Circon- stance, No. 60, 27 pp. Rasen, E. 1910. Wissen. Meeresuntersuchungen, Kieler Komm., Bd. 11, pp. 111, 303, 305. Sorensen, 8. P. L. 1909. C. R. Lab. Carlsberg, Kopenhagen, Bd. 8, pp. 1-168; also Biochem, Zeitschrift, Bd. 21, pp. 130-304. SanpstroOm, W. J. 1919. Canadian Fisheries Expedition, 1914-1915, pp. 221-343, 60 text-figs., 15 pls. We tts, R. C. 1918. U.S. Geological Survey, Professional Paper No. 120-A, pp.1-16. EXPLANATION OF THE CHARTS. The charts show the Pu, temperature in degrees centigrade, and salinity of the sur- face water in grams of total salts in 1,000 grams of sea-water. Thus 8.05-11.9°-33.48 June 18 means that the Pa was 8.05, temperature 11.9° C., salinity 33.48 grams of total salts in 1,000 grams of sea-water, and date June 18. Currents are indicated by arrows. These charts represent results obtained on four voyages over the Pacific between Samoa, or Fiji, and San Francisco, or Vancouver, British Columbia, and five voyages over the Atlantic ranging from Nova Scotia to Trinidad, British West Indies. They are intended to give a graphical representation of the characteristic temperature, Px, and salinity in these regions. 60 75 NewYork. Wor (98.04 -6.7 - 3/.42-Dec. 28 08. Washington, WAM J | @ 5.05 @7 95-5.7* Feb./3 8/3-8.2~-30.9-Jan, 26 §4.05-/0.2°- 32.39-Dec.29 108 710.2 31.8-Jan.26 DG 14-14. .04-6.7- DEC.28 -2.8°32.72-Feb.s3 gine = Ba * @Y.95-/45-3/.67-Mar 26 @795027°3/.46-Mar. 26 @8.-3.5°32.36-Marl 25 @ 8.-4.2-32.54-Mar. 25 ”8.03-4.3-3/.73-Mar.25 07- 5.72 31. 51-Jen.27 ee & Newfoundland % 6./-/./-34.37-Mar 8 £33.44-Dec. 24 23-17.2°35.97-Van.25. Charleston gf F735), 44~Jan.25 06.23-/9.5~36.18-DEC.30 OB/T-M.3- 35.71-Feb/S '@6/- 13.5" 32.07-Jan. 24 ©8/7-20.2-36.15-Feb.15 18.22 -/7. F-35.9-Feb. 1G ape Canhveral Wo8/7-2/./536.36-Dec. 3 "| 8.2-22°36.2-Feb.16 ae 18.23 -22.5°36.//-Jan.2/ te @GL3-54.45 36.4-Van./ G22 -22.5° 35.9-Feb./8 SS ©8025} 7.8°30.77-Feb./4 Be cri 13. (98-25-23.55°36.78-Mar./0 AB AFP Porto Rico ius e © Saba.” ‘ pares 8.23-25.3~ 36.08 Mar |. Ay o i) o 60° 75' 8.23-26735. 28-Mar 1 = Pom Ae Op Grenada ut - 2 al Trinidad VOYAGES © Feb.13-19,1918. ® March 8-20,1918. © Dec. 28-Jan. 21,1919. @ Jan.24-27,1919. ® March 25-26,1919. 70° 65° ey) 98.1}-26./731-85-Mar 20 60° “o Cuart 1.—Voyacss IN ATLANTIC OcEAN, 1918 anv 1919. SS SS ee ee ee ae o ° 2 70 180 s . | | | 10 ..Marshall ,lslands ne ° Gilbert, Islands « Ellice*, Islands . 7.85 - 0.5 33.33-May / o pvp aer 3p QF-05-12.3-Feb.NY 0 8./7-15.4°-Feb. 22 @/-16.6~33.89 - Hor. 29 é 8.2-/9.8%35.05- Apr. 28 e°? .2-17.6- Feb. 23 ° 8.p3-21.6° 35.23-Aprl27 @ ROXIE 08.25+2/.6% Feb.25 8.2/- 25.6% 34179 -Apr. 26 @* HA 1.21 - 23.75°- ; ise B.25-24.2~- e427 ol. 8.235-24.05-ADr. 24 24.5° 34.58-Apr.24 @.23- 2¢.55°Apr. 24 <08)25-24.0-Feb.28 6.22- 25.35|- 34.58 -Apr. 23° panpzeray a2 i ipere ans 8./-25.9~ 3 05 -Apr. 22 panes ; Al 5 8./7-24.2~\mar2 o| Gilbert. 8.18 - 24.65" Mar 2% 8.12-24.957 35.26 -Apr. 2l 0° Phoenix Islands 08.2- 264°Mar3 ar 08.15 -26.1° 35, Apr 20 Ellice*, Islands 4 130° 120° 110° © VOYAGE OF S.S.SIERRA ,FEBRUARY 21-MARCH 5,1917.) @ SS.VENTURA, APRIL 19- MAY 1, 1917. Cuartr 2.—VoyaGEs IN Paciric OcEan IN 1917. a / D! re 7 M ie + Oke ve , puters a = ai es hens «i eagle aed Dea eh sas a, - a | ete et sl oeecneneane . = =. — aa 2 8 23-2.83°34./3-Sef 8. 23-29. 3-33.06 -Sé ft wJalvit .23-29.7234.3/ - Sept.9 | satut 8.23-29: 5939-67 - Sent.9+@ Gilbert. 823-29./°35.44-Sept.8 Phoenix Islands —¢ -2 5. 6/-Sept.7 @ islands Union’ : Islands 6.2, 8.25-27°-35.6 Bae e+ | Samoa Islands <0 825- = i595. -s 0 8'25-"26.6~ 35.41-, Fiji ot LHP. 8.25-26.4-Aug./2. Islandse 306.2 25- 25.4~ 35./7-Sept.6 ‘Tonga 170° 180° 170° O VOYAGE OF S.S.SONOMA, JUNE | Wo 180 170° 160° 150° 140° 130" 8.05-13.3-32.3 4 8/7-/8-|33.68-Sept. 17. ° | Se eae Sen Ke 0 06.05-1.9* 33.48-June./8 8./7- 22°2 -34,64 Sept./5 ° 078 /7-18.3+33.53-June 20 é 82 pel" 35.28-Sept 4 @! © 8/8 -20-34.4 ~june © 8.2-22°35.37-June 22 623-25.25°35.28-SeptiS® \, 8.23-24./2.34.92-June 23 HA Wa R23-25.6-35.0/~ Sept.l2. 0"8.23-26.2°- 34.67-June 25 8.23-26.$° 34.67 -Sept.// +® 823-27.4°-35./7-Sept.// *@ * 823-28.2-Sept@ * 8.23-|26.6-Sept./0 @. 823-2.83°34./3-Sepr./0 Ow 6.23-29. 3-33.26 -Sept./0 bd © 8|23-27.6- 34.74 Sie 26 *o 8.23} 28.8-June 27 8.23- 29.5" Sept.9 ty +0 823-286. 8.23-29.7234.3/-Sept.9 @ 0 6)3\- 263-34. 58-June 27 8.23-29.5°34.67- Sept3+@ O+ G/9\- 26.45% 34.79 yune 27 >: % ‘Jaluit “ 9 8:23-26|5~ June 26 oO Baye 3 8.2-28.5°, ip Gilbert. 823-29./°35.44-Sept.8 B= 2719 Tslands « * 6.18 27)|7°-35.28-June 26 P8.9-35.5-Sept.8+@ Phoenix , islands | _. 98.18 -28,3°- 34.7/-June.29 @ 8.23.-28.3- Ellice’. Islands 5. 6/-Sept.7 @- Union : Islands 40 623-26 3- als 30 +o 825-275" 35.41- Aug.I/ bs © 8'25-"26.6~- 35.41 - Aug. /2 6.25-26.4 ae Ie 5-25.4°- 35.7-Sépe.6 / Tonga Fiji 6 < 160° 170° 160 O VOYAGE OF S.S.SONOMA, JUNE 18-30,1918. © S.S.TELUNE, AUGUST II-12. @ S.S.NIAGARA, SEPTEMBER 6-18,1918, CuarT 3.—Voyaces IN Paciric OcEan IN 1918. IV. CARBON-DIOXIDE CONTENT OF SEA-WATER AT TORTUGAS. By ROGER C. WELLS. One figure. 87 CARBON-DIOXIDE CONTENT OF SEA-WATER AT TORTUGAS.’ By Rocer C. WELLS. INTRODUCTION AND SUMMARY OF RESULTS. It is generally considered that the carbon-dioxide content of sea- water may be increased by accessions from the air, by animal life, by the decay of organic matter in the sediments on the bottom or elsewhere, by the solution of carbonate rocks, by the contributions of rivers, and by gas vents beneath the sea. Although all rivers carry carbon dioxide into the sea, most of them actually dilute the sea-water, and should, therefore, as a matter of fact, not be classed as increasing the concentration of the carbon dioxide in sea-water. On the other hand, sea-water may lose carbon dioxide to the air, to plants, and in the formation of carbonate rocks and the carbonaceous parts of organisms. Mere evaporation and precipitation also alter the carbon- dioxide concentration somewhat if other conditions remain un- changed. It is obvious that the actual condition of the water at any given time and place depends on a complex set of factors whose evaluation requires many observations as well as a knowledge of the previous history of the water, including information concerning the currents, flora, fauna, and other agencies that may affect it. With the hope of gaining information on some of these points, the writer has made determinations on sea-water from various localities, which supplement to some extent the work of Schlosing (1880), Dittmar (1884), Murray (1889), Fox (1909), McClendon (1918), and Mayor (1919). Determinations were made at Tortugas, Florida, in June 1919,? on water taken directly from the sea at various points about Loggerhead Key, which reveal unmistakable diurnal variations, first noted by McClendon in 1916. The water has sufficient contact with plants and sea-weeds to show the effect of photosynthesis on its CO, content. There is a loss of CO, by day and a gain by night. The respiration of the animals is not sufficient to keep the equilibrium steady during the day. It is impossible to say to what extent the balance is maintained by the atmosphere, but in this locality, at least, where the water is 1 Published by permission of the Director, United States Geological Survey. 4 Carnegie Inst. Wash. Year Book for 1919, p. 195. ? Carnegie Inst. Wash. Publication No. 252, p. 216 (1918). 89 90) Carbon-Dioxide Content of Sea-Water at Tortugas. relatively shallow, plant life appears to be the chief agency in causing a daily variation in the CO, content, and also a slight deficit in the average content from the theoretical. Even here, however, the departure from the equilibrium conditions required by the equation of Fox, which is based entirely on the physical factors of temperature and salinity, is not great. It is to be inferred that the slight deficit in CO, is accompanied by an excess of oxygen in the water, increased activity in the animal-vegetable cycle, and increased precipitation of calcium carbonate. The diurnal changes caused by photosynthesis are in the same direction as those caused by the diurnal temperature changes, assum- ing complete adjustment of the equilibrium between the carbon diox- ide in the water and that in normal air. This equilibrium can scarcely reach a full adjustment each day, however, even in the relatively shallow water of the lagoon, but if it did it would not account for all of the observed variation in carbon-dioxide content. Theoretically, all surface sea-water should show slight diurnal changes in its gas content, but the mixing of the water and experimental errors have heretofore prevented the demonstration of such changes. I have attempted to discover whether Dole’s figures’ on total carbon dioxide at Tortugas reveal diurnal changes, but unfortunately the data were gathered solely with reference to the tides and do not give the time of day exactly, so that it is impossible to use them for this purpose. As far as can be determined, there is no general cor- relation with the tidal flow, but if the effects of photosynthesis were considered, it is possible that some relationship to the tides could be determined. However, as photosynthetic action extends to considerable depths, it appears doubtful whether unaltered water gains access to Tortugas by tidal action, the keys being situated on the western part of the Florida platform. There is thus no opportunity for an up-welling of deep water rich in CO,, as noted by Mayor in the open water of the Pacific. The source of the water in the flowing tides should be determined, if possible, at the time further samples are taken to test these points. Wood-Jones has shown how markedly the whole development of the Cocos-Keeling Islands is affected by the prevailing winds and currents of the open Indian Ocean. The average diurnal variation in CO, found around Loggerhead Key was about 4.3 per cent of the total CO. There was greater variation than this on some days, as much as 5.6 per cent. Moreover, the absolute quantities of CO. found varied from day to day, the mean being 0.0895 gram per liter at 27.2° C. On one rainy, cloudy day the CO, found averaged 0.091 gram per liter, whereas on a fine day it averaged 0.0885. This is as would be expected with regard to the photosynthetic effect of sunlight, but the data are still too few 1 Carnegie Inst. Wash. Publication No. 182, p. 73 (1914). Carbon-Dioxide Content of Sea-Water at Tortugas. 91 to establish the relationship definitely. Even wider differences are shown in Dole’s figures on different days. No diurnal variations were found in the excess base, and it is doubtful whether any will be, as solu- tion and precipitation of carbonates from sea-water are not thought to be an action that reaches equilibrium rapidly. Even the final CO, distribution attained between sea-water and air appears to be reached slowly, according to the experimental evidence at present available. Determinations of CO, should probably be made soon after the time the samples are collected, on account of the possibility of the decay of organic matter, such as alge, in preserved samples. More- over, the writer’ has shown that sea-water preserved for a long time in glass loses part of its COz, owing to the precipitation of aragonite brought about by the slow solution of alkali from the glass. This was ordinary glass. It is well known that ‘“‘nonsol”’ glass is attacked more slowly. The other change found accompanying the decrease in CO, was a decrease in the titration alkalinity of the water, corresponding to the decreased quantities of carbonate and bicarbonate, but the Pu value was not greatly changed, as sodium had merely taken the place of calcium. The average ‘‘excess base”’ found at Tortugas corresponds to a normality of 0.00239. This titration includes everything that con- sumes acid; it represents chiefly bicarbonate, about 0.00183, some carbonate, about 0.00041, and other substances that contribute to the alkalinity, about 0.00015. The last figure, however, was not deter- mined at Tortugas, but with Gulf Stream water that had been shipped to Washington. This water was collected November 30, 1919, tem- perature 17.94° C., lat. 24° 24’ 20” north, long. 81°31’ 15” west, about 7.5 miles from American Shoal Light, depth 105 meters. Its titration alkalinity, or excess base, was 0.00258, total CO. 0.0968 gram per liter, PH 8.08 at 25°C. These figures give on calculation bicarbonate alkalinity 0.00197, carbonate alkalinity 0.00046, other alkalinity 0.00015. The observations here reported are too few, however, to warrant discussion. The methods used in arriving at these figures are given below. METHODS. Carbon dioxide—The total CO, was determined by adding an excess of hydrochloric acid to 500 c.c. portions of the water and boiling about 15 minutes, while a current of CO, free air was passed through the water, then over calcium chloride, and finally through two soda- lime tubes, each having its last third part filled with calcium chloride. The soda-lime tubes were weighed with a counterpoise to minimize errors likely to be caused by the high humidity. The second tube served as a check on the absorbing power of the first. 1 Jour. Wash. Acad. Sci., 10, 249 (1920). 92 Carbon-Dioxide Content of Sea-Water at Tortugas. Chloride-—The chloride was titrated with silver nitrate, using potassium chromate as indicator, and the density of the water was calculated with the aid of Knudsen’s tables. ng :) i 7 a -_ = 22s i RENIN : : i i | ; et et ar 7 ES fil i ‘ Cloudy N.wind Cloudy Cloudy Rough Fair Fair Rai a Rain Fig. 1— Total carbon dioxide and time of day. H-ion concentration.—The Pu values were estimated colorimetri- cally by comparison with a set of standard tubes very kindly loaned by Professor J. F. McClendon. Excess base-—The excess base was obtained by titrating 100 ec. ec. portions with 0.02 normal sulphuric acid, using methyl red as indi- cator and blowing out the carbon dioxide by means of pure air for TaBLE 1.—Determinations on Sea-Water at Loggerhead Key, Tortugas, June 1919. [t, temperature, °C.; Cl, chlorine, grams per kilogram; D, density; Pu, hydrogen-ion concentra- tion expressed as —log [H+]; Alk., excess base, in terms of a normal solution, or alkalinity titrated with acid, using methyl red and blowing out CO2; COse, total, gram per liter.] | Date, time, and condition of water. t Gi D Pu Alk. CO2 | 14) June) 10; /3230= p.m) cloudy... 2656) | 202010} MhO28 7 Shia sacs alee see 0.0890 Sy Tie U2noon tains cer Ze Gu | s20 e008) MeO02339Fe ence 0.002370 .0925 3 11, 5530™p. m.; cloudy...| 28.2 | 20.05 | 1.02326 |...... ACPA ee a as ae 4 12, 12 noon; after rain....| 27.4 | 19.90 | 1.02332 | 8.23 .002398 .0902 5 13, 6545™ a.m.; after rain.| 27.1 | 19.28 | 1.02258 | 8.19 . 002394 .0924 6 13, 950 a.m.; cloudy...| 27.1 | 19.90 | 1.02342 | 8.19 .002394 .0913 if 13, 12) 50) p.m:; cloudy... .|° 27.5 | 20.07 || 1.02352) || 8-21 . 002410 .0900 8 13 cA eOl spsmeemiens. eine 27.6 | 19.96 | 1.02334 | 8.20 .002402 .0897 9 14, 945 a.m.; after rain Chalo Wallonia a Melaie a bic 27.0 | 19.89 | 1.02344 | 8.19 . 002406 .0881 10 14, 3545™ p.m.; wind..... 27.4 | 19.87 | 1.02328 | 8.22 .002402 .0863 11 14, 20415. pemss wind... eles | 20800) ol 02333elescer .002412 0873 12 Mie) SiO) Elganlgs yebetole 5 Ate 26.5 | 19.94 | 1.02366 | 8.21 .002435 .0910 13 Sy asi sme oe eee er a oe 26054 LOPS e023 | Seon 002424 .0869 14 16, 7510™ a.m.; fair...... 26.8 | 19.99 | 1.02368 | 8.07 .022368 .0904 15 V6" (S05 asm hair. a,c 27.0 | 20.00 | 1.02359 | 8.19 .002414 .0896 16 AGS Satmenhatinrtes oo eley ewe 27.0 | 19.99 | 1.02357 | 8.20 .002440 .0888 ize 16, 4515™ p.m.; fair...... 280 203059) 102830) |s8e23 .002374 .0873 18 16) (5.45. *p-mey tain... cen. 2128) | LOGS) LIO2380 Sot .002410 .0873 19 eNOUSOl CALI setae eras 27.1 | 19.99 | 1.02354 | 8.20 .002414 .0928 20 17,1015 a.m; slightly ecloudyert ee tacae te re 27.3 | 20.09 | 1.02361 | 8.18 .002396 .0910 21 17, 4545™ p.m.; rainy....| 26.8 | 19.16 | 1.02239 | 8.20 . 002306 .0857 22 iv, 7 p.m.; aiter Traine... 27.6 | 19.77 | 1.02308 | 8.19 . 002326 .0893 23 18, 7%15™ a.m.; after SHOWOL. eLearn 26 8 | 20.04 | 1.02370 | 8.18 . 002418 .0918 24 PSN MU ALT ERIM oc se tore ay sie PA Covad AoE TEMPS Sah oe 5 S .002400 .0928 25 Ss) depim-arsine ei cites: PATA MAE HOGS | CORB BY yee .002374 .0879 Carbon-Dioxide Content of Sea-Water at Tortugas. 93 15 to 20 minutes. This method gives sharp results, but the alka- linity thus found probably includes a small amount due to substances other than carbonates. For the alkalinity due to carbonates alone the method at present used by the writer is as follows: A slight excess of 0.02 normal sul- phuric acid is added from a burette to 100 c. c. of sea-water in a non- sol flask. The mixture is then boiled 15 to 20 minutes, while a gentle stream of pure air is passed into it to assist in blowing out the carbon dioxide. (This step might be done under reduced pressure.) The solution is then titrated back with 0.02 normal sodium hydroxide, using cresol red (o-cresolsulphonephthalein) as indicator. This indi- cator is not entirely satisfactory, but is used in order to arrive at an end point of approximately the same Pu value as sea-water. If this end point is not used the titration will include in part the acidifi- cation of certain substances besides carbonates that are present in sea-water. The water is first more than acidified in order to assure the removal of all carbon dioxide. The record of determinations made at Tortugas is given in table 1. Figure 1 shows the relation between the carbon-dioxide content of the water and time of day. GENERAL REFERENCES. Dirrmar, W. 1884. Challenger Rept., Physics and Chemistry, vol. 1, p. 215. Fox, C. J. J. 1909. Faraday Soc. Trans., vol. 5, p. 82. McCEnpon, J. F. 1918. Carnegie Inst. Wash. Pub. No. a pp. 213-258. Mayor, A. G. 1919. Proc. Amer. Philos. Soc., vol. 58, p. 150. Morray and Hyort. 1912. The depths of the ocean. Scuiésine, T. 1880. Compt. rend. 90, p. 1410. We ts, R. C. 1918. U.S. Geol. Surv. Prof. Paper 120-A. Woop-Jonegs, F. 1910. Coral and atolls. Vi: ANALYTICAL SEARCH FOR METALS IN TORTUGAS MARINE ORGANISMS. By ALEXANDER H. PHILLIPS, Princeton University. 95 ANALYTICAL SEARCH FOR METALS IN TORTUGAS MARINE ORGANISMS. By ALEXANDER H. Pariuies. This is the second instalment of analyses of marine organisms collected at the Tortugas. The first was printed in the report of the Carnegie Institution of Washington for 1917, where also the general method of analyses was described. Owing to the smaller amounts of both copper and zinc in these specimens, the method was modified and the copper was determined by the ferrocyanide colorimetric method, and zine was determined by the terbidimetric method described by Victor Berckner.' Weight Weight Manga- Specimen. Name. ficchs dried |Copper.| Zinc. | Iron. nese > | at LLO. as Mno. No. gms. gms. 614-O....} Plexaura homomala..... 1055 398 |0.0006 |0.0016 |0.0021 |0.00021 614-S....| Gorgonia flabellum...... 550 288 .00025} .0025 | .0032 | .00024 614-T....| Pseudoplexaura crassa... 1205 418 .00022| .00023| .0043 | .00029 614-U....| Plexaura flexuosa....... 400 172 .00024| .00024) .0048 | .00015 614-A-B..| Eunicea crassa ......... 110 98 .00014) .00052} .0026 | .00012 614-A-R..| Eunicea rissoe ......... 560 260 .00018) .00066|} .0034 | .00008 614-@) |) Brisriumys.: cence as oe 1235 461 .00009| .00045} .0036 | .00014 614-B-H..| Xiphigorgonia amceps... 445 190 .00008} .00148|} .0032 | .00011 614-A-W .| Gorgonia acerosa....... 480 141 .00012} .00208} .0060 | .00019 GLASWie) ciel TeV OZO Aner ms cracls ec: cielcrs ans .00014| .00084} .00196} .00021 614-C-A..| Toxopneustes .......... .00016} .00046) .0026 | .00009 B42 ASC Mellatay. i: olsen o Ronee 1205 717 .00032| .00042} .00015; .00065 614-A-E...| Clypeaster............- 1345 698 .00026| .00027} .0032 | .00072 614-A-D..| Clypeaster............ 5 1955 956 .00022) .00034! .0036 | .00046 614—B-W,. Diadema’..4. 02.46.00. * 97 -00021} .00006) .0028 | .0002 614-B-A .| Astrospecten........... 255 127 .00029} .00092) .0032 | .00041 614-C....| Cassiopea xamachana...} 10370 472 .00021} .00062} .00016|} .00013 614-B-E; ;|) Pentaceras: 2.0. 22.3% 1175 402 .00036} .00056} .0029 | .00037 614-A-1f.| Holothuria bermudiana. . 1945 404 .00012| .00034| .00072| .00021 614-A-2f.| Holothuria bermudiana. . 725 74 .00011} .00064) .0031 | Trace. 614-AA-2.| Loggerhead sponge...... .00013| .00094| .0015 | .0030 G20ra 4 al) Umi Ose. erste tues Renlele alee: « 3623 280 .00026| .0032 615-A-1..| Mud from Marquesas,Fla. .0032 | .0018 | .0261 | .0058 * Not weighed. + Composed of the body walls, muscles, etc. t Composed of organs and intestine only. The weight of the sample taken for the determination of copper and zine was in each case 20 grams of the material, dried at 110° C. to 1 Jour. Bio. Chem. Soc., June 1919. 97 98 Analytical Search for Metals in constant weight, and the amounts of all the metals, reported in the table above, are in each case expressed in grams per 20-gram sample. The four metals sought occur in all the samples analyzed, but not in as large amounts as in those forms where it is possible to separate the soft tissues from the calcareous skeleton. In some of the present material the calcareous skeleton was by far the larger part, by weight, of the sample taken for analysis, thus reducing the amount of each metal reported in the 20-gram sample, as there is no doubt that the metals here reported are associated to a much greater extent with the soft tissues than with the calcareous skeleton. From a consideration of the amounts of each metal reported in the various organisms, there seems to be no ratio of occurrence or quanti- tative relation of one to the other, but zinc is present in larger amounts than copper. Sample No. 620-a—4 was composed of the soft parts of a number of fresh-water mussels taken from the Millstone River east of Prince- ton, at a point where all the water of the river is collected from a low, level, sandy area far removed from any possible metallic veins. It is interesting to note that this fresh-water form collects both copper and zine from such a natural water and in about the same amounts as do the salt-water organisms. This may be true of other fresh- water forms, but as yet this sample of Unio is the only one analyzed. Iron and manganese in this case were not determined, as it was diffi- cult to completely eliminate the silt contained in the intestines, and as this silt would contain both iron and manganese, their determina- tion under the circumstances would be of no interest. In the note‘‘ A possible source of vanadium in sedimentary rocks it was stated that the conditions for the fixation of these small quanti- ties of metal in the mud and slime at the bottom of the shallows and lagoons are ideal, about the Tortugas at least, as the constant libera- tion of hydrogen sulphide by the mud in the slightly alkaline sea- water would precipitate all four of the above metals as sulphides, even though they were present in very small quantities. To test this, the sample No. 615-A-1 was analyzed for these metals. The sample consists of a mud collected in the shallow lagoon of the Marquesas, Florida, and taken by means of a special sampling apparatus at a depth of 2 feet below the surface of the mud. The results of this analysis are shown in the table. It is not surprising to find iron and manganese, but zinc and copper are both present and in larger quantities than the analyses show them to be present in the organisms; therefore these metals are concentrated in this bottom mud. Unfortunately, no other samples of muds were taken and the extent of concentration of copper and zine can not be determined at present. x71 1 Amer. Jour. Sci., vol. XLvI, p. 471. Tortugas Marine Organisms. 99 Copper has been reported as a component of the surface mud of the ocean bottom at other localities, and both zine and copper have been reported as being present in small amounts in many limestones and dolomites. The calcareous mud of the Marquesas is limestone in the making, and when the conditions under which it accumulates are considered there can be very little doubt that the copper and zinc content is derived from organisms which have concentrated these metals from sea-water, and at death the metal content of their decaying tissues is fixed as sulphides and becomes a part of the limestone or sedimentary rock thus formed. Even though the percentage (0.016 per cent for copper and 0.009 per cent for zinc) is seemingly small, the actual weight of metal present when calculated for large areas of limestone is considerable, as each cubic meter of rock would contain 4382 grams of copper and 243 grams of zinc—an amount quite sufficient to produce metallic deposits of commercial value after secondary concentration by natural agents. VI. THE TRACKING INSTINCT IN A TORTUGAS ANT. By ALFRED GOLDSBOROUGH MAYOR 101 THE TRACKING INSTINCT IN A TORTUGAS ANT. By Aurrep Go.LpsporouGH Mayor. In the preparation of this paper it is a pleasure to acknowledge my indebtedness to Professor William M. Wheeler for his identifica- tion of the ant in question and for references to the literature. All papers previous to 1910 are referred to in Wheeler’s masterly work “Ants,” published by the Columbia University Press, while the studies of later authors, such as Pieron, Turner, Santschi, ete., are referred to by R. Brun (1914, Rie Raumorientierung der Ameisen und das Orientierungs problem in allgemeinen, 234 pp., 51 fig. Jena). Another important paper is by V. Cornetz (Les exploration et les voyages des fourmis, 192 pp., 83 fig. Paris, 1914). In this brief paper we will not attempt to review the already voluminous literature, but refer to it only as it relates to our observations. Monomorium destructor Jerdon, a tropicopolitan ant of East Indian origin, was identified in Florida by Wheeler (1906, Entomo- logical News, vol. 17, p. 265). It is a small, reddish-brown ant, and is a great pest in the wooden buildings of the Tortugas laboratory, making its nests in crevices of the woodwork. So voracious are these insects that we are obliged to swing our beds from the rafters and to paint the ropes with a solution of corrosive sublimate, while all tables must have tape soaked in corrosive sublimate wrapped around their legs if ants are to be excluded from them. These pests have the habit of biting out small pieces of skin, and I have seen them kill within 24 hours rats which were confined in cages. The experiments herein described were made on the flat wooden floor of the laboratory, this flatness having possibly prevented the ants from orienting themselves with respect to conspicuous objects or unevenness in the ground, although I have no evidence that they do this under any conditions. In order to attract the ants, a number of recently killed house- flies were impaled upon a pin; and then, upon looking over the floor, one soon found an ant wandering in a tortuous course over the flat surface. The pin with its lure of flies was then thrust into the floor in front of this foraging ant, which would often pass within 0.25 inch of the lure without perceiving the flies; but if its course were such that it came appreciably nearer than 0.25 inch, the ant suddenly turned toward the flies, and without apparent excitement appeared to ‘‘inspect”’ them, spending a half minute or more crawl- ing over them and stroking them with its antenne. ‘This applies to 103 104 The Tracking Instinct in a Tortugas Ant. dead flies, for if a fly be wounded and moving the ant usually be- comes much excited and proceeds to bite it, and it is remarkable how efficacious the bites are in quieting the fly. In any event this ‘‘finder ant’? soon leaves the flies without carrying off any piece of them, but instead of moving off in the erratic and tortuous path it was pursuing before it found the flies, it now goes in a fairly straight path toward some crevice in the floor, out of which there soon pours an excited swarm of its nest-mates, who proceed toward the flies in a fairly straight path, but which is not necessarily identical with that taken by the ‘‘finder ant” in returning from the flies to the nest. Often the path of the return swarm is not quite right in direction, and thus the ants would pass to one side or the other of the flies; but, curiously, when the right distance has been made and the ants are about to pass the flies, the swarm suddenly breaks up into individuals coursing in random fashion in all directions (the ‘““Turner’s curves” of authors). It is very dramatic to see the straight path of the once orderly file of ants suddenly break into this random wandering, but so accurately do they gage the distance that I have never seen them miss it by more than 2 inches in a journey of 8 feet, while often the direction may be so much in error that in going 8 feet the ants may tend to pass as much as 4 inches to one side or the other of the flies. Of course, in cases when the path of the main swarm does not happen to “‘strike” the flies, but passes to one side and then breaks up, many of the ants will not succeed in finding the flies, but must wander in erratic curves over the floor. A considerable number, however, do find the flies, and within a few minutes a fairly straight swarm-path is established between the nest and the flies. Cornetz, observing ants in Algiers, finds that when an ant returns to the nest it pursues a fairly straight path which is more or less right in direction, but in any event, when the ant has gone the correct distance, it begins to wander in more or less tortuous courses until it finds the nest. EXPERIMENTS. In studying the behavior of the Tortugas ants I would usually place a few recently killed house-flies upon the floor in order to draw out the ants, so that many of them would be constantly moving over the floor in all directions and the entrance to the nest would be well defined by crowds of moving ants in its neighborhood. Often paper was pinned down to the floor in order that the paths of the ants might be drawn accurately in pencil. I. When an ant has discovered a dead fly and is engaged in “Inspecting” it, we may draw a circular line of a solution of cor- rosive sublimate in 35 per cent alcohol about a foot in diameter a The Tracking Instinct in a Tortugas Ant. 105 around the fly. Then, when the ant leaves the fly and proceeds more or less in the direction of the nest, it soon meets with the barrier of corrosive sublimate. This arrests it. It then crawls around close to the inner edge of the barrier, then suddenly goes straight back to the dead fly, and again starts out for the nest, to be again arrested by the barrier of corrosive sublimate. Finally, after re- peating these movements a number of times, it crosses the barrier and goes in a more or less direct path toward the nest; but when it meets other ants and rubs antenne with them they are not excited and no swarm comes back to the dead fly. The fact that the “‘ finder ant”’ has crossed the corrosive sublimate seems to have destroyed its power to excite other ants or to draw them back to the lure it has found. II. Conversely, if after a “finder ant”’ has gone back to the nest, and the swarm is well established and the fly is being torn to pieces, a circle of corrosive sublimate be drawn around the fly, the ants coming from the nest are at once arrested when they reach the outside of the ring of poison. A block occurs and in about a minute nearly every ant between the outside of the ring and the nest is seen to be returning straight to the nest, so that the swarm vanishes in a short time. The ants caught within the ring usually show some hesitation in crossing the poisoned area, and once having crossed, they rarely return to the fly, so that the numbers attacking the fly constantly decrease, due to lack of new recruits. III. On one occasion an ant which was carrying a grain of sand in its mandibles found a dead fly and “‘inspected”’ it in the usual fashion, but did not drop the grain of sand. It then crawled off toward the nest, carrying the sand, but no return swarm came. No inferences can be drawn from this observation, however, for it is based on only a single case, although it seems possible that under certain conditions a “‘finder ant”’ does not produce a return swarm. IV. If after having found the fly the ‘‘finder ant” is allowed to go toward the nest and to rub antennze with several of its nest- mates, and is then gently brushed up from the floor with a camel’s- hair brush and removed, the ants it has encountered show normal excitement; and this excitement spreads by contact to others in their neighborhood, but no return swarm occurs. The excited ants rushing to and fro often cross the path the “‘finder ant”’ traversed in going from the fly toward the nest, but none of them attempts to follow the trail back to the fly. V. If the “‘finder ant,” after having ‘‘inspected” the fly and started toward the nest, is brushed up and carried through the air to the nest-crevice, the ants it falls among may at times display some excitement, but of this I am uncertain. Certainly, however, no return swarm comes back to the fly. Apparently, having been 106 The Tracking Instinct in a Tortugas Ant. carried through the air, the “finder ant’’ becomes incapable of con- ducting a return swarm to the fly. Indeed, it appears probable that the ‘‘finder ant”’ has lost its sense of orientation, for if we brush up an ant from the midst of the crowd moving in the neighborhood of the nest-crevice and carry it through the air to a dead fly, say 8 or 10 feet away, the ant “inspects” the fly in a normal manner, but instead of starting on a fairly straight path back to the nest- crevice, it courses widely over the floor in all directions, every now and then turning and going straight back to the fly, and then starting out again. If in the course of these wanderings it meets with several of its fellows and rubs antenne with them, it then returns to the fly, while its mates follow it in much excitement and a swarm starts; but apparently the ‘‘finder ant’ has lost its sense of the direction of the nest after having been carried through the air. But while this may apply to Monomorium destructor, it seems not to be true for certain other ants. Thus, Brun found that Formica rufa and several other species of ants have a remarkable sense of direction which it is difficult to confuse. This sense, according to Brun, is complex and composed of perceptions which are chemical, topographical, tactile, gravitational, and (as Santschi showed) a perception of the direction of light. They seem also to be able to remember the “‘lay of the land,” even after an interval of 3 weeks between visits to a given region. It is possible that the flat floor of the laboratory at Tortugas presented no topographical features of sufficient definiteness to serve as guides to the ants, who became lost, much as a good woodsman might be lost at sea. It is also possible that different species of ants differ widely in their sense of orientation, and that in Monomorium destructor this sense is some- what poorly developed. VI. If the abdomen of an ant be slightly notched or split with a pair of fine dissecting scissors, so as to mark the ant, she does not seem to be rendered abnormal in behavior by the operation, although such ants are apt to die after a few hours, apparently through injury to the tracheal system. If such an ant finds a dead fly it ‘“‘inspects”’ it in the normal fashion, and then starts off normally, but the other ants pay no attention to it and are not excited if it rubs antenne with them. At times, indeed, the normal ants may interfere with the maimed ant, seizing it by the legs, so as to arrest its progress, but generally they wholly ignore its presence. In any event, when the marked “finder ant’? has met several of her nest-mates and rubbed antenne with them, she starts back in a straight course to the fly, but none of the nest-mates follow it, and thus no swarm occurs. In one experiment such a maimed ‘“‘finder ant” repeated this return journey eight times, each time after having rubbed antenne with its nest-mates, but none of them followed her back The Tracking Instinct in a Tortugas Ant. 107 to the fly. It seems that her instinctive behavior as a “‘finder ant”’ is unimpaired by her injury, but due to this injury her nest-mates no longer recognize her. VII. It is difficult to prove that the ‘‘finder ant” actually con- ducts a swarm of her nest-mates back to the lure she has found, and I tried many experiments to demonstrate or refute this hypothesis. Most of these were unsuccessful, due to the ‘‘finder ant’? becoming indistinguishable from the numerous ants crowding around her. Several instances, however, seem to give a positive result, leading me to infer that the ‘‘finder ant” actually takes the lead and con- ducts the swarm back to the dead fly. These successful experiments were made with unusually small ants, so small that they can be distinguished even among a crowd of their nest-mates, unless, indeed, another equally small individual enters the swarm. ‘These small ants are only about half the size of the average worker. Moreover, workers of normal size are much more numerous, outnumbering these small ants perhaps 50 to 1. These small ants appear to be normal in behavior, for if one of them finds a dead fly it goes through with the usual ‘‘inspection,”’ and then starts off deliberately more or less in the direction of the nest. When it meets a cluster of its nest-mates it rubs antennze with them, and this causes intense excitement, which spreads rapidly by contact from ant to ant. As soon as this is accomplished, the small ant starts back toward the fly in a fairly straight course, but which is rarely or never identical with the path she took from the fly to her mates. The nest-mates crowd around the “finder ant,” and others follow these, so that a moving file of ants is seen rushing toward the fly as if an army were moving along its length with the small “finder ant” in the lead. Of course, if other dwarfed workers were seen in this army the observation was thrown out as non-convincing; but in several instances I clearly saw one of these dwarfed ants lead a small swarm, composed entirely of its larger nest-mates, back to the dead fly, and am thus inclined to think that it is the normal function of a ‘‘finder ant’’ to “personally conduct’ her nest-mates back to the food she has discovered. } ’ Tees ] orn 7 ‘ ’ ; y) ae y J Poa rT ; 7 at ‘ ¢ * a , ot Vent os iff ts fowl be A) . yee "Tr? vr ae vi: is ¢ f f ‘ Ui, ia i roa é i] t a aT \ ’ “4 ‘ VT A COLLECTION OF FISHES FROM SAMOA. BY HENRY W. FOWLER, Academy of Natural Sciences of Philadelphia, AND CHARLES F. SILVESTER, United States Army. Two figures. 109 A COLLECTION OF FISHES FROM SAMOA. By Henry W. Fow.er AND CHARLES F.. SILVESTER. The specimens forming the basis of the present paper were col- lected at Pago Pago in the spring of 1917 by the Carnegie expedition to Samoa. Efforts were made chiefly to secure small or incon- spicuous forms, and, though the collection embraces only 53 species, several are rare and one species is described as new. The collection consists of five lots of small fishes taken from the following localities. First lot, April 5, 1917, from the cove just south of Aua village and 100 feet northwest of Dr. Mayor’s ‘‘Aua line.’”?’ These were taken by lifting bunches of coral from the bottom and then breaking the coral. The second lot has the same data, except a few specimens screened at the bottom with wire and mosquito screening. The third lot, taken April 6, consists of specimens shaken from coral in the reef in front of the hospital, Pago Pago Harbor, Tutuila. The fourth lot was obtained March 20, 1917, from tide-pools near Double Point, just west of the entrance to Pago Pago Harbor. The fifth lot is simply labeled Pago Pago. The collection is now contained in the Museum of the Academy of Natural Sciences, Philadelphia. The ichthyology of Samoa has claimed the attention of several investigators. The most important general account is the ‘ Fische der Siidsee”’ by Giinther.!. This was founded largely on the colored drawings of fishes from various Polynesian Islands made by Andrew Garrett. After the first few parts were published the work was discontinued for a number of years, though in 1910 it was finally completed. Previously some of the fishes collected by the Godeffroy firm, which also financed Ginther’s ‘‘Fische der Siidsee,’”’ were sent to the Vienna Museum and described by Kner and Steindachner.? Later a collection from Savaii and Upolu was made by Rev. S. J. Whitmee and sent to the British Museum. The percoids from this collection are published in Boulenger’s Catalogue. Streets made a small collection about 1876, which he later described. In 1900 Fowler® reported a small collection made at Apia, Upolu, by Dr. 1 Jour. Mus. Godeffroy, I (Heft 1), 1873, pp. 1-24, pls. 1-20; II-III (Heft v-v1), 1874, pp. 25-96, pls. 21-60; IV, 1875, pp. 96-128, pls. 61-83; V (Heft x1), 1876, pp. 129-169, pls. 84-100; VI (Heft x1), 1877, pp. 169-216, pls. 101-120; VII (Heft xv), 1881, pp. 217-256, pls. 121-140; VIII (Heft xv1), 1909, pp. 261-388, pls. 141-160; IX (Heft xvir), 1910, pp. 389-519, pls. 161-180. 2 Sitz. Ak. Wiss. Wien, 54, 1866, pp. 356-395, 5 pls.; l. c., 58, 1868, pp. 26-31, 293-356, 9 pls. Cat. Fish. Brit. Mus., I, ed. 2, 1895, pp. 1-394, pls. 1-xv. 4Bull. U. S. Nat. Mus., VII, 1878, pp. 43-102. 5 Proc. Acad. Nat. Sci. Phila., 1900, pp. 524-528. 111 112 A Collection of Fishes from Samoa. H. C. Caldwell, of the U. S. Navy, and received at the Academy of Natural Sciences of Philadelphia in 1857. The most complete work appeared as ‘“‘The Fishes of Samoa,’’ by Jordan and Seale,! though its scope is widened to include a list of all the species then known from Oceania. Finally Steindachner,? under the title ‘Zur Fischfauna der Samoa-Inseln,”’ reports a collection made by Dr. Rechinger in 1905. OPHICHTHYIDE. Chlevastes colubrinus (Boddaert). One example, 683 mm. Aua Reef, Pago Pago Harbor, June 14, 1920. Head 8.4to vent. When fresh in alcohol grayish white generally, lower surface of tail slightly tinted with pale cream-color. Blackish-brown cross-bands broad, nearly or quite half width of pale interspaces, most all complete, little narrower below, and about edges of each narrow whitish border, Beginning at vent, 10 interspaces with rounded, black blotch within each along fin edge. Also along side large, round, black blotch in each of interspaces, of which several may be dorsal, or some absent and extend for some extent as small blotches. Chlevastes fasciatus (Ahl). One example, 549 mm. Same locality as preceding. Head 8.87 to vent. Differs in dark cross-bands, much narrower, at least much less than one-third width of pale interspaces. Also, all along dorsal surface of trunk pale interspaces, each with small, round blackish blotch but little larger than eye. These extend only on first four interspaces of tail. All dark cross-bands interrupted below, except last three on tail and no dark blotches on anal, which uniform whitish, except last three dark cross-bands. MURANID. Gymnothorax punctatus (Schneider). Head about 8; depth at vent about 19; head width about 3.66 in its length; snout 5.5; mouth 3.5; interorbital 5.5; eye 2in snout. Body moderately long, well compressed, rather slender with convexly flattened sides; tail long, slender, and tapers largely from vent. Combined head and trunk about 1.75 in rest of body. Head rather small, compressed, with slightly swollen pharynx, apparently rather blunt in front. Snout (damaged above) apparently conic and about as broad as long. Eye rounded, little backward in mouth length, without eyelid. Mouth rather small, horizontal. Teeth uniserial in jaws, entire, compressed, attenuate. First 7 teeth each side in front above little larger than others. Vomer in front with 2 similar teeth, front one smaller. No tongue. Row of very small and rather wide-set cutaneous points, minute, along lower lip. Upper lip (damaged) not examined. Jaws apparently equal,® lower jaw with low rami, convex and strong. Front nostril in short, fleshy tube near snout tip. Interorbital convex. Occipital region well swollen or convex. Gill-opening, little below median body axis, little inclined from horizontal, length about equals snout. Pharynx smooth. Skin smooth, tough, rather thick. Along each side of mandible 5 pores. Lateral line obscure, with row of indistinct rather wide-set pores along side medially. Dorsal origin apparently about last third in space between posterior edge of eye and front of gill-opening, fin high, especially on last half of tail, and narrowly contin- 1 Bull. Bur. Fisheries. U. S., XXV, 1905 (Dec. 15, 1906), pp. 173-455, pls. 33-53. * Sitz. Ak. Wiss. Wien, CXV (1), 1906, pp. 1369-1425. * This specimen has the head slightly damaged and due allowance should be made in these proportions, en ee he) ee A Collection of Fishes from Samoa. 113 uous with very small obsolete caudal to anal. Caudal length less than eye. Anal less than half high as dorsal. Vent directly in front of anal origin. Color in alcohol very pale grayish white generally, everywhere marked with small, pale brownish, irregularly crowded dots or specks of variable size and density. Spots pale or obsolete along fin borders, but distinct on basal portions of fins. Under surface of head and belly pale, nearly immaculate. Gill-openings very inconspicuous, pale, same as general color. Iris slaty. Teeth whitish. Length about 173 mm. Only the above example from Pago Pago. Probably pale yellowish generally in life, with dark specks. Gymnothorax goldsborought Jordan and Evermann, a synonym of the above species, differs from our example in coloration, as it is marked with very many minute whitish or pale spots and has a distinct white fin edge, not seen in our specimen. Gymnothorax pictus (Ahl). Head about 7.75; depth at vent about 15.4; head width 2.4 in its Jength; head depth 2; snout 6; eye 8; mouth 2.8; interorbital 6. Body moderately long, well compressed, moderately deep, and with convexly flattened sides, long tail tapering largely behind. Combined head and trunk length equals rest of body. Head moderate, compressed, pharynx scarcely swollen, flattened sides but slightly approximate below, front rather robust and upper profile little concave over eye. Snout conic, tip and surface convex, length seven-eighths its width. Eye rounded, little nearer upper profile than mouth, about midway in gape of latter, without eyelid. Mouth moderate, horizontal, closing completely. Lips rather tough, fleshy, and row of minute papille or filaments around edge of each. Teeth conic, entire, uniserial along jaw edges. Front teeth in each jaw enlarged as patch of several (6 to 8), strong, erect. Single row of small, erect conic teeth down vomer. No tongue. Upper jaw slightly protrudes. Mandible rather low, strong, surface convex. Front nostril in short, fleshy tube about half of eye. Posterior nostril simple pore nearly over middle of eye within interorbital space. Interorbital convex. Occipital region well swollen convexly. Gill-opening near median body axis, slightly inclined from horizontal, about 0.66 of eye. Pharynx smooth. Skin smooth, tough, of about uniform texture. Along each upper lip at least 6 distinct pores well above edge, first slightly in front of nasal tube. Pore directly above upper anterior eye edge in front of posterior nostril. Pair of pores little above bases of front nasal tubes, and another pair well up about midway in snout length. Four distinct pores along each mandibular ramus, well below edge of lip. Lateral line not developed. Dorsal origin about midway between posterior eye edge and front of gill-opening, fin moderately high, though more elevated posteriorly, where confluent with small caudal. Caudal rounded, about long as eye. Anal similar to dorsal, though much lower. Vent about an eye diameter in front of anal origin. Color in alcohol, olive-brownish generally, washed with pale lilac-gray, producing a more or less uniform tint. Though visible to the naked eye as very fine reticulations or specks, under a lens body seen to be everywhere marked with dusky to blackish- brown vermiculations, extremely minute, though well defined. End of tail and muzzle tinged slightly more brownish. No dusky blotch at gill-opening, or at mouth corner; latter pale inside. Iris dull slaty. Teeth pale. One 115 mm. long, from Pago Pago. It differs from any example of the species we have seen in its very minute, dark vermiculations. Among the many figures of Bleeker is none of the small size of our own example or with its color pattern. 114 A Collection of Fishes from Samoa. Anarchias allardicei (Jordan and Seale). Head 6.87; depth at vent about 15.25; head width 3.16 in its length; head depth 2.87; snout 5.75; eye about 9; mouth 3.12; interorbital 5.75. Body moderately short, well compressed, rather deep, with convexly flattened sides and tail tapering rather abruptly behind. Combined head and trunk 1.2 in rest of body. Head moderate, compressed, depth slender and pharynx not swollen, about even in width, convex above and below. Muzzle rather obtuse, upper profile slightly con- cave over eye. Snout obtuse, convex at tip and on dorsal surface, length three-fourths its width. Eye rounded, about median in depth over mouth, little backward in gape length, without eyelids. Mouth rather small, horizontal, completely closes. Lips rather tough, fleshy, entire along edges. Teeth conic, entire, subequal, strong. Upper teeth with one series small, mostly uniform and erect all around outer edge of jaw and inner series of enlarged, depressible, wide-set sharp-pointed teeth on both sides. Lower jaw with similar dentition. Front of vomer with two large fangs and row of few small teeth down its shaft behind. Notongue. Upper jaw tip slightly protrudes, and mandible with strong rami. Anterior nostril short, fleshy tube about as long as pupil, near snout tip. Posterior nostril simple pore over eye center within interorbital space, which is convex. Occipital region not especially swollen, convex. Gill-opening close to ventral profile, as simple pore, size of posterior nostril. Pharynx smooth. Skin smooth, tough, 5 pores along each side of upper lip, first in front of anterior nostril, second close behind base of anterior nostril, third nearer eye than second, fourth below front pupil edge, and fifth close behind eye. Pair of pores above, close to and within front internasal space, second pair midway on snout above, third pair adjoin hind nostrils over eyes. Lower edge of mandible with 5 pores each side, grad- ually more distant from one another backward. No lateral line. Dorsal begins as very slight ridge over gill-opening; extends back also as very slight fold to caudal, where it is a little broader. Caudal rounded, about as long as eye. Anal developed only as low fold on under surface of tail about last two-elevenths of its length, continuous also with caudal. Color in alcohol uniform dusky brown above. Under surface of head, belly, and end of tail tinted brownish; hind edge of latter whitish. Iris pale slaty. One example, 116 mm. long, from Pago Pago. Varies from the original account in the presence of two large anterior vomerine teeth, and no smaller posterior vomerine teeth are mentioned by its describers. The figure of A. allardicei shows the dorsal origin beginning apparently nearer the mouth corner than the gill-opening, while in our example it begins over the gill-opening. A. allardicei has been united! with A.knighti Jordan and Seale, but its mottled coloration and more elevated dorsal doubtless renders it distinct. HEMIRAMPHID. Hyporhamphus pacificus (Steindachner). Head from upper jaw tip 4.6; depth 9.33; D. 1m, 14; A. 11, 16; P. 1, 10; V. 1, 5; scales 66 in lateral series from shoulder to caudal base and 6 more on latter; about 7 scales above lateral line to dorsal origin; 2 scales above anal origin to lateral line; snout about 2.66 in head without beak; eye 3.33; maxillary 4.5; interorbital 4.33; pectoral 2.25; first branched dorsal ray 2.87; first branched anal ray 3.5; least depth of caudal peduncle 5.25; lower caudal lobe 1.25; ventral 3.2. Body elongate, rather robust, slightly compressed, though sides are convex and not flattened, deepest medially. Caudal peduncle compressed, least depth half its length. 1 Giinther, in Jour. Mus. Godeffroy, XVII, 1910, p. 421. A Collection of Fishes from Samoa. 115 Head compressed, flattened sides approximated below where width is half that of cranium, well attenuated forward. Snout long, depressed, width 1.75 in its length. Eye elongately ellipsoid, close to upper profile, slightly advanced in head (without beak). Free portion of upper jaw nearly an equilateral triangle as seen from above, its length 2.4 in snout. Maxillary 1.5 to eye, broadly vertical, width equals pupil. Lower jaw long, slender, so rest of head from upper jaw tip only 0.75 of remainder of beak. Teeth fine, simple, in narrow bands in jaws. Upper buccal fold narrow, lower broader. Tongue elongate, depressed, smooth, free. Nostrils rather large, together, their depression as long as pupil along upper snout edge close before eye. Interorbita] depressed to very slightly concave. Opercle broad, smooth, width 1.25 eye diameters. Preorbital slightly less than eye. Gill-opening forward to front eye edge. Rakers 10+23, lanceolate, 1.5 in gill- filaments and latter 1.75 in eye. Isthmus narrowed, trenchant frenum in front. Seales deciduous, all well imbricated and above computations, largely according to pockets. Dorsal and anal mostly covered with small scales, at least basally, also caudal base. Scales with basal circuli 19. Lateral line complete, apparently low along side, touches at ventral origin, tubes simple and each well exposed. Dorsal origin well posterior, much nearer caudal base than ventral origin or little behind last third in space between caudal base and pectoral origin, anterior rays longest, though their tips extend only to middle of fin when depressed, and entire depressed fin three-fourths to caudal base. Anal inserted opposite dorsal, similar. Caudal well forked, lower lobe much longer than upper (damaged). Pectoral base high, fin 4 to ventral origin, latter midway between pectoral origin and caudal base, fin short or but 2.25 to anal. Vent close before anal. Color in alcohol brownish on back, paler on under side, apparently whitish in life. Down middle of back well-marked dusky line with narrow one each side and parallel, also scale edges same tint. From shoulder, narrow silvery-white band to caudal base, widest below dorsal, where about two-thirds vertical eye diameter, and its upper border tinted slaty narrowly or with deeper line. Fins all pale brownish, vertical ones and pectoral above tinted little with grayish. Iris silvery white, also side of head. Inside gill-opening marked with dusky dots. One 205 mm. long, from Pago Pago. Agreement was found with Hawaiian examples, which have rakers 9+ 22. Hyporhamphus samoensis Steindachner,! as suggested by Giinther, is probably the same. This species is doubtless identical with Hemiramphus dussumieri Valenciennes. MUGILIDZ. Neomyxus chaptali (Hydoux and Souleyet). Head 3.4; depth 3.4 to 3.5; D. IV-I, 9; A. III, 9; scales 37 to 39 in median lateral row to caudal base and 5 more on latter; 12 or 13 scales transversely between dorsal and anal origins; about 21 or 22 predorsal scales; snout 3.5 in head; eye 3.25 to 3.33 in head; mouth width 2.87; interorbital 2 to 2.4. Body compressed. Head broad above, constricted below, upper profile nearly straight. Snout broadly obtuse as seen dorsally, length two-fifths its width. Eye large, posterior edge midway in length of head, rim free. Premaxillaries concealed. Upper front lip thick, width slightly over half of eye. Edges of lips with single row of rather large fleshy papille. Mandible included in upper jaw. Nostrils small, close, near upper edge of snout. Interorbital broadly convex, with slight depression in front. Rakers 22+36, slender, lanceolate, little less than filaments, the latter 1.66 in eye. Scales large, firm, in even longitudinal rows; basal radiating strie 4 to 6, with 3 to 5 incomplete accessory ones; circuli rather coarse. Dorsal, anal, and caudal largely 1Sitz. Ak. Wiss. Wien, CXV (1), 1906, p. 1418, Upolu. 116 A Collection of Fishes from Samoa. covered with small scales. Spinous dorsal origin about opposite pectoral tip. Soft dorsal inserted little behind anal origin. Second anal spine but little shorter than third. Pectoral reaches half-way to anal. Ventral inserted about opposite middle of depressed pectoral. Vent close before anal. Color in alcohol brownish on back, sides and below silvery white. Dorsals and caudal tinted with dusky, also pectoral, the latter with small dark spot at origin. [ris whitish. Length 73 mm. (caudal damaged). Two small examples from Pago Pago. HOLOCENTRIDZ. Holotrachys lima (Valenciennes). One example, 68 mm. long. Holocentrus punctatissimus Cuvier. Head 2.6 to 2.75; depth 2.66 to 2.8; D. XI, 14 or 15; A. IV, 9 or 10; scales in latera; line 35 or 36 to caudal base and 2 more on latter; 4 scales above lateral line to soft dorsal origin; 7 scales below lateral line to spinous anal origin; 7 predorsal scales snout 3.8 to 4.2 in head; eye 2.66 to 2.75; maxillary 2.75 to 2.66; interorbital 4.12 to 4.2. Head about half as long as wide. Snout length two-thirds its width. Pos- terior edge of pupil about midway in head length. Jaws about even. Maxillary two-fifths in eye, expansion 2.75. Bands of villiform teeth in jaws, on vomer and palatines. Interorbital level. Cranial bones striate. Preopercle spine long, strong, reaches back slightly beyond gill-opening, length two-fifths of eye. Preorbital narrow, with several strong marginal spines. Suborbital chain about equally wide as pre- orbital, its serrated edge finer. Edges of suprascapula, preopercle, opercle, and subopercle serrate. Preopercle ridge entire. Rakers m 2+8 11, lanceolate, nearly long as filaments, which one-third of eye. Scales largest on flanks, smaller on predorsal and vertical fin bases. Cheek with 4 rows of scales. Scales with basal parallel vertical strie 18 to 25 (more numerous in larger examples); 7 to 13 strong, broad apical spines. Color when fresh in alcohol pale orange, generally as ground-color. Back with two and a half longitudinal rows of dusky brown, narrow, and not sharply defined, parallel with lateral line above. Below lateral line six and a half broad longitudinal rows of similar color, lower much narrower. Head brownish above, tinged with pale orange below, and each scale on cheek and opercles with pale dusky spot. Spinous dorsal grayish, with first three and last membranes jet black. Other fins all pale orange to whitish. Iris silvery, fading slaty. Four examples, all young, shaken from coral from reef in front of hospital, Pago Pago Harbor, 42 to 45 mm. long. Also 3 examples 64 to 71 mm. long, from tide-pools near Double Point, just west of the entrance to Pago Pago Harbor. These largely in agreement with Giinther’s figure. Two small examples from cove just south of Aua village. These have a conspicuous dark blotch in the front of the spinous dorsal. Holocentrus diadema Lacépéde. Head 2.87; depth 3.33; D. XI, I, 12, 1; A. IV, 9; scales 50 in lateral line to caudal base and 3 more on latter; 5 scales above lateral line to origin of soft dorsal; 9 scales below lateral line to origin of spinous anal; 9 predorsal scales; snout 4.25 in head measured from upper jaw tip; eye 3; maxillary 2.87; interorbital 4.25. Width of head half its length. Length of snout four-fifths its width. Eye with posterior edge of pupil midway in head-length. Closed lower jaw slightly projects. Maxillary reaches beyond anterior edges of eye, not quite to pupil, expansion about one-third in eye. Bands of villiform teeth in jaws, on vomer and palatines. Interorbital level. Cranial 1 Proc. Zool. Soc. London, 1871, p. 660, pl. 60, 2 figs. A Collection of Fishes from Samoa. 117 bones striate. Preopercle spine reaches back only to bony edge of infraopercle. Preorbital very narrow, and suborbital chain but little wider, its edge more weakly serrate than finely serrated preorbital edge. Rakers 1 2+9 m1, lanceolate, about three-fourths of filaments, which one-third of eye. Scales largest on flanks, smaller on predorsal and breast. Cheek with 5 rows of scales. Scales with parallel vertical strie 60 to 70 basally; 8 blunt, short basal denticles; 16 to 18 broad, strong apical spines. Color when fresh in alcohol bright rosy-red generally, with 3 rows of narrow, dark longitudinal bands above the lateral line and 5 broad ones below. Later these faded out below and made up of brownish dots, as seen under a lens. Upper surface of head washed with pale brownish. Iris silvery, fading slaty. Spinous dorsal pale rosy generally, fading whitish, except large median black blotch on first two membranes, then black blotch submarginally after each dorsal spine, and from fourth spine basally black band back to last spine. Other fins all uniform pale rosy, fading whitish. One example, 78 mm. long, from coral in reef in front of the hospital, Pago Pago Harbor. It differs from examples in the Academy of Natural Sciences of Philadelphia in the much shorter preopercular spine and the coloration. It has more whitish on the spinous dorsal, lacks entirely the blackish on the front part of the ventrals and anal, besides having a pale or whitish pectoral axil. Holocentrus praslin Lacépéde. Small examples from tide-pools near Double Point, just west of entrance to Pago Pago Harbor. Holocentrus sammara (Forskal). One example from Pago Pago, 100 mm. long. It agrees with Bleeker’s figure in the anterior dark blotch on spinous dorsal median and lengthwise. Jordan and Seale! describe four examples from Samoa. First has “‘spinous dorsal broadly edged with blood red.” Second has “dorsal maroon, whitish spots at base, tips white, and front of fin with large, black, red-washed blotch.” Third with “large black blotch on front of spinous dorsal.” Fourth with “front of soft dorsal with very large blotch of maroon- black, fin otherwise flesh-color, tips white.” In a Hawaiian example Jordan and Evermann show lengthwise lines made up of dark spots. Also 3 small examples from cove just south of Aua village, April 5, 1917. In two of these the front of the spinous dorsal has a large black blotch and succeeding mem- brane with less distinct dark blotches. Remaining example with spinous dorsal uniformly pale or whitish. CHEILODIPTERID, Amia savayensis (Giinther). Five from reef in front of hospital, Pago Pago Harbor. Fourteen from cove just south of Aua village. These agree in every way with the large series of Philippine examples in the Philadelphia Academy. Amia novemfasciata Cuvier. Adult and young example from tide pools near Double Point, just west of entrance to Pago Pago Harbor, March 20, 1917. Two also from Pago Pago. Fowleria marmorata (Alleyne and Macleay). Head 2.5; depth 3, D. VIII-I, 9; A. IT, 9; scales 23 in lateral line to caudal base and 2 more on latter; 2 scales above lateral line, 6 below; 8 predorsal scales; snout 4.16 in head; eye 3.87; maxillary 2; interorbital 5.5. Body elongate, compressed, deepest at spinous dorsal origin, profiles alike. Caudal peduncle compressed, least depth 1.12 its length. Head large, compressed, profiles 1 Bull. Bureau of Fisheries, XXV, 1905, p. 227. 118 A Collection of Fishes from Samoa. alike. Snout convex on dorsal surface, slightly so in profile, length about three-fifths its width. Eye large, impinging on upper profile, posterior edge about midway in head length. Mouth large, well inclined, lower jaw slightly included. Maxillary extends beyond posterior edge of pupil, not quite to posterior edge of eye, expansion little less than pupil. Villiform bands of teeth in jaws and on vomer, none on palatines. Tongue elongated, rounded tip free. Nostrils together, directly in front of eye. Interorbital level. Preopercle edge entire. Rakers 1+6 short points, about equal to filaments, which one-third of eye. Scales large, little smaller along body edges, and 2 rows on cheek. Basal radiating strie 13 or 14; apical denticles 80 to 90; circuli fine. Lateral line of 5 well-exposed, simple tubes first, then only as row of pores to caudal base, one in center of each scale exposure, and all concurrent largely with dorsal profile. Third dorsal spine longest, reaches back to soft dorsal origin. Soft dorsal and anal alike, opposite, rather elongate, height of former slightly less than half of head. Caudal rounded. Pectoral reaches anal origin, ventral little shorter. Color in alcohol deep brick-brown, with 9 vertical cross-bars, twice width of pale interspaces. Head mottled brownish, with pale lilac tint on mandible and_ branchi- ostegal region. Large jet-black, round blotch, little less than eye, though larger than pupil and margined narrowly with golden-brown. Small, black crescent above opercular spot. Pale bar from eye to preopercle angle, lower edge dusky. Each scale on caudal peduncle with median dusky blotch, rather small, though distinct. Two rows of scales between pectoral and ventral bases on side of abdomen, with slightly oblique, narrow dusky line. Fins all dusky-red. Iris brownish. Length 47 mm. Also smaller examples same locality. Head 2.5; depth 2.87; D. VII-I, 9; A. I, 8; scales about 21 in lateral line to caudal base and 2 more on latter; snout 3.75 in head; eye 3.33; maxillary 1.8; interorbital 4; length 33 mm. This approaches A pogonichthys isostigma Jordan and Seale, in the more spotted appearance, which possibly may not be distinct from A. marmoratus Alleyne and Macleay, as the black spots on the trunk seem to be the chief character of distinction. Both from cove Just south of Aua village, April 5, 1917. SERRANIDZ. Epinephelus merra Bloch. Two young examples from cove just south of Aua village. It differs from the adult stage in the much larger dark blotches. Pharopteryx nigricans Riippell. Two small examples, from tide-pools near Double Point, just west of entrance to Pago Pago Harbor. One example from Pago Pago. All show D. XII. Length 48 to 57 mm. Pharopteryx melas (Bleeker). Two from cove just south of Aua village. In alcohol, body dusky-brown generally, clouded with blackish. Head same, little paler below. Iris slaty. Bases of vertical fins pale or largely whitish, all broadly blackish about outer or terminal portions. Spinous dorsal edge, together with upper soft dorsal edge, especially in front, orange. Pectoral and ventral brownish. Length 50 to 55 mm. One 35 mm. long, same locality. All have D. XI. OPISTHOGNATHID&. Gnathypops samoensis new species. Fig. 1. Head 2.75; depth 3.33; D. VII, 20; A. III, 17; P. 15; V. I, 5; scales from shoulder to median caudal base about 50, and 10 more on latter; 31 tubes in lateral line; 4 scales above lateral line to soft dorsal origin; 22 scales in vertical series below lateral line to ee a ee oe i — A Collection of Fishes from Samoa. 119 spinous anal origin; about 40 predorsal scales; head width 1.8 its length; head depth at occiput 1.4; mandible 2.1; sixth dorsal spine 3.8; sixth dorsal ray 2.75; second anal spine 5; sixth anal ray 2.75; least depth of caudal peduncle 3; caudal 1.87; pectoral 1.6; ventral 3.12; snout 5 in head, measured from upper jaw tip; eye 5; maxillary 2; interorbital 7.5. Body oblong, compressed, deepest at front of spinous dorsal, edges rounded con- vexly. Caudal peduncle well compressed, length about seven-eighths its least depth. Head compressed, upper profile little more inclined than lower, flattened sides slightly approximated above. Snout convex over surface and in profile, short, length half its width. Eye small, advanced, posterior pupil edge near first third in head length. Mouth large, oblique, lower jaw prominent and slightly protrudes, rami robust and moderately high inside mouth. Maxillary extends back slightly beyond posterior edge of eye, though not quite halfway in head length, expansion equals eye. Lips fleshy, moderately broad. Teeth fine, pointed, in bands in jaws and on vomer and palatines. Tongue rather slender fleshy point, free and smooth. Anterior nostril in short tube near front end of snout, posterior one simple pore close to anterior edge of eye medially. Interorbital narrow, nearly level. Preorbital narrow, less than half of eye. Preopercle edge uneven, largely convex. 0 oe.) Soren oo aS 7m = ) ; BTR re Pep ee. bap Oe eee a1 8 BOR SD: EAL. one ay : g WU BSS rf : Ss Fic. 1—Gnathypops samoensis Fowler and Silvester. Type. Gill-opening in front of posterior edge of eye. Rakers rv 1+6 11, broad, asperous knobs, longest three-fourths of filaments and latter one-third of eye. Pseudobranchize large as gill-filaments. Isthmus moderately broad, slightly constricted forward. Scales moderate, smooth on front part of body, as on head, predorsal, breast, and trunk toward end of depressed pectoral, after which finely ciliated entirely. Scales on trunk in even longitudinal rows, small and crowded along edges of body, though less on breast than at most areas. Snout, front preorbital, maxillary, lips, chin, and branchiostegal region naked, head otherwise scaly. Several pores on suborbital chain close to eye, others on mandible. Fourteen scales across widest extent of cheek. Scales on opercle largest on head. Fins all with small scales basally, extending well out on dorsals and anals. Scales with basal parallel marginal strie 11 to 13; circuli parallel laterally and same end as 4 or 5 apical denticles of small size. Lateral line only as superior branch from shoulder along back near upper profile, and not extending beyond soft dorsal. Tubes simple, large, well exposed. Spinous dorsal inserted over origin of pectoral, spines all slender, low, pungent, graduated up to third and then about uniform. Soft dorsal inserted about midway between posterior edge of preopercle and base of caudal, fin uniformly high, rounded behind, and like soft anal posterior rays extend back to caudal base. Second anal spine longest, first little longer than third, origin much nearer origin of pectoral than base of caudal. Soft anal like soft dorsal. Caudal rounded, with median rays longest. Pectoral elongate, pointed median rays longest, reaching anal. Ventral origin slightly 120 A Collection of Fishes from Samoa. in front of pectoral origin, not quite reaching halfway to anal, spine half of length of fin. Vent directly in front of anal. Color when fresh in alcohol rich blackish-chocolate, largely uniform, with slight lilac tinge on branchiostegal region. Fins all largely blackish; also blackish blotch on opercle. Iris dark brown. Length 61 mm. Type No. 50,563 A. N.S. P. Cove south of Aua village, 100 feet and northwest of Dr. Mayor’s “Aua line.” Taken by lifting bunches of coral from the bottom, April 5 A91 7. Also No. 50,564 paratype, A. N.S. P., same data: Head 2.5; depth 3; D. VII, 20; A. IIT, 17; scales from shoulder to median caudal base 50 and 11 more on latter; snout 5.2 in head from upper jaw tip; eye 4; maxillary 2; interorbital 7; length 55 mm. This interesting species has no close allies and is the first occurrence of the family in Samoan waters. (Named for Samoa.) POMACENTRID&. Pomacentrus melanopterus Bleeker. One from Pago Pago, 86 mm., differs from Bleeker’s figure,! as he shows the pre= orbital with a spine, the suborbital rim serrate, and the posterior edge of the preopercle almost entirely serrate. The black pectoral basal blotch is shown as a dark bar within a crescent. Bleeker says,? however, ‘‘ossibus suborbitalibus alepidotus—non vel vix denticulatis; osse preorbitali . . . incisura plus minusve profunde ab ossibus suborbitalibus ceteris distincto, postice rotundo vel in spinullum desienta.”’ Pomacentrus nigricans (Lacépéde). Two young and one adult from tide-pools near Double Point, just west of entrance to Pago Pago Harbor. Apparently the same as Jordan and Seale’s material, as the squamation extends much further forward than shown by Gunther.* All our examples show a black blotch at pectoral axil and another at last dorsal ray bases. Pomacentrus albofasciatus Schlegel. Our material includes 4 examples from coral in the reef in front of the hospital at Pago Pago Harbor; 10 from cove just south of Aua village; 2 from tide-pools near Double Point, just west of entrance to Pago Pago Harbor. Abudefduf ccelestinus (Cuvier). Young example with 6 transverse dark bars, largely reflected on fins, though dorsals and anals largely and caudal completely whitish. Length 18 mm. Pago Pago. Abudefduf glaucus (Cuvier). Four small examples from cove just south of Aua village, and 24, all dull and uniform in color, from tide-pools near Double Point, just west of entrance to Pago Pago Harbor. Abudefduf zonatus (Cuvier). Seventeen from Pago Pago. No trace of the white lateral bar, though head and back are thickly spotted with pale blue. Bleeker’s figure‘ does not show the bluc spots as distinct and variegated as in our examples. Glyphidodon brownriggit Ginther’ has been referred to the present species, but none of his figures show spots, and though his figure A is perhaps closer, it has the dorsals and anals broadly dark. 1 Atlas Ich., IX, 1877, pl. 42, fig. 6. 2 Nat. Verh. Hollands. Maatsch. Wetensch. (Mem. Pomacent.) (3), Deel. 2, No. 6, 1877, p. 55. ? Jour. Mus. Godeffroy, VII (Heft. xv), 1881, pl. 124 f.y. ‘ Atlas Ich. IX, 1877, pl. 407, fig. 3. ¢ Jour. Mus. Godeffroy, VII (Heft. xv), 1881, pl. 127, figs. a, c, e. a A Collection of Fishes from Samoa. 121 Dascyllus aruanus (Linnzus). Three small examples from cove just south of Aua village. Chromis czruleus (Cuvier). Two adults from Pago Pago. Chromis isomelas Jordan and Seale. Head 3.16; depth 1.87; D. XII, 13; A. II, 14; P. I, 15; V. I, 5; tubes 14 in upper arch of lateral line and 7 porous scales in horizontal section before caudal base; 3 scales above lateral line to origin of spinous dorsal; 9 scales below lateral line in vertical row to origin of spinous anal; 19 predorsal scales; width of head 1.5 its length; head depth at occiput 1; snout 4; eye 2.75; maxillary 3.2; interorbital 2.4; fourth dorsal spine 2.12; ninth dorsal ray about 1.75; second anal spine 1.9; ninth anal ray 1.4; least depth of caudal peduncle 2. Body strongly compressed, deeply ellipsoid, deeper midway in combined head and trunk. Edges all convex. Caudal peduncle well compressed, long as deep. Head deep, profiles about evenly inclined, with upper very slightly concave over eye, compressed and flattened sides slope evenly above and below. Snout surface convex, also profile, length half its width. Eye, also pupil, slightly ellipsoid, little advanced or with posterior edge about midway in head length. Mouth small, oblique, terminal. Lips narrow, rather thin. Teeth conic, in rather broad bands in jaws, outer row slightly enlarged, also extend all along premaxillary edge. Mandible even with upper jaw tip when closed, rather shallow and rami well elevated behind inside mouth. Buccal membranes (breathing valves) present inside mouth, upper broader. Tongue pointed, free, smooth, rather elongate. Nostril small, simple pore, about midway on side of snout. Interorbital evenly convex. Preorbital narrow, about two-fifths of eye. Suborbital and preopercle edges entire. Gill-opening forward to anterior edge of eye. Rakers 7+20, lanceolate, but little shorter than filaments and latter slightly less than half of eye. Pseudobranchize large as gill-filaments. Isthmus narrowly constricted in front. Seales large, minutely ctenoid, rather narrowly imbricated, smaller along edges of body. Cheek with 4 rows of scales. Single row of scales on preorbital and infra- orbital. Small scales crowded densely over bases of vertical fins, though on spinous portions forming a sheath basally, row of scales up behind each spine on membrane. Small scales at pectoral base. Ventral with pointed axillary scale about one-third of fin-length, median flap between fins about three-fourths length of axillary flap. Scales with basal radiating strie 7 to 10, sometimes 2 or 3 auxiliaries; small apical denticles 98 to 110; circuli fine. Upper arch of lateral line extends back opposite eleventh dorsal spine base. Tubes large, simple, each well exposed or over first three-fifths of scale; also continued irregularly as 4 pores, then drops a scale and 2 more pores below soft dorsal. Horizontal section of lateral line of simple pores, begins below soft dorsal opposite third pore of upper section, skips 1 or 2 scales, then continues to caudal base. Spinous dorsal inserted immediately after pectoral base or much nearer snout tip than origin of soft dorsal, spines graduated up to third and fourth, the longest, others posteriorly but slightly shorter, edge of fin notched and little cutaneous flap behind each spine tip. Soft dorsal inserted about last third in space between origin of spinous dorsal and base of caudal, fin pointed, median rays longest. Spinous anal inserted opposite ninth dorsal spine base or little nearer pectoral origin than caudal base, second spine longer, first two-fifths its length. Soft anal like soft dorsal, only larger. Caudal deeply forked and outermost rays of each lobe produced in long slender points, length 1.87 in combined head and trunk. Pectoral reaches anal, upper rays longest, and fin about 3 in combined head and trunk. Ventral inserted slightly behind origin of pectoral, first ray ends in filament reaching soft anal origin. Ventral spine three- fifths of fin. Vent directly in front of anal. 122 A Collection of Fishes from Samoa. Color when fresh in alcohol, the front half of entire body is deep blackish brown, hind portion white, line of demarcation very striking or exactly midway between eye center and caudal base. Pectoral base and dorsal jet-black. Iris blackish-brown, golden circle around black pupil. Length 75mm. Pago Pago. Another with same locality shows: Head 3.16; depth 1.87; D. XII, 14; A. II, 18; 14 tubes in upper arch of lateral line; snout 4 in head; eye 2.5; maxillary 3.25; inter- orbital 2.25; length 60 mm. Concerning C. dimidiatus, Jordan and Seale state: ‘It is very close to our Chromis isomelas, but according to the figure by Dr. Giinther, and the description of Dr. Klun- zinger, the posterior boundary of the black area is at the front of the anal fin.” Turn- ing to Giinther’s figure of Heliastes dimidiatus,! one finds such is not the case, as Ginther shows the dark anterior area extending almost to the caudal peduncle, at least over a good portion of the soft dorsal and certainly over more than half of the anals. No dark pectoral blotch is indicated, though Giinther says that the pectoral base is black. It thus appears that his figure represents a variation of C. dimidiatus, and it is quite likely C. isomelas is simply another variation. Klunzinger says? that the dark anterior color extends to origin of anal, bases of pectorals and ventrals black, pectoral hyaline. He mentions only one example, 60 mm. long, and states that its caudal has elongate points. LABRIDZ. Platyglossus notopsis (Valenciennes). Three young examples from Pago Pago, quite unlike the adult in coloration. In alcohol our specimens are generally dull brown. Five longitudinal bands, expanded medially, each bordered broadly with blackish-brown so as to form 10 bands all together. Bands on head much narrower and pale areas thus wider. On trunk inter- vening dark areas of each pair of dark bands mottled or blotched obscurely darker. Dorsals and anals black. Middle of spinous dorsal with large black ocellus, edge narrowly whitish, equal to 1.5 eye diameters. Caudal base with blackish bands extending short space, then end abruptly, rest of fin white. Pectoral with dusky base, fin otherwise gray-white. Ventral dusky. Iris slaty-brown. Largest example 44 mm. Smaller examples show pale brown, general color paler or more whitish, also pale bands much broader. In very young each dorsal white, with 2 black blotches, side with 2 broad longitudinal blackish bands and parallel short band on back and breast. Also bands end on caudal base as black blotch to each caudal lobe. Median blackish band on head above and another below upper forks at interorbital to form band each side of back, and lower extends back to join abdominal band of each side behind. Otherwise coloration whitish or pale. Also, small example from cove just south of Aua village. Cheilinus fasciatus (Bloch). Young example from cove just south of Aua village. SCARICHTHYIDE. Callyodon rubro-violaceus (Steindachner). Head 2.33; depth 2.8; D. IX, 10; A. III, 9; scales 19 in upper arch of lateral line, 5 in lower section to caudal base and 2 more on latter; 1 scale above lateral line and 6 below; snout 3 in head; eye 4.25; mouth 6.5; interorbital 3. Three rows of scales on cheek, of which lower row on preopercle limb. Body elongately ellipsoid, com- 1 Jour. Mus. Godeffroy, VII (Heft. xv), 1881, p. 237, pl. 125, fig. B. 2 Verh. Zool. Bot. Ges. Wien, X XI, 1871, p. 29. A Collection of Fishes from Samoa. 123 pressed. Head rather pointed. Eye large, slightly advanced. No posterior canines, Lips wide, upper covering greater part of teeth. Caudal slightly convex behind. Color in alcohol dull olive-brownish generally, scarcely paler below, and without conspicuous markings, though center of each scale little paler. Dark line on upper lip. Dorsals and anals mottled brownish medianly. Caudal pale brownish, with about 4 very obsolete or faint brownish cross-bars. Iris slaty. Length 46 mm. From coral reef in front of hospital, Pago Pago Harbor. Head 2.33; depth 2.33 to 2.87, D. IX, 10; A. ITI, 9; scales 19 in upper arch of lateral line, 4 in lower section to base of caudal and 1 more on latter; 1 scale above lateral line and 6 below; snout 3.25 to 3.33 in head; eye 3.33 to 3.5; mouth 4.5 to 4.66; inter- orbital 2.75 to 3. Three rows of scalesoncheek. Body elongately ellipsoid, compressed. Head pointed. Tye large, slightly advanced. No posterior canines. Lips moderate, teeth broadly exposed. Caudal slightly convex behind. Color in alcohol pale brownish-olive with 4 broad dark-brownish longitudinal bands, expanded medially and broader than pale interspaces, but not so on head. Pale-brown bar down from lower anterior edge of eye, another from posterior edge. Lips dusted brownish. Head mottled brownish above. At caudal base each median dark body-bar ends as blackish blotch at base of each lobe, fin otherwise whitish. Dorsals and anals largely deep dusky, at least basally, rest with other fins pale. Length 27 mm. Two from cove just south of Aua village. Giinther unites this species with C. ruberrimus Jordan and Seale, and questionably includes Pseudoscarus rubro-violaceus Steindachner and Scarus paluca Jenkins. CHZTODONTID. Chetodon trifascialis Quoy and Gaimard. Two examples, 20 to 31 mm. long, from coral in reef in front of hospital, Pago Pago Harbor. Chetodon pelewensis Kner. One from Pago Pago. A comparison with Giinther’s figure shows how crude his representation really is. The seales in our specimen are all much finer on the fins, pale ocular bar has dark border above the eye, ends of upper jaw dusky, 7 oblique dark bars but little curved and lowest nearly straight, with one on anal sub-basally, the other close to the body edge, though not extending on caudal peduncle. Row of dark spots between each defined dark bar and parallel. Chetodon melannotus Schneider. Young example, 26 mm. long, from coral in reef in front of hospital, Pago Pago Harbor. It agrees largely with Day’s figure, except that the ocular bar is broader, no black submarginal dorsal, anal, and caudal line, and the black on caudal peduncle encompasses most all of the fin, leaving only narrow white crescent across caudal base. Two other examples, 24 mm. long, from cove just south of Aua village. Chetodon miliaris Quoy and Gaimard. Four from cove just south of Aua village, largest 23 mm. Close to the young of C. melannotus, but differ in slightly less inclined lines on sides of body and presence of blackish ventral and anal edge, last more broad anteriorly. Holacanthus nicobariensis (Schneider). One small example from same locality as last. 1 Fishes of India, I, 1875, pl. 28, fig. 1. 124 A Collection of Fishes from Samoa. ACANTHURID&. Hepatus atrimentatus Jordan and Evermann. Small example from coral in reef in front of hospital, Pago Pago. Traces of longi- tudinal blue lines, better separated and fewer than in the original figure.!. Also only trace of black blotch at bases of last dorsal rays. Hepatus triostegus (Linnzus). Four small examples from tide-pools near Double Point, just west of entrance to Pago Pago Harbor. Jordan and Seale say: “This seems like Hepatus sandwichensis, but lacks one cross-bar and is very pale, only four bands on sides.” Such is not the case with our material, as all are like Jordan and Evermann’s Hawaiian figure,? except that they all have the dark cross-bar on caudal peduncle above and below, but broken medianly on each side of caudal peduncle. Jordan and Evermann do not show it on the lower surface of the caudal peduncle. Black bar at pectoral base in our examples extending below only slightly, if at all. MOWNACANTHIDE. Oxymonacanthus longirostris (Schneider). Small examples from cove just south of Aua village. TETRODONTIDE. Canthigaster solandri (Richardson). Three examples, largest 32 mm., from cove just south of Aua village, tide-pools near Double Point, just west of entrance to Pago Pago Harbor and Pago Pago. SCORPANIDZ. Sebastopsis guamensis (Quoy and Gaimard). Two from Pago Pago, larger 84 mm. Six from cove just south of Aua village, largest 75 mm. Four from coral reef in front of hospital, Pago Pago. These agree with Giinther’s figure,* except that he does not show the supraorbital cirrhus. Sebastopsis scabra (Ramsay and Ogilby) is said to differ in its longer anal spine, though this is no longer than in our examples if Jordan and Seale’s! figure is correctly identified. S. parvipinnis (Garrett) is alleged to differ in its minute dermal flaps and rather low, uniform dorsal, both characters possibly due to variation. Sebastapistes laotale Jordan and Seale. One 53 mm. from coral in reef in front of hospital, Pago Pago. Two, 64 and 46 mm., from cove just south of Aua village. Also another, same locality, screened at the bottom. GOBIESOCIDE. Crepidogaster samoensis Steindachner. Two small examples from cove just south of Aua village. GOBIIDZE. Eviota zonura Jordan and Seale. Two small specimens, same locality as last. 1 Bull. U.S. Fish. Comm., XXIII, 1903 (1905), p. 393, fig. 171. 27.\¢., D. 395, fig. 172; 8 Jour. Mus. Godeffroy, IV, 1875, pl. 76, fig. c. ¢ Bull. Bur. Fisher., XXV, 1905, p. 375, fig. 71. A Collection of Fishes from Samoa. 125 Eviota afelei Jordan and Seale. Six from coral in reef in front of hospital, Pago Pago; cove just south of Aua village; tide-pools near Double Point, just west of entrance to Pago Pago Harbor. Probably #. smaragdus Jordan and Seale is identical. Eviota distigma Jordan and Seale. Two from cove just south of Aua village. Small black spots, two in number, on each pectoral base distinctive. Two from tide-pools near Double Point, just west of entrance to Pago Pago Harbor. Pseudogobiodon citrinus (Riippell). Twenty examples from cove just south of Aua village. Variably pale or dark. Some with first dorsal olive, border bright orange, edge narrowly black. Second dorsal olive with yellow border, narrowly edged blackish in some examples; others show pectorals with yellowish tints. The darker examples mostly uniform slaty and without the brilliant borders to the fins. Eleven examples, same locality, screened at the bottom, are variably light and dark, some yellowish, others with orange-bordered dorsal. Three from coral in reef in front of hospital, Pago Pago Harbor, and one from Pago Pago. BLENNIDA. Enneapterygius tusitale Jordan and Seale. One from cove just south of Aua village and 5 from tide-pools near Double Point, just west of entrance to Pago Pago Harbor. Salarias variolosus Valenciennes. One example from cove just south of Aua village. Jordan and Evermann! say: “The fish figured and described by Giinther in Fische der Siidsee as Salarias variolosus from Tahiti? is a different species.” It is also further inferred that the specimen in the Academy of Natural Sciences of Philadelphia from the “Sandwich Islands,” collected by Thomas Nuttall, is not identical with Giinther’s fish. A comparison of Nuttall’s specimen with our Samoan leaves no doubt as to their identity. Salarias gibbifrons Quoy and Gaimard. One from cove just south of Aua village. Alticus biseriatus (Valenciennes). (Fig. 2). Head 3.33 to 4.12; depth 3.66 to 5; D. XIII, 18 to 20; A. 22 to 24; P. 15; V. 2; head width 1.5 its length; head depth (without crest) 1.4; eye 2.5 to 3.5; mouth width 2; first dorsal spine 2; fifth dorsal ray 1.33; fourth anal ray 2.33; least depth of caudal peduncle 2.33; caudal 1; pectoral 1.25; ventral 2. Body elongate, slender, tapers back gradually and evenly from head to caudal peduncle. Latter not free, compressed. Head small, robust, checks and lower sides little swollen. Snout very obtuse, front profile vertical and slightly convex, breadth opposite front of eyes about equals twice its length to upper jaw end medially. Eye large, antero-lateral, moderately elevated, posterior edge near first third in head. Mouth broad, with short gape, inferior, so front of lower jaw about opposite eye center. Each side of lower jaw with large posterior canine. Teeth minute otherwise, very close-set, pointed, in single narrow flexible row. Interorbital narrow, not one-third of eye, level. Anterior nostril level with and close before lower eye edge and with short fleshy tentacle about one-third of eye. Posterior nostril close above anterior or lower nostril, also nearer eye, simple pore. 1 Bull. U. S. F. Com., XXIII (pt. 1), 1903 (1905), p. 498. 2 Jour. Mus. Godeffroy, VI (Heft. x1), 1877, p. 203, pl. 116, fig. a. 126 A Collection of Fishes from Samoa. Gill-opening forms free fold across broad isthmus. Rakers at least a dozen short points, about one-third of filaments and latter one-third of eye. Body covered with smooth skin. Head with median cutaneous keel or crest in largest individual only, arises opposite posterior edge of pupil and not quite to dorsal origin, its length 1.6 in head. The smaller individuals show a pair of short nuchal tentacles. Long, pointed, fleshy flap above eye 1.5 eye diameters, each edge with several (3 to 5) short tentacles. Row of pores behind eye along suborbitals, another down preopercle, third one on mandible. Spinous dorsal origin slightly in front of gill-opening edge, only last few spines graduated down short, as deep notch before soft dorsal, and fin largely uniform with entire edge. Soft dorsal with entire edge, large, uniform in height, last ray joined by membrane to upper caudal peduncle edge, but not to caudal fin. Anal origin about opposite tenth dorsal spine base, fin uniform in height, free from caudal peduncle behind, membrane behind each ray tip notched. Caudal rounded. Lower median pectoral rays longest, fin almost reaches anal. Ventral inserted slightly in front of pectoral origin, inner ray slightly longer, halfway to anal origin. Color in alcohol dull lavender-brown generally on back, lower and under surface pale or whitish. Trunk with a dozen dusky-brown vertical blotches somewhat arranged as if in pairs, joined above alternately on back with small, dusky, vertical Fia. 2.—Alticus biseriatus (Valenciennes). blotches extending up somewhat on bases of dorsal fin. On head dusky clouded blotch behind eye, one on opercle, one on cheek below, one from edge of lower eye to mouth, and one on snout. Number of indistinct, small brownish ocelli, much less than pupil, on head. Crest dusky, mottled paler. Trunk mottled with scattered paler dots and obscure marblings. Fins largely grayish, spinous dorsal with membranes medianly anteriorly, or until seventh, with jet-black round blotch little less than eye, and on rest of fin gradually become pale dusky behind. Soft dorsal, except base, with many even oblique pale gray-blue lines, up and backward. Caudal with dark blotch little less than eye at bases of median rays, lower and submarginal part of fin dusky. Anal with long submarginal dusky band. Dark obscure transverse streak at pectoral base. One specimen, 50 mm. in length, from tide-pools near Double Point, just west of entrance to Pago Pago Harbor, March 20, 1917, and three specimens, 21 to 30mm., same locality. Salarias rivulatus Riippell. Eleven dark examples, largest 98 mm. Pago Pago. Enchelyurus ater (Giinther). Two from cove just south of Aua village. Jordan and Seale state that “Giinther describes the ventrals as reaching the anal, but in his figure the fins are much shorter.” This is probably a variation with age. FIERASFERIDZ. Jordanicus parvipinnis (Kaup). One, same locality as last. Vit. LEODICIDA FROM FIJI AND SAMOA. By A. L. TREADWELL, Professor of Zoology in Vassar College. Eight plates and 68 text-figures. CONTENTS. Page lntroductloni er: ater ee ene ae 129 Systematic descriptions............... 130 Arabellay Grube secmeies sem beastie ete. 160 Arabella dubia Treadwell............... 160 Dorvallea Partitbs + sy. ccs see leie te taser 166 Dorvillea australiensis McIntosh........ 166 Dovalleinss si cece So ee ek Realtor 166 Drilonereis;Claparedes. es 4-- sss oe 161 lumbricus Treadwell......... 161 paucidentata Treadwell ..... 162 Meadice i Savigny.n ceva oce: eile eevee aie 130 Leodice aciculata Treadwell............ 143 antennata Savigny............. 136 aphroditois' Pallas. .2). 22... 2: = 134 armillata Treadwell............ 144 biformi-cirrata, Treadwell...... . 148 coccinea Gruber ssa eemas senses 142 crassi-tentaculata Treadwell..... 146 flava-punctata Treadwell....... 136 gracili-cirrata Treadwell ........ 149 suviensis Treadwell............ 138 tubicola Treadwell............. 139 VATIGISI GLY te ciel cin ena aie tee eae, © 131 Page Leodice viridis Gray, var. vernalis Tread- wells Shier eee see 133 CO CI CIAD ooh hoe eRe 130 TiCOCICINEE sepsis) aa eha ei arse Dhoea eee 130 Lam brinereine. asec. s cs ite oe weenie 157 Lumbrinereis de Blainville.............. 157 Lumbrinereis brevicirrata Schmarda.... . 158 japonica v. Marenzeller.... 159 sphzerocephala Schmarda... 158 yaidice Savignyy senior ace oeee 154 Lysidice fusca Treadwell............... 154 parvaclreadwelll; ss... 15 seinem: 155 IMarphysa Savignyenry eetisc : < Bay Sera TF) aN Cae < s; CMe os eae aieys SS 2 ° a % “A 0 +) uf Mar EX R $ Np ‘SITA eo Sina - Nz 7 wed y s Mt. MATAFAO> = ee 2% 7 AWN Ry te] o ee Bmwoe TUTUILA ay 1S ‘ WEST CAPE